Invited Review

International Harmonizationof Nomenclature and Diagnostic
Criteria (INHAND): Nonproliferativeand Proliferative Lesions of the Minipig

Toxicologic Pathology
2021, Vol. 49(1) 110-228ª The Author(s) 2021
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DOI: 10.1177/0192623320975373
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Mikala Skydsgaard1 , Zuhal Dincer2, Wanda M. Haschek3, Kris Helke4,
Binod Jacob5, Bjoern Jacobsen6, Gitte Jeppesen7, Atsuhiko Kato8,
Hiroaki Kawaguchi9, Sean McKeag2, Keith Nelson10 , Susanne Rittinghausen11,
Dirk Schaudien11, Vimala Vemireddi12, and Zbigniew W. Wojcinski13

Abstract
The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/
inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan(JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative
lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying
microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of thelesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is alsoavailable electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases fromgovernment, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as
lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely
accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common
language among regulatory and scientific research organizations in different countries and increase and enrich international
exchanges of information among toxicologists and pathologists.
Keywords
minipig, pathology, toxicopathology, nomenclature, background findings, INHAND

1

Novo Nordisk A/S, Måløv, Denmark
Pathology Department, Covance Laboratories Limited, Harrogate, United
Kingdom3
Department of Pathobiology, University of Illinois, Urbana, IL, USA
4
Medical University of South Carolina, Charleston, SC, USA
5
Merck & Co, Inc, West Point, PA, USA
6
Roche Pharmaceutical Research and Early Development, Pharmaceutical
Sciences, Roche Innovation Center, Basel, Switzerland
7
Charles River Laboratories Copenhagen, Lille Skensved, Denmark8
Chugai Pharmaceutical Co, Ltd Research Division, Shizuoka, Japan
2

9

Kagoshima University, Kagoshima, JapanCharles River Laboratories, Mattawan, MI, USA
11
Fraunhofer Institute for Toxicology and Experimental Medicine ITEM,
Hannover, Germany
12
Covance Laboratories, Chantilly, VA, USA
13
Toxicology & Pathology Consulting, LLC, Ann Arbor, MI, USA
10

Corresponding Author:
Mikala Skydsgaard, Novo Nordisk A/S, Måløv, Denmark.
Email: mqsy@novonordisk.com

Skydsgaard et al.

Table of Contents
Chapter 1.
Introduction
Chapter 2.
Systemic Pathology
Chapter 3.
Cardiovascular System
Chapter 4.
Digestive System
Chapter 5.
Endocrine System
Chapter 6.
Hematopoietic and Lymphoid System
Chapter 7.
Hepatobiliary System
Chapter 8.
Integumentary System
Chapter 9.
Mammary Gland
Chapter 10. Nervous System
Chapter 11. Reproductive System—Female
Chapter 12. Reproductive System—Male
Chapter 13. Respiratory System
Chapter 14. Skeletal System
Chapter 15. Soft Tissue
Chapter 16. Special Senses
Chapter 17. Urinary System
Figure Legends
References

Chapter 1. Introduction
The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria) is a joint initiative of the societies of toxicologic pathology from Europe (European Societyof Toxicologic Pathology [ESTP]), United Kingdom (British
Society of Toxicological Pathologists [BSTP]), Japan (Japanese Society of Toxicologic Pathology [JSTP]), and North
America (Society of Toxicologic Pathology [STP]) to update
the existing World Health Organization/International Agency
for Research on Cancer (WHO/IARC) and Society of Toxicologic Pathology/Standardized System of Nomenclature andDiagnostic Criteria (STP/SSNDC) nomenclature systems. The
INHAND nomenclature and the related diagnostic criteria represent a consensus of experienced toxicologic pathologists andwere reviewed by the INHAND-Global Editorial and Steering
Committee (INHAND-GESC) for compliance with INHAND
principles. Members of the societies of toxicologic pathology had
the opportunity to comment on the draft versions of INHAND
documents during a 60-day review period. The initial series ofnomenclature publications was focused on lesions in rats andmice. With interest of the United States Food and Drug Administration (FDA) in the use of published terminology standards andthe decision of the Clinical Data Interchange Standards Consortium (CDISC) initiative on Standard for the Exchange of Nonclinical Data (SEND) to model the controlled terminology (CT)based on the INHAND nomenclature, the INHAND project was
extended to other laboratory animal species including the nonhuman primate, rabbit, minipig, dog, and fish.
Although the INHAND nomenclature and diagnostic criteria
represent a preferred international standard nomenclature forlesions identified in nonclinical studies, recommendations fordiagnostic criteria and preferred terminology may not be applicable in all situations. The purpose of specific experiments or the111
specific context of a given study may require deviation from thisstandardized nomenclature and diagnostic criteria. The appropriate diagnoses are ultimately based upon the scientific judgment of the study pathologist.
The present publication provides standardized terms anddiagnostic criteria for histopathologic observations to be usedin nonclinical toxicology studies conducted in the minipig (Sus
scrofa), which is increasingly used as a nonrodent species in
nonclinical toxicology studies. The different breeds of minipigs
used in the field of nonclinical safety assessment include theGöttingen, Hanford, Yucatan, Wuzhishan, and Sinclair minipig, with the Göttingen as the most commonly used breed in
North America, Japan, and Europe. The nomenclature, observations, and comments included in this article cover all thesebreeds, but most data are derived from the Göttingen minipig.
The focus of the present publication is on standardized terms
and diagnostic criteria for histopathology findings that occur innonclinical safety studies conducted in the minipig and will notinclude all background lesions of domestic pigs. References todomestic pigs have been included, where appropriate.
Throughout this publication, findings applicable for use in general toxicology studies in minipigs are tabulated by organ system. The nomenclature and tables in this publication build on
the existing INHAND rodent nomenclature. In most instances,the definition and description of the rodent lesion applies to theminipig and is not described further. This publication focuses
on findings that are unique to the minipig and are not observedin rodents, findings in the minipigs that share the same terminology with a rodent finding but display different morphologic
features, or findings for which additional commentary specificto the minipig is needed. Findings that are unique to rats ormice and are not found in minipigs are denoted accordingly inthe tables in this article. The tabulated findings are categorizedaccording to the following characteristics: “common,”“uncommon,” “not observed but potentially relevant,” and “notapplicable.” The distinction between common and uncommon
findings is based on the authors’ experience regarding theoccurrence in untreated minipigs and, when available, published references are included. The uncommon category is
reserved for changes only observed sporadically as spontaneous findings in minipig studies or those that are almost exclusively induced by xenobiotics. “Not observed but potentiallyrelevant” are changes that have not been described or observedin minipigs; however, the use of these terms has been considered permissible should a lesion meet the diagnostic criteria.
The category “not applicable” refers to rodent-specific findings
and terms, and the use of these terms in minipigs is consideredinappropriate; examples include chronic progressive nephropathy of the kidney or fibro-osseous lesion of bones. It should be
noted that minipigs used in toxicologic studies are usually
young in age and are kept on study only for a relatively shorttime frame, a fraction of the normal life span of the minipig.
Prior to study initiation, the health status of individual minipigs
is usually carefully checked and the individual minipigs
selected for a study are in good health. For these reasons, thespectrum and frequency of changes are different from those112

Toxicologic Pathology 49(1)encountered in domestic pigs and diagnostic laboratories, andcommon age-related findings including neoplasms are rare.
Neoplasms described in this publication are limited to thoseobserved by the authors or described in the literature. Whenever possible, the equivalent rodent term/SEND terminology
should be used for any tumors not specifically addressed in thisarticle, as appropriate.
In addition to this journal publication, the nomenclature anddiagnostic criteria for the minipig are also available online(www.goreni.org). The online version contains additional
images and useful links to differential diagnoses as well as
trimming guides, making it a practical tool for toxicologic
pathology and experimental work. In addition, all INHAND
publications are available at www.toxpath.org and www.euro
toxpath.org/nomenclature.
Across the INHAND publications, the recommended nomenclature is generally descriptive rather than diagnostic. The criteria used are based only on standard hematoxylin and eosin(H&E)–stained paraffin-embedded sections. Histochemical or
immunohistochemical staining, and occasionally electron microscopic, characteristics may be addressed in the comments section for the respective finding. Such special techniques may berequired in some situations, but a comprehensive discussion ofthese methods is outside the scope of this publication. Systemic
nonproliferative findings that occur across organ systems and are
not specific to an organ are reviewed in the section on Systemic
Pathology. Instead of “synonyms” for each term, the nonrodent
publications have used the notation “other terms.” While thesesynonyms or other terms have been used historically, the primary listed term is the preferred term and will link to the controlled terminology (CT) in SEND.
Findings included in this nomenclature system may be further
specified by modifiers. Criteria are given for modifiers that are ofparticular relevance. These modifiers should be consistently
applied. Additional modifiers not provided in this nomenclaturesystem may describe the location, tissue type, or duration, among
others. General principles of the INHAND nomenclature have been
published separately.1 As new information becomes available, newterms and modification of current terms will be needed from time totime and a request for this new term or modification will be appliedby “change control” (see goRENI and STP websites).
The minipig is a good animal model for the human withexceptional relevance in several organ systems, including skin,cardiovascular system, gastrointestinal tract, and kidney. In the
introduction to each of the following organ systems, a short
description of, or reference to, any specific features of thesystem that can potentially contribute to risk assessment isincluded, as consideration of features common to minipigs andhumans can be important in the design or risk assessment of
nonclinical safety studies.
Recommended general references regarding background
pathology of minipigs include the following:
Dincer Z, Skydsgaard M. Spontaneous/Background Pathology of Göttingen Minipig. The Minipig in Biomedical
Research. CRC Press; 2012:305-320.
Glerup P, Grand N, Skydsgaard M. The use of minipigs in
non-clinical research. In: Haschek, Wanda M. C. G. Rousseauxand M. A. Wallig. Haschek and Rousseaux’s Handbook of
Toxicologic Pathology. 3rd ed. Vol 2. Academic Press;
2013:461-475. Chapter 13.
Helke KL, Nelson KN, Sargeant AM, et al. Pigs in toxicology: Breed differences in metabolism and background findings. Toxicol Pathol. 2016;44:575-590.
Jeppesen G, Skydsgaard M. Spontaneous background pathology in Göttingen minipigs. Toxicol Pathol. 2015;43:257-266.
Mclnnes E. Minipigs. Background Lesions in Laboratory
Animals. A Color Atlas. Mclnnes E, eds. Saunders Elsevier;2012. Chapter 6.

Chapter 2. Systemic Pathology
There are a number of microscopic findings that may be seen
across several organs and/or tissues and are not specific to justone organ system. There are also a number of different microscopic findings that are present across several organs and/or
tissues that together constitute a syndrome. Those findings that
occur in multiple tissues are listed here, and they are also
described under the organ systems in which they occur if they
have unique features. Syndromes specific to the minipig are
mentioned in individual chapters, but their definitive descriptions are presented here.
The table gives an indication of how frequently the changes
may be observed in the minipig, associated diseases, conditions,
etiologies or inducing agents, and a list of tissues where they
may be found. Where further explanation is deemed useful,selected lesions are discussed in more detail below the table.

Table 2.1. Microscopic Findings of Systemic Pathology (Generally Used Preferred Terms): Minipig.
Systemic pathology
Congenital
Aplasia
Ectopic tissue
Hypoplasia
Nonproliferative
Abscess
Accumulation, adipocyte
Accumulation, adipocytes, interstitial

Common Uncommon Associated diseases/conditions
X

Developmental anomaly
X

X

Developmental anomaly
X

X
X

Tissues commonly reported in:Gall bladder
Accessory adrenal cortical tissue,
accessory spleen, ectopic thyroid
Gall bladder
Mandibular lymph nodePancreas, skeletal muscle
Kidney(continued)

Skydsgaard et al.

113

Table 2.1. (continued)
Systemic pathology

Common Uncommon Associated diseases/conditions

AmyloidX

Angiectasis
Apoptosisa
Basophilic granulesb

X
X
X

Congestion
Ectopic tissue

X
X

Edema
Extramedullary hematopoiesis(EMH)a,b
Fibrosis

X

Hemorrhage—Multiple organs

X

Infiltrate, eosinophila

X

Infiltrate

X

X

X
X

Inflammation—Multiple organs

X

Metaplasia, osseous—Multiple organs
Metaplasia, squamous cell

X
X
X

Necrosis—Multiple tissues
Pigment—Multiple tissuesa

Regeneration
Serous atrophy of adipose tissuea
Vacuolation, macrophagea
Infectious
Bacteriab
Parasite
Neoplastic
Lymphomaab

Bleeding disorders (hemorrhagic syndrome),
very young animals (<6 weeks)

X

Infiltrate, vascular/perivascular
Inflammation, vascular/perivasculara

Mineralization—Multiple tissues

Antisense oligonucleotides

X
X

X
X
X

Tissues commonly reported in:
Kidney, thyroid gland, adrenal gland,
mandibular lymph node, liver,spleen, stomach, duodenum,
jejunum, ileum, cecum
Bone marrow, lymph node, spleen
Multiple tissues
Hepatocytes, macrophages, renal
cortical tubules
Multiple organs
Accessory adrenal cortical tissue,
accessory spleen, ectopic thymus,
ectopic thyroid
Skeletal muscle and soft tissues
Adrenal, liver, spleen
Catheter-associated tissues, lung,bone marrow, periosteum
Bone marrow, bronchi, bronchioles,
heart, joints, larynx, skeletal
muscle, soft tissues, testis, thymus,
thyroid, trachea
Brain, lungs, mesenteric lymph node,
renal pelvis, suburothelial, skin,gastrointestinal tractb

Modifiers: Eosinophil; lymphocyte; plasma cell; Multiple tissues
macrophage; monocyte; mononuclear;neutrophil; mixed cells
Lung, nasal cavity, stomach, meninges
Individual small to medium-sizedarteries, especially lung,mesentery, renal pelvis, stomach
Neutrophil; mononuclear cell; lymphocyte;
Multiple tissues
monocyte; mixed cells; lymphoplasmacytic;
pyogranulomatous; granulomatous; acute;subacute; chronic; chronic active
Lung
Oviduct, sublingual salivary gland,
uterusOvary, kidney, cerebral andcerebellar leptomeninges, pineal
gland, pituitary, blood vessels,lung, salivary glands
Multiple tissues
Exogenous: Tattoo ink, inhaled particulate
Adrenal, bone marrow, dermis,
matter, test article-associated inert orkidneys, liver, lung lymph node,
insoluble pigments, endogenous:
meninges, pineal gland, spleenhemosiderin, lipofuscin, ceroid, melanin
Skeletal muscle
Asymptomatic
Bone marrow of femur and sternum,
coronary groove, and subcutis
Phospholipidosis
Kidneys, liver, lungs, lymph nodes,
spleenX
X

Coccidiosis (Isospora, Eimeria)

Injection/infusion sites, skin
Intestine

X

Lymphocytic

Liver, small intestine, multiple tissues

Terminology with diagnostic criteria or comments described below.
Lesion may be induced.

114

Toxicologic Pathology 49(1)media or wall, artery, inflammation, media or wall, artery
fibrosis, perivascularAmyloid
Eosinophilic, homogenous, fibrillar material that stains orangeor red with Congo Red and exhibits apple green birefringence
under polarised light has been observed in aged (8 years orolder) Microminipigs. Interstitial deposition was noted in kidney, thyroid gland and adrenal gland, and in the splenic cords
and the walls of arterioles of several organs.2

Diagnostic features
In the minipig, inflammation of the blood vessels usually has
one of two appearances:

Apoptosis
For a full discussion, see Elmore S. Apoptosis: a review ofprogrammed cell death. Toxicol Pathol. 2007;35(4):495-516.

Ectopic Tissue

A fibrinoid necrotic form, where inflammatory cell
infiltrates associated with necrosis and fibrin deposits
in the vessel wall are present in all layers of theartery.
A chronic form, where the vessel wall is thickened by
fibrosis and extends into the surrounding tissues withassociated mononuclear cells.

Comments
Accessory adrenal cortical tissue may be found adjacent to the adrenal gland or attached to it and surrounded by a fibrous capsule.3
Ectopic spleen has been recorded in the pancreas of a cloned Yucatan minipig, and accessory spleens may be found in the gastrosplenic
ligament.4 As the parathyroid is often found embedded in the cervical thymus, it is not considered an ectopic tissue in this location.

Comments

Extramedullary hematopoiesis is generally greatest at around
14 days postpartum, is decreased or absent by 35 days, and isabsent after 63 days5 or 6 weeks.6,7 In older pigs, extramedullary hematopoiesis may be seen in response to severe anemia.
The INHAND term for rodents is “extramedullary hematopoiesis, increased”; however, in minipigs over 6 weeks old, as this is
not considered a normal feature of the spleen, the modifier“increased” has been dropped.

Arteritis/polyarteritis is an occasionally observed spontaneously occurring background change in Göttingen minipigs. It can be present in a small- or medium-sized single
artery of a single organ or several organs in an animal or inseveral animals in a study. The severity is generally minimal or mild but can occasionally be observed at moderatelevels with no age or sex predilection. The prevalence of thefinding ranges from 0.06% to 0.29%; it is most commonlyrecorded with descending frequency in the cardiac andextracardiac blood vessels, vagina, oviduct, rectum, epididymis, spinal cord, pancreas, urinary bladder, kidneys, andstomach.9
The rodent terms “degeneration/necrosis, media or wall,artery,” “necrosis/inflammation, media or wall, artery,”“inflammation, media or wall, artery,” “infiltrate, inflammatory cell, perivascular”, and “fibrosis, perivascular” may bereferred to for a detailed description.3,7,10

Infiltrate, Eosinophil (Figures 2.1-2.3)

Pigment

Diagnostic features

Comments

Infiltration of a relatively pure population of eosinophils into
the tissue with no other histological criteria of inflammation.

Endogenous pigments

Extramedullary Hematopoiesis
Comments

Comments
This finding has been highlighted due to the prominence of thiscell type in certain tissues, especially the mesenteric lymph
node, in minipigs.8 This is also commonly seen in brain, lungs,
renal pelvis, suburothelial, skin, and gastrointestinal tract. It is
recommended to record this routinely when seen.

Inflammation, Vascular/Perivascular
Other term(s)Arteritis, vasculitis, periarteritis, perivasculitis, degeneration/necrosis, media or wall, artery, necrosis/inflammation,

The Göttingen minipig has unpigmented skin, but is
not albino, so brown-black pigment (melanin) may
be seen, for example, melanin has been recorded in
the pineal gland. Pigmented breeds are also used in
toxicology studies (eg, Sinclair, Yucatan, Micro
Yucatan) and may contain melanin in various
tissues.
Lipofuscin is a yellow-brown, finely granular cytoplasmic
pigment found predominantly in macrophages and can be
confirmed with Schmorl’s stain.
Ceroid is a golden cytoplasmic pigment, similar to lipofuscin, but does not stain with Schmorl’s.
Hemosiderin is found within macrophages as golden brown
granules.

Skydsgaard et al.

Exogenous pigments
Tattoo ink is a black pigment localized to the skin and draining lymph node and is usually found in macrophages.
The appearance of other exogenous pigments will vary with
their nature.

Serous Atrophy of Adipose Tissue
Other term(s)Gelatinous bone marrow transformation

115
showing this alteration are typically clinically unaffected. Serous
atrophy is less commonly seen in minipigs in North America thanin Europe13; however, incidence appears to be decreasing in Europe. In humans, serous atrophy is observed in patients with anorexia nervosa, alcoholism, and AIDS and patients suffering withgeneralized severe illness (cachexia).14 Serous atrophy in minipigs as described above is limited to the bone marrow, or in
extreme, but still asymptomatic cases, the fat in the coronary
groove and subcutaneous fat, whereas in humans, serous atrophy
is usually systemic in the situation mentioned above. This finding
may also be observed in nonhuman primates. In domestic pigs,
serous atrophy of fat can be found in cases of emaciation or
inanition associated with illness.

Pathogenesis/cell of origin
This spontaneous lesion is most commonly observed in the femur
and tibia of Göttingen minipigs used as control animals in toxicology studies. This lesion may occur in other marrow cavities;
however, femur and tibia are the bones sampled as standard in
most studies. It can occur as a physiological response to a normal
degradation of adipose tissue under conditions where the minipig
needs additional energy.7,11,12

Diagnostic features

Focal or diffuse depletion of atrophied or degenerated
adipocytes, often starting in the epiphysis. If only the
epiphysis is affected, it is graded as minimal or slight.
With increasing severity, the metaphysis and diaphysis
are affected and the assigned grade would be moderate
or marked.
Reduced cellularity of the hematopoietic cells.
Interstitial accumulation or total replacement of adipose
tissue by homogenous eosinophilic gelatinous tissue(hyaluronic acid, mucopolysaccharides).

Differential diagnoses

Adipocyte atrophy in the absence of eosinophilic gelatinous tissue.
Decreased hemopoietic cells.

Comments
Serous atrophy is characterized by minimal to marked serous
atrophy of the adipose tissue in the bone marrow. Serous atrophy
can also be observed in the marrow of the sternum, in the adipose
tissue in the sulcus coronarius in the heart, and in the subcutaneous adipose tissue. This is a well-described change seen especially in male Göttingen minipigs but is also observed in femaleGöttingen minipigs.11,13 The etiology of the serous atrophy seen
as background pathology is thought to be nutritional, likely
related to a restricted feeding regimen. Microscopically, serous
atrophy of the adipose tissue in the bone marrow is seen as atrophyof the adipocytes accompanied by increased interstitial acidic
mucopolysaccharides which stains with Alcian blue.3 Decreased
cell density of the hematopoietic cells of the bone marrow often
accompanies this change. It is important to state that minipigs

Vacuolation, Macrophage (Figure 2.4)
Other term(s)Phospholipidosis.

Comments
Similar to the other laboratory species, the minipig is susceptible to phospholipidosis. Affected cells usually have pale,finely vacuolated cytoplasm and eccentric nuclei.15 The roleof type II cells in phospholipid metabolism makes them vulnerable to phospholipidosis induced by amphophilic cationicdrugs.16,17

Infectious Diseases
Comments
Minipigs are bred under barrier conditions, are microbiologically defined, and are kept in strictly controlled/biosecured
facilities when on study, so infectious disease (parasitism, bacterial and viral diseases) is unlikely. Isospora and Eimeria spp
have been observed in the past but have not been observed forseveral years. Domestic pigs, which are often used in medical
device studies, are, however, susceptible to a plethora of infectious disease so one must be aware of the possibility of infectious disease. For the purposes of histology data collection,
only entering the observation of “parasite” or a description of
the inflammation present, and so on, is appropriate and an
etiologic or disease diagnosis (eg, coccidiosis) is not appropriate as a morphologic term.

Syndromes
Syndromes that may occur in the minipig include hemorrhagic
syndrome, porcine stress syndrome (PSS), and phospholipidosis, which are described below. The syndrome should not berecorded as a finding but should be mentioned as a histonote in
the data capture system and/or in the pathology narrative. The
individual findings that together make the syndrome should berecorded in their own right using INHAND terms where
possible.

116

Toxicologic Pathology 49(1)Hemorrhagic Syndrome

Porcine Stress Syndrome

Other term(s)Other term(s)Thrombocytopenic purpuraPathogenesis/cell of origin
The etiology of hemorrhagic syndrome is unknown but may be dueto thrombocytopenia due to an immune complex-associated disorder (type III hypersensitivity, although a type II reaction cannot
be excluded.). Animals between 7 weeks and 1 year old have been
affected. There does not appear to be a hereditary etiology.

Diagnostic featuresOn hematology, regenerative anemia and thrombocytopenia (20 000/μL) are consistent findings.
Macroscopically, generalized petechial to ecchymotic
hemorrhages or hematomas in the skin, periosteum,muscle, mesentery and mucosal and/or visceral surfacesof skin, heart, urinary bladder, intestine, kidney, andlung, but also of other tissues, are seen.
Microscopically, findings consist of multifocal interstitialhemorrhages in multiple organs, edematous inflammationof the bladder, interstitial nephritis, membranoproliferative glomerulonephritis and interstitial fibrosis, hemorrhagic parenchymal necrosis of liver, reactive lymphoid
hyperplasia in lymph nodes, apoptotic and immaturemegakaryocytes, and increased hematopoiesis in bonemarrow and extramedullary hematopoiesis in liver andspleen. Complement (C1q) and IgG and IgM may bepresent in glomerular capillaries and mesangium. A
wide range of degenerative to proliferative vascularlesions in small to medium muscular arteries may beseen, particularly in epicardial and intramural coronary
arteries and pelvis and medulla of the kidney; however,this is not a consistent feature. Lesions range fromendothelial hypertrophy, medial thickening, and vacuolation of myocytes to perivascular and/or vascularinflammation, necrosis of the tunica media, and concentric laminar thickening of the media.18,19

Differential diagnoses

Exclude infectious diseases that cause vasculitis, forexample, classical swine fever, porcine reproductive andrespiratory virus, and erysipelas.
Exclude other hemorrhagic diatheses, for example, neonatal alloimmune thrombocytopenia, von Willebranddisease, disseminated intravascular coagulation.
Xenobiotic.

Comments
All microscopic observations should be recorded individually,
and a diagnosis of hemorrhagic syndrome made as an animalcomment in the data capture system and report text.

Malignant hyperthermia; back muscle necrosis; pale
soft exudative pork; transport myopathy; fulminant
hyperthermia stress syndrome

Pathogenesis/cell of origin
In susceptible pigs, a defective ryanodine receptor gene (RYR1)
leads to defective ryanodine receptors which allow Ca2þ leakage into sarcoplasmic reticulum resulting in sudden increased
sarcoplasmic calcium concentration on exposure to a trigger,with resulting skeletal muscle pathology. A single-nucleotide
polymorphism in the dystrophin gene (DMD) has recently been
identified in commercial breeds that causes decreased dystrophin in cardiac and skeletal muscle and PSS-like symptoms on
exposure to isoflurane or stress. Pathology due to the DMD
mutation is limited to cardiac myocytes.20-23

Diagnostic features










Histopathology and clinical history are diagnostic.
Rapid rigor mortis (within 5 minutes) is characteristic.
Macroscopically, pale, wet, swollen muscles, and signsof cardiac failure including hepatic congestion, pulmonary edema and congestion, hydrothorax, and hydropericardium may be seen.
Microscopically, swollen myofibers, myocyte segmental hypercontraction, degeneration (of a floccularnature), necrosis, and edema between myofibers, particularly in the longissimus, psoas, and semitendinosus(predominantly type 2 fibers), are characteristic.
Subepicardial hemorrhage, cardiac myocyte degeneration, necrosis, and intramural hemorrhage may be present in the heart, particularly in the left ventricle.
In the kidney, acute tubular necrosis and hemorrhage(acute renal failure, shock kidney) may be seen.
There may be marked hepatic congestion and congestion, hemorrhage, and edema in the lungs.

Comments
All microscopic observations should be recorded individually,
and a diagnosis of PSS made as an animal comment in the data
capture system and report text.
Porcine stress syndrome is an autosomal recessive pharmacogenetic disorder usually seen in commercial herds,
although PSS has also been reported in Vietnamese Potbellied pigs. The Göttingen minipig has German Landrace
and Vietnamese Pot-bellied pig in its genetic makeup, andthis is presumably where the susceptibility to PSS arises. It
is extremely uncommon.
Triggers include exposure to halothane or isoflurane,increased physical activity, tension/stress, handling, transport,
fighting, climatic conditions, high environmental temperature,
low energy levels in diet, vitamin D deficiency, and caffeine.

Skydsgaard et al.

117

One of the triggers causes an increased concentration of
intracellular channel gating agents and a sudden sustained
increase in cytosolic Ca2þ due to hypersensitive RYR1. This
causes sustained muscle contraction leading to depletion of
ATP by calcium homeostasis mechanisms and a switch toanaerobic metabolism. This leads to increased CO2 and lactic
acid and results in metabolic and respiratory acidosis, thermogenesis, and peripheral vasoconstriction. Cytokine release may
be seen, which is pyrogenic. Increased temperature, acidosis,
and ATP depletion lead to rhabdomyolysis. Enzymes and electrolytes are released from damaged cells, and high potassium
levels eventually lead to cardiac arrest.
Clinically, muscle tremors, dyspnea and open-mouth
breathing, tachycardia, elevated body temperature, and alternate areas of blanching and erythema of skin are seen. Mortality is associated with signs of cardiac failure.
Clinical chemistry perturbations may include creatine phosphokinase elevation, metabolic and respiratory acidosis, hemoglobinuria, myoglobinuria, renal failure, or impaired blood
coagulation.
Susceptible pigs may be identified by exposure to halothane(homozygous only) or by conducting a DNA polymerase chain
reaction test for the defective gene (HAL 1843) available fromcommercial laboratories.

Chapter 3. Cardiovascular System
Anatomically the heart and great vessels of the pig are similar
to human, the main exception being the presence of a left
azygos vein draining the intercostal system into the coronary

sinus. The coronary artery’s blood supply to the heart is almost
identical to that of human in anatomy and function. As inhumans, the pig has no collateral vessels in the myocardium,
leading to increased susceptibility to cardiac infarcts.3 A
characteristic of the pig heart is fully differentiated and large
Purkinje fibers (Figure 3.1) that can be found both subendocardially and intramurally within the heart.24,25 Similar to humans,
the porcine aorta also has vasa vasorum.
At necropsy, the heart is sampled with the root of the large
vessels and fixed in buffered-formalin solution. The heart shouldbe opened prior to sampling and immersion in fixative in orderto ensure adequate fixation and eliminate large blood clots in the
cardiac chambers. Several samples are collected from the heart
including a section from left and right ventricles, atria and either
tricuspid or bicuspid valves, left and right auricles, left and right
papillary muscles, and interventricular septum. In addition, thepulmonary valve and pulmonary trunk, thoracic aorta, and caudal vena cava are also sampled.26 This allows an accurate identification of changes in the cardiomyocytes, extracellular matrix,
conduction system, and the vascular structures within the myocardium and the adjacent epicardial tissue. Special dissection
procedures may be required to systematically evaluate the coronary arteries.
For detailed description of the cardiovascular system
lesions, refer to the rodent publication.27 While the type andclassification of pathological findings of the blood vessels,
heart, and heart valves are identical or very similar acrossspecies, their frequency in untreated individuals may differ
substantially among various species.

I. Heart
Table 3.1. Microscopic Findings of Cardiovascular System: Heart; Minipig.
Heart
Nonproliferative
Amyloid
Apoptosis, cardiomyocyte
Cardiomegaly
Cartilaginous metaplasia, epicardium
Degeneration, cardiomyocyte
Degeneration/necrosis, cardiomyocytea,bEdema, myocardium
Fibrosis, myocardium
Hemorrhagea b
Hypertrophy, cardiomyocyte
Infarct
Infiltrate,c endocardium or epicardium
Inflammation, endocardiuma
Infiltratea
Mineralization, cardiomyocyte, or myocardium
Infiltrate/fibrosis
Necrosis, cardiomyocytea,bCommonUncommonNot observed, but
potentially relevant

Not applicable

x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
(continued)

118

Toxicologic Pathology 49(1)Table 3.1. (continued)
Heart
Necrosis/infiltrate
Pigment, cardiomyocyte, or myocardium
Rodent progressive cardiomyopathy
Thrombus, atrium
Vacuolation, cardiomyocytea,bProliferative (nonneoplastic)
Hyperplasia, mesothelium, epicardium, or pericardium
Hyperplasia, Schwann cell, subendocardium

CommonUncommonNot observed, but
potentially relevant

Not applicable

x
x
x
x
x
x
x

a

Terminology with diagnostic criteria or comments described below.
Finding more frequent as an induced change.
cTerminology addressed in Systemic Pathology section.
b

Degeneration/Necrosis, Cardiomyocyte,
Heart (Figures 3.2 and 3.3)
Species
Minipig.

Comment
Treatment with the vasodilating antihypertensive drug, Minoxidil, caused left ventricular papillary muscle necrosis.3,9,28 The
lesion may be observed with PSS, which is further described in
Chapter 2, Systemic Pathology.13,29

intravenous catheter.7 The Hanford breed commonly has subacute to chronic myocardial inflammation.13,31

Infiltrate, Heart (Figure 3.3)
Species
Minipig.

Other term(s)Infiltration; infiltrate, inflammatory cell; cell infiltration.

Modifier(s)Hemorrhage, Heart (Figure 3.3)
Minipig.

Type of inflammatory cell that represents the predominant cell
type in the infiltrate: lymphocyte; plasma cell; mast cell; monocyte/macrophage; mononuclear; neutrophil; eosinophil; basophil; mixed.

Comment

Comment

Treatment with the vasodilating antihypertensive drug, Minoxidil,
caused diffuse atrial epicardial hemorrhage as well as myocardial
necrosis in miniature swine,9,28 while radiation caused multi-organ
hemorrhages including in the heart of Göttingen minipigs.30
The lesion may be observed with hemorrhagic syndrome
and PSS, which is further described in Chapter 2, Systemic
Pathology.3,10,18,19,29

Randomly distributed focal mononuclear infiltrates, predominantly lymphocytes are found primarily within the interstitial tissue. The mononuclear infiltrates can be in association with necrosisof myocytes. These lesions are usually focal and minimal.3
Inflammatory cell infiltrates have been reported in the heartof both Göttingen and Yucatan minipigs as a backgroundlesion.7,10,13,31

Inflammation, Endocardium, Heart(Figure 3.4)

Necrosis, Cardiomyocyte, Heart (Figures 3.2
and 3.3)

SpeciesSpecies

Minipig.

Minipig.

Comment

Comment

In intravenous infusion studies, focal subacute endocardial
inflammation and thickening of the intima/endocardium in theright atrium are considered to be related to the method oftreatment representing a local irritant effect at the tip of the

Occasional foci of myocardial necrosis have been observed as a
background lesion. Focal myocarditis was also sometimes
present.3,7 The lesion may be observed with PSS, which isfurther described in Chapter 2, Systemic Pathology.19

SpeciesSkydsgaard et al.

119

Vacuolation, Cardiomyocyte, Heart(Figure 3.5)

Comment
Cardiomyocyte vacuolation and myofibrillar loss was inducedin miniature swine by chronic doxorubicin administration.
Vacuolation may also be observed spontaneously.32

Species
Minipig.

II. Heart Valves
Table 3.2. Microscopic Findings of Cardiovascular System: Heart valves; Minipig.
Valve

CommonUncommonNonproliferative
Angiectasis, valve
Degeneration, myxomatous, valve
Inflammation, valve
Proliferation, stroma, valve

Not observed, but potentially relevant

Not applicable

x
x
x
x

III. Blood Vessels
Table 3.3. Microscopic Findings of Cardiovascular System: Blood Vessels; Minipig.
Blood vessels
Nonproliferative
Amyloid, media or wall, artery
Aneurysm, artery or aortic
Angiectasis
Atherosclerosisa,bDegeneration/necrosis, media or wall, artery
Embolusa,bFibrosis, perivascular
Hemorrhage, media or wall, artery
Hypertrophy, endothelium
Hypertrophy, media or wall, artery
Inflammation, vascular/perivascularc
Inflammation, media or wall, artery
Infiltrate, perivasculara
Intimal thickening, acellular, artery
Intramural plaque, artery
Mineralization, media or wall, artery
Necrosis/inflammation, media or wall, arterya,bThrombus
Vacuolation, media or adventitia, artery
Proliferative (nonneoplastic)
Hyperplasia, angiomatous
Hyperplasia, hemangioendothelium
Proliferation, intimal, artery or veinabcCommonUncommonNot observed, but potentially relevant

Not applicable

x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

Terminology with diagnostic criteria or comments described below.
Finding more frequent as an induced change.
Terminology addressed in Systemic Pathology section.

Atherosclerosis, Blood Vessels(Figures 3.6-3.10)
Species
Minipig.

Pathogenesis/cell of origin
Atherosclerosis is a degenerative change that occurs along thetunica intima of large and small arteries, and several induced
pig models were reported. Yucatan minipigs expressing
human, liver-specific D374Y-PCSK9 exhibited reduced low-120

Toxicologic Pathology 49(1)density lipoprotein (LDL) receptor levels, resulting in impaired
LDL clearance and increased plasma LDL levels. The administration of high-fat/high-cholesterol diet for 46 weeks led togreatly increased cholesterol levels. At approximately 1 year ofage, complex progressive human-like atherosclerotic lesions in
the aortae and iliofemoral and left anterior descending coronary arteries were observed.33
Feeding a high cholesterol diet to Göttingen minipigs for 3
months produced atherosclerotic lesions in the abdominal
aorta, aortic arch, celiac artery, right coronary artery, and paracoronal interventricular branch.34 A high-fat/high-cholesterol
diet fed to microminipigs also causes this lesion.35,36
A diabetes mellitus and high-fat/high-cholesterol diet atherosclerosis model has been developed in Yorkshire or Hanford
pigs, and a metabolic syndrome-induced atherosclerosis model
has been developed in Ossabaw pigs.33,37–39

Diagnostic features

In atherosclerosis models, macroscopically, areas ofyellow-white discoloration may be observed grossly on
the inner surface of vessels, some of which may be raised
plaques (especially remarkable in abdominal arteries).
Microscopically, intimal thickening, infiltration of lipidladen foamy macrophages, destruction of elastic layers,
increased extra cellular matrix and spindle-hapedsmooth muscle cells, calcification, cholesterol clefts,
fibrous cap, and necrotic core may be seen.

Special techniques for diagnostics
Basophilic granular material visible in H&E sections, confirmed with special stains: Von Kossa (mineral), Oil-Red-O(lipid), Elastic van Gieson (extracellular matrix and elastic
layer changes), or a-smooth muscle actin.

Comment
Mineralization, proliferation (intimal), and thrombus may all
be seen in conjunction with atherosclerosis. These terms aredefined further in the rodent cardiovascular INHAND
manuscript.27

Embolus, Blood Vessels (Figures 3.11-3.13)

In some animals, the lipid changes are accompanied by
focal alveolar edema and hemorrhage. In the kidney ofsame animal model, variable-sized vacuoles in glomeruli is observed and the glomerular capillaries are dilated
due to lipid and sometimes erythrocytes are seen to be
displaced in the capillaries.40

Special techniques for diagnostics
Oil-Red-O (lipid).

Comment
Obesity is induced by gradually offering increasing amounts offood approaching ad libitum level after 9 to 10 months, aiming
for a body weight of approximately 80 to 85 kg at study start. In
general, the ad libitum fed pigs consumed 1 to 1.5 kg per day,which correspond to 2 to 3 times the recommended amount.
The lipid embolism observed at approximately 6 to 12 months
after initiation of the study.40

Infiltrate, Perivascular, Blood Vessels(Figures 3.14-3.16)
Species
Minipig.

Comment
Arteritis/polyarteritis is an occasionally observed spontaneously occurring background change in Göttingen minipigs.
It can be present in a small- or medium-sized single artery of asingle organ or several organs in an animal or in several animals in a study. The severity is generally minimal or mild but
can occasionally be observed at moderate levels with no age orsex predilection. The prevalence of the finding ranges from
0.06% to 0.29%; it is most commonly recorded with descending frequency in the cardiac and extracardiac blood vessels,
vagina, oviduct, rectum, epididymis, spinal cord, pancreas,
urinary bladder, kidneys, and stomach.9 Mononuclear inflammatory cells have also been reported in the aorta.41
The lesion may be observed with hemorrhagic syndrome,
which is further described in the Chapter 2, Systemic
Pathology.18,19

Species
Minipig.

Necrosis/Inflammation, Media or Wall,
Artery, Blood Vessels (Figures 3.14-3.17)

Modifier(s)Species

Lipid.

Minipig.

Diagnostic features

Diagnostic featuresIn the obesity Göttingen minipig model, lipid embolism
is reported. In the lung of this model, thickening of thealveolar septa with variable-sized vacuoles is observed.

In spontaneous cases, inflammatory cells infiltrate the media
and/or all other layers of the artery, associated with necrosis
and fibrin deposits of the vascular wall.3,7,9,10

Skydsgaard et al.

121

Comment
Necrotizing vasculitis characterized by fibrinoid necrosis of thevascular wall and often associated with lymphocytic exudates
or thrombosis was observed in Yucatan minipigs following
endovascular radiation from 32P sources resulting in exposure
between 6 and 40 Gy.42
Treatment with the vasodilating antihypertensive drug, Minoxidil, caused endothelial injury, intramural accumulations oferythrocytes and platelets, periadventitial hemorrhage, fibrin
deposits, and inflammatory cell reaction in epicardial small
arteries.9
Pigs treated with immunomodulating drugs, such as hydrocortisone or betamethasone, caused medial degeneration withfibrinoid necrosis in small/medium-sized arteries of intestines,
peritoneum, spleen, pancreas, mesenteric plexus, liver, kidney,
and adrenals.9
The lesion may be observed with hemorrhagic syndrome
and PSS, which is further described in Chapter 2, Systemic
Pathology.18,19

Chapter 4. Digestive System
For detailed description of the digestive system lesions, refer tothe rodent publication.43 While the type and classification ofpathological findings of the digestive system are identical orvery similar across species, their frequency in untreated individuals may differ substantially among various species.
The minimum recommendation for examination of the digestive system is to examine stomach (nonglandular and glandular),small intestine (duodenum, jejunum, ileum), and large intestine(cecum, colon). For minipig/pig nonclinical safety studies, it isrecommended that the same samples of digestive system as inrats and mice are examined to adequately evaluate the digestive
tract in routine studies. Considering the length of the porcine
esophagus and intestine, the collection of additional samples isrecommended.26 However, in nonclinical safety studies, thenumber of samples examined is the same as in rodents. In investigational studies, additional sampling could be considered.26 A

detailed step-by-step sampling protocol of all organ systems
including digestive system is presented by Albl et al.26
The human and pig digestive systems are very similar toeach other in many respects and are classified as monogastric(ie, having one stomach) or nonruminant. Pigs and humans areboth omnivores and have comparable metabolic functions, pHchanges, gastric cell types, secretion, intestinal transport times,
and nutrient absorption characteristics. Therefore, pigs havebecome very useful in basic nutritional research.44,45
Xenobiotic-induced toxic injury to the gastrointestinal system often results in degeneration, ulceration, or hemorrhage inmany laboratory species. However, the porcine gastrointestinal
tract does not appear to be as sensitive to the ulcer-inducing
properties of nonsteroidal anti-inflammatory drugs as the gastrointestinal tract of dogs.44

I. Upper Alimentary Tract (Oral Cavity,
Tongue, Pharynx, and Esophagus)
Oral Cavity
The oral cavity is not routinely examined in nonclinical safety
studies microscopically and therefore is not included in this chapter.

TonguePorcine nonclinical safety study protocols recommend thesame sample generation as in rats and mice to adequately evaluate the tongue: one transverse section through the mid-portionof the tongue.26
On microscopic examination, the basal parts of the epithelium of the tongue appear quite basophilic, often with vacuolated and swollen cells; focal differences in this basophilia canbe present within the same animal and care should be taken not
to misinterpret this as a test article-related finding.7
Most often lesions in the tongue are found in studies withoral administration. The lesions are linked to mechanical damage, as the minipig is prone to biting the tongue during thedosing procedure (gavage).7

Table 4.1. Microscopic Findings of Digestive System: Tongue; Minipig.
TongueCongenital
Malformation
Nonproliferative
Amyloid
Atrophy, squamous epithelium
Apoptosis, squamous epithelium
Apoptosis/necrosis, single cell
Cyst
Degeneration/necrosis, muscle
Edema
Erosion/ulcera
Hemorrhage
Hyperkeratosis

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
(continued)

122

Toxicologic Pathology 49(1)Table 4.1. (continued)
TongueCommonb

Infiltrate
Inflammation
Inflammation, foreign body
Mineralization
Necrosis, squamous epitheliuma
Pigment
Vacuolation, squamous epitheliuma
Proliferative (nonneoplastic)
Hyperplasia, basal cell
Hyperplasia, squamous cella,cUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.
cUncommon as a background finding; commonly induced.
b

Erosion/ulcer, tongue (Figures 4.1 and 4.2)
Pathogenesis/cell of origin
Nonkeratinized stratified squamous epithelium.

Diagnostic features

Focal or multifocal.
Basal lamina may be intact under the erosion.
Ulcer is associated with mixed inflammatory cell infiltrates in the submucosa.

Differential diagnoses

Necrosis, squamous epithelium.

Comment
Erosion/ulcer in the fungiform papilla of nonkeratinized stratified
squamous epithelium is commonly observed, generally focal andof slight to moderate severity. The lesion is caused by trauma due
to biting the tongue during the dosing procedure (gavage). It canbe associated with minimal to slight focal inflammation of themuscle and occasionally with focal necrosis.3,7Hyperplasia, squamous cell, tongue (Figure 4.3)

and characterized by deaths of individual epithelial cellswith fragmented nucleus (karyorrhexis or karyolysis), paleeosinophilic cytoplasm, and absence of inflammatory
cells. Necrosis is generally present in the vicinity ofvacuolated epithelial cells of the mucosa (Jeppesen,
unpublished data).

Vacuolation, squamous epithelium, tongue(Figure 4.5)
Comment
Vacuolation of squamous epithelium in the tongue is acommonly observed background finding in minipigs. It
is characterized by enlarged, fine or coarsely vacuolated
epithelial cells with central or peripheral nucleus locatedpredominantly in the basal parts of the epithelium andgenerally seen at minimal level.7 The basal parts of theepithelium of the tongue appear quite basophilic oftenwith vacuolation. Focal differences in this basophilia canbe present within the same animal and care should betaken not to misinterpret this as a treatment-related
finding.7

Comment
Hyperplasia of the squamous epithelium of the tongue is notobserved in minipigs as a background change. However, hyperplasia of the squamous epithelium has been seen as an inducedchange with antioxidant inflammatory modulators (AIMs)(Jeppesen, unpublished data).

Necrosis, squamous epithelium, tongue(Figure 4.4)
Comment
Necrosis of the squamous epithelium of the tongue is acommonly observed in minipigs as a background change

Pharynx
The pharynx is not routinely examined in nonclinical safety
studies. However, the pharynx can be occasionally sampled
for examination of mucosal changes. The palatine tonsils
which are located on the dorsolateral side of the pharynx canalso be sampled. 26 When sampling of the pharynx isrequired, the complete nasopharynx is primarily sampled andthe soft palate (the “roof” of the oropharynx) is included. A
transverse section of the sampled circular structure (nasopharynx and the soft palate) is processed (Figure 4.6). Sincepharynx is not routinely evaluated, information on lesion
incidences is limited.

Skydsgaard et al.

123

Table 4.2. Microscopic Findings of Digestive System: Pharynx; Minipig.
Pharynx

CommonUncommonCongenital
Malformation
Nonproliferative
Amyloid
Apoptosis, squamous epithelium
Apoptosis/necrosis, single cell
Atrophy, squamous epithelium
Cyst
Degeneration/necrosis, muscle
Edema
Erosion/ulcer
Hemorrhage
Hyperkeratosis
Infiltratea
Inflammation
Inflammation, foreign body
Mineralization
Necrosis, squamous epithelium
Pigment
Proliferative (Nonneoplastic)
Hyperplasia, basal cell
Hyperplasia, squamous cella

Not observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in Systemic Pathology section.

Esophagus
In spite of the length of the porcine esophagus, sampling from one
location of the esophagus as in rats and mice is considered adequate for evaluation of the porcine esophagus in nonclinical toxicity studies: one transverse section through the esophagus and thetrachea at the level of the thyroid gland is recommended.26

The most commonly observed background change in theesophagus is focal inflammatory cell infiltrates, predominantly
mixed cells, at minimal severity.7
Periesophageal hemorrhage/inflammation occurring as aresult of blood sampling procedures is often observed in toxicity studies.7

Table 4.3. Microscopic Findings of Digestive System: Esophagus; Minipig.
Esophagus
Congenital
Dilatation, esophagus
Diverticulum, esophagus
Malformation
Nonproliferative
Amyloid
Apoptosis, squamous epithelium
Apoptosis/necrosis, single cell
Atrophy, squamous epithelium
Cyst
Degeneration/necrosis, muscle
Edema
Hemorrhage
Erosion/ulcer
Hyperkeratosis
Infiltratea
Inflammation
Inflammation, foreign body
Mineralization
Necrosis, squamous epithelium
Pigment

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

124

Toxicologic Pathology 49(1)Table 4.3. (continued)
Esophagus

CommonUncommon

Proliferative (nonneoplastic)
Hyperplasia, basal cell
Hyperplasia, squamous cellb,cNot observed, but potentially relevant

Not applicable

X
X

aTerminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.
cUncommon as a background finding; commonly induced.
b

Hyperplasia, squamous cell, esophagus(Figure 4.7)
Comment
Squamous cell hyperplasia in the esophagus is uncommon as a
background change. However, squamous cell hyperplasia has
been observed as an induced change with AIMs (Jeppesen,
unpublished data).

II. Stomach
The stomach is typical of monogastric species except for a
prominent muscular protuberance, the torus pyloricus at thelevel of pylorus, and a more prominent cardia (Figures 4.8 and
4.9). Among domestic mammals, pigs and ruminants are theonly species that have a pyloric torus. The pyloric torus is apedunculated structure, forming a protuberance at the lesser
curvature of the pylorus of the stomach. The histomorphologic
features of the pyloric torus include a simple columnar mucus
secretory cell lining, simple branched tubular mucous glands
arranged into lobules by connective tissue septa in the lamina
propria, large vascular spaces devoid of a muscular layer at thebase of the lamina propria, an internal fibromuscular layer
surrounding the glandular lamina propria, an inconstant layerof adipose tissue of variable thickness between the internal andexternal muscular layers, and serosa.46
As in rodents, the minipig stomach consists of 2 parts:
glandular and nonglandular. The nonglandular portion of thestomach, the pars esophagea, is well defined from the glandular
portion as in the rodent, however, is less extensive comparedwith rodents. The glandular mucosa of the stomach is divided
into 3 parts: the cardiac gland region (the largest), the fundic
gland region, and the pyloric gland region. The stomach produces mucus, acid, peptic secretions, and gastric hormonesfrom its glandular mucosa. The pH in the nonglandular parsesophagea and in the cardiac gland region is maintained above
5, while in the fundic and pyloric gland regions, where acid andpepsinogen are secreted, the luminal pH of 3.5 or lower is
optimum for the proteolytic activity of pepsin, the hydrolytic
product of pepsinogen.47 The anatomic fundus in swine is lined
by cardiac-type mucosa with mucous glands and not by true
gastric glands (with zymogen and oxyntic cells). True gastricglands are present only in the body of the stomach, nearer thepylorus than the fundus.48
For nonclinical safety studies, the same sampling of gastricregions as in rats and mice is recommended in minipigs: (1)junction between nonglandular and cardiac gland region, (2)fundic gland region, and (3) junction between pyloric glandregion and duodenum.

Table 4.4. Microscopic Findings of Digestive System: Stomach (Nonglandular); Minipig.
Stomach (nonglandular)
Nonproliferative
Apoptosis, squamous epithelium
Apoptosis/necrosis, single cell
Atrophy, squamous epithelium
Cyst, squamous
Erosion/ulcera
Helicobacter
Hemorrhageb
Hyperkeratosisa
Infiltrateb
Inflammation
Inflammation, vessel
Necrosis, squamous epithelium
Vacuolation, squamous epithelium
Yeast

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

125

Table 4.4. (continued)
Stomach (nonglandular)

CommonUncommon

Proliferative (nonneoplastic)
Hyperplasia, basal cell
Hyperplasia, squamous cellab

Not observed, but potentially relevant

Not applicable

X
X

Terminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.

Erosion/ulcer, stomach (nonglandular)(Figure 4.10)

Pathogenesis/cell of origin
Comment

Keratinization of the squamous epithelium

Diagnostic features

Erosion/ulcer in the nonglandular stomach is generally an
uncommon background finding in nonclinical safety studies
in minipigs.44 However, it should be mentioned that theincidence may increase depending on the stress status of
the animals during handling and dosing procedures. Erosions and ulcers are seen in the nonglandular portion of thestomach in Gottingens and Yucatans, whereas the glandular
portion of the stomach in the Hanford displays erosions andulcers.7,13







Diffuse.
Increase in the thickness of the keratin layer with nonnucleated or nucleated keratinized cells on the luminal
epithelial surface.
Hyperkeratosis, orthokeratotic: thickened keratin layer
with nonnucleated keratinized cells.
Hyperkeratosis, parakeratotic: thickened keratin layer
with nucleated keratinized cells.

Differential diagnoses
Hyperkeratosis, stomach (nonglandular)(Figure 4.11)



ModifiersHyperplasia, squamous cell: proliferation and thickening of the stratum spinosum (Figure 4.11)

Comment

Orthokeratotic
Parakeratotic

Hyperkeratosis is a commonly observed background change in
the absence of hyperplasia.

Table 4.5. Microscopic Findings of Digestive System: Stomach (glandular); Minipig.
Stomach (glandular)
Congenital
Ectopic tissue
Nonproliferative
Amyloid
Atrophy
Cyst, glandular
Dilatation, gland
Diverticulum
Erosion/ulcera
Globules, eosinophilic
Helicobactera
Hemorrhageb
Hypertrophy, mucous cell
Infiltrateb
Inflammation
Inflammation, arterya
Mineralization
Necrosis, mucosa
Secretion, decreasedc
Vacuolation, epithelium
Yeast
Proliferative (nonneoplastic)

CommonUncommonNot observed, but potentially relevant

Not applicable
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

126

Toxicologic Pathology 49(1)Table 4.5. (continued)
Stomach (glandular)

CommonUncommonNot observed, but potentially relevanta,c

Not applicable

X
X
X

Hyperplasia, mucosa, diffuse
Hyperplasia, mucosa, focal
Hyperplasia, neuroendocrine cellaTerminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.
cUncommon as a background finding; commonly induced.
b

Erosion/ulcer, stomach (glandular)(Figures 4.12-4.14)

Hyperplasia, diffuse, mucosa, stomach (glandular)

Cell of origin

Diffuse mucosal hyperplasia of the glandular stomach has notbeen noted as a background change in minipig nonclinicalsafety studies. However, it has been recorded as an inducedchange with an AIM (Jeppesen, unpublished data).

Surface epithelial cells.

Diagnostic features

Loss of mucosal epithelium with preservation of muscularis mucosa (erosion) or with penetration of muscularis mucosa (ulcer) in association with inflammatory
cell infiltrates and/or fibrosis in the lamina propria.
Focal or multifocal.
Acute or chronic.

Differential diagnoses

Autolysis: loss of cells at the luminal surface andabsence of inflammatory infiltrate.

Comment
Erosions or ulcers are quite commonly seen in the glandular
part of the stomach. They are generally graded as minimal orslight. Focal ulcers can be of moderate severity. These changes
are predominantly observed at the pyloric–duodenal junction,
possibly due to constant pH changes in the area, but can also beseen in the cardiac region. Administration of the xenobiotics
and/or stress can increase the incidence and severity of suchchanges.3,7

Helicobacter, stomach (glandular)
Comment
Production pigs are commonly infected with the zoonotic
pathogen Helicobacter spp, particularly Helicobacter suis. This
pathogen mainly colonizes the fundic and pyloric regions of the
porcine stomach inducing inflammation and a decrease in daily
weight gain. Alterations in hydrochloric acid production in theglandular region of the stomach, associated with chronic H suis
infections, may play a role in the pathogenesis of swine gastriculceration.49 Helicobacter infections have not been observed ordemonstrated in nonclinical safety studies; however, gastricerosion/ulcers are commonly observed and attributed to stressor constant pH changes in the area.3

Comment

Inflammation, artery, stomach (glandular)
Comment
Inflammation of the arteries is commonly seen in the muscularis
mucosa of the glandular stomach (see Cardiovascular section).

III. Small and Large Intestines
The small intestine is approximately 10% duodenum, 80% jejunum, and 10% ileum. In humans and nonhuman primates, theduodenum is relatively shorter and the ileum tends to be similarin length to the jejunum or even larger. The mesenteric vessels of
the small intestine form vascular arcades in the muscularis
mucosa of the intestine and not in the friable mesentery, as inother mammals. The presence of duodenal Brunner’s glands andjejunal Peyer’s patches allows easy histologic differential identification of these structures as it does in other species. In the pig,
unlike other domestic animals, Peyer’s patches occur in a continuous band along much of the length of the small and large
intestine and form a 2-cm-wide, thickened focus around theileocecal opening referred to as the cecal tonsil. The large intestine of the pig is quite different anatomically from that of othercommon laboratory animals. The cecum, the ascending andtransverse colon, and the proximal portion of the descending
colon are arranged in a series of centrifugal and centripetal coilsin the left upper quadrant of the abdomen, collectively forming
the spiral colon. The cecum has 3 longitudinal muscular bands(tenia), and the proximal portion of the spiral colon has 2 bands.
These result in a series of sacculations (haustra).44
Pigs develop intestinal crypts prenatally, similar in timing to
the human fetus.50 However, presence of Paneth cells in thecrypts of the small intestine is still debatable. Underwood statesthat pigs normally do not develop Paneth cells.50 Gonzalez et al
could not identify Paneth cells in the crypts of porcine small
intestine by TEM and immunostainings, however a Paneth cell
equivalent (Paneth-like cell), similar to that found in the mousecolon, may be present.51 Therefore, background changes

Skydsgaard et al.

127

associated with Paneth cells in the rodent are recorded in minipig as occurring in Paneth-like cells.
Sampling the intestines for nonclinical safety studies is similar in minipigs as in rats and mice: one transverse (cross)section from the duodenum, jejunum, ileum, cecum, and colonand one longitudinal section from the rectum.26 Albl et al rec-ommended collection of additional samples in investigational
studies (1 mid-segmental sample taken from the duodenum, 1
from the ileum, and 1 from the cecum. Two samples are prepared from the jejunum, 3 from the colon [1 from a centripetal
turn, 1 from a centrifugal turn, and 1 from the descending
colon], and 1 from the rectal mucosa).26

Table 4.6. Microscopic Findings of Digestive System: Small and Large intestines; Minipig.
Small and large intestines
Congenital
Ectopic tissue
Nonproliferative
Amyloid
Apoptosis
Apoptosis/necrosis, single cell
Atrophy
Atrophy, Brunner’s gland
Cyst, squamous
Degeneration, neuron, myenteric plexus
Degeneration/necrosis, Brunner’s gland
Dilatation
Diverticulum
EdemaaErosion/ulcera
Hypertrophy
Hypertrophy, Paneth cell-like
Infiltrateb
Inflammation
Inflammation, vessel
Intussusception
Lymphangiectasis
Metaplasia, osseous
Metaplasia, Paneth cell-like
Mineralization
Necrosis, mucosaa,cPaneth cell, decreased
Parasitea
Prolapse
Reduction, Paneth cell
Syncytia, epithelium
Tissue, ectopic
Vacuolation, mucosa
Proliferative (nonneoplastic)
Hyperplasia, Brunner’s gland
Hyperplasia, mucosaa,cNeoplastic
Lymphomaa

CommonUncommonNot observed, but potentially relevant

Not applicable
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.
cInduced change.
b

Edema, small and large intestine

Erosion/ulcer, small intestine (Figure 4.15)

Comment

Diagnostic features

Edema without inflammation/inflammatory cells is occasionally observed, predominantly in the large intestine. Edema canbe accompanied by hemorrhage.



Focal loss of mucosal epithelium (enterocytes) withpartial mucosal penetration (erosion) or with full

128






Toxicologic Pathology 49(1)penetration of the mucosa including muscularis
mucosa (ulcer).
Epithelial cells are necrotic or absent from the superficial mucosa, and muscularis mucosa is intact; noedema or hemorrhage are present (erosion).
Epithelial cells are necrotic or absent and muscularis
mucosa is destroyed (ulcer).
Erosion/ulcer is associated with an inflammatory cell
infiltrate.

Differential diagnoses

Necrosis, mucosa, small and large intestine
Comment
Necrosis of intestinal epithelial cells is a common backgroundfinding in minipig nonclinical safety studies. Intravenous administration of diacetoxyscirpenol (anguidine, a mycotoxin) to swine
at 0.5 and 1.0 mg/kg resulted in enterocyte damage (cytotoxic
effect), mimicking radiation poisoning. Enterocytes in different
anatomical regions of the gastrointestinal tract show differing
susceptibilities to the toxic effects of anguidine. Mitotically andmetabolically active tissues are predominantly affected.53

Artifacts or autolysis

Parasite, small intestine (Figures 4.18 and 4.19)

Comment
Erosion/ulcer is a commonly observed background finding in
nonclinical safety studies. Erosion is seen more commonly thanulcer and is usually focal or multifocal at minimal to slightseverity (occasionally can be of moderate severity) and is present most frequently in the duodenum. Focal ulcers can be ofmoderate severity.3,7Hyperplasia, mucosa, small intestine
Comment
In juvenile minipigs, treatment with Gattex (Teduglutide, a 33
amino acid recombinant analog of the human glucagon-like
peptide-2 [GLP-2]) can result in microscopic changes in the small
intestine (mucosal hyperplasia) as observed in mice and monkeys.52

Lymphoma, small intestine (Figures 4.16
and 4.17)
Comment
Lymphoma has been diagnosed in the ileum (see Hematolymphoid section) (McKeag, unpublished data).

Comment
Eimeria spp and Isospora spp can occasionally inhabit thesmall intestine but may also be detected in the large intestinein nonclinical safety studies.

IV. Salivary Glands
Salivary glands of the pig are large and consist of paired sets of
buccal (seromucous), sublingual (mucous), parotid (serous),and mandibular (seromucous) glands.47,54
For minipig nonclinical safety studies, similar sampling andtrimming as in rats and mice is recommended. The left parotid,
mandibular, and sublingual glands are routinely sampled andexamined microscopically. After removal of the tongue andtrachea with their adjacent structures, the parotid gland is harvested. This gland is triangular, lobulated, light brown in color,and located under the skin directly caudal to the mandibularbone. Next, the mandibular gland, located medial and slightly
cranial to the parotid gland, can be accessed. Sublingual glandis sampled through an incision to the left of the base of the
tongue. One longitudinal section through the largest surface ofthe glands is examined microscopically.26 Salivary gland background findings were rare in the Yucatan minipigs comparedwith Gottingen breed.13

Table 4.7. Microscopic Findings of Digestive System: Salivary Glands; Minipig.
Salivary glands
Congenital
Tissue, ectopic
Nonproliferative
Accumulation, adipocytes
Amyloid
Apoptosis
Apoptosis/necrosis, single cell
AtrophyCalculus, duct
Ectasia, duct
Edemaa,b
Fibrosis
Focus, hypertrophic, basophilic
Hemorrhage
Hypertrophy, acinar cell

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X

(continued)

Skydsgaard et al.

129

Table 4.7. (continued)
Salivary glands
Infiltratec
Inflammation
Metaplasia, acinar cell
Metaplasia, squamous cell
Mineralization
Necrosis
Pigment
Secretion, decreased, acinar cell
Vacuolationa
Proliferative (nonneoplastic)
Hyperplasiaa

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Macroscopic lesion.
c
Terminology addressed in Systemic Pathology section.
b

Edema, salivary glands
Pathogenesis/cell of origin
Accumulation of tissue fluid in the interstitium resulting
from increased vascular permeability.

Diagnostic features

Eosinophilic, interstitial fluid within the tissue.
No involvement of inflammatory cells.
No tissue or vascular damage.

Comment
Macroscopically clear, thick, gelatinous material (edematous
appearance) is occasionally noted surrounding the mandibular
glands. However, this is often not visible on microscopic examination of the gland.3 Recording the presence of edema using amacroscopic, rather than a microscopic, term is recommended
in minipig nonclinical safety studies.

Hyperplasia, ductal, salivary glands
Pathogenesis/cell of origin
Ductal epithelium of the salivary glands.

Diagnostic features

Focal, multifocal.
Small ductal cells with increased basophilia.
Minimally altered ducts with basophilic cytoplasm andhyperchromatic nuclei.
Associated with interstitial fibrosis.

Differential diagnoses

Hypertrophy, adenoma, adenocarcinoma.

Comment
Isolated occurrences of focal ductular hyperplasia can be seen
as a background finding in nonclinical safety studies. Focal

ductular hyperplasia is associated with interstitial fibrosis.7
Potential differential diagnoses (including hypertrophy, adenoma, adenocarcinoma) observed in other species are notobserved in minipig. Therefore, criteria for hypertrophy, adenoma, and adenocarcinoma cannot be detailed.

Vacuolation, acinar cell, salivary glands
Comment
Vacuolation of the acinar cells has not been observed in Göttingen minipig. However, it has been observed in Yucatan
minipigs (unpublished data).

V. Pancreas (Exocrine)
The pancreas is an extensive thick gland with an irregular outline
and is divided into 3 parts, the head (right lobe, duodenal portion:
upper right corner in Figure 4.25), the body (including the neck:down middle in Figure 4.25), and the tail (left lobe, splenic portion: upper left corner in Figure 4.25; Figure 4.25: pancreas, indicating the locations and orientations of the histology (black lines)
and molecular analyses (black rectangles), samples for investigated studies).26 The head, which is in contact with the gastrointestinal tract from the end of the pylorus to the proximal
duodenum, extends to the left and connects to the body. The body
separates into 2 (anterior and posterior) portions that encompass
the portal vein making the pancreas appear to be “ring-shaped.”
The posterior portion extends caudally, ventral to the right kidney.
The tail is located on the left of the body and extends caudosinistrally ventral to the left kidney, and it terminates near the hilus of
the spleen. The 3 parts of the pancreas in minipigs can also be
regarded more simplistically as two lobes, the right lobe or head
(duodenal portion) and the left lobe or tail (splenic portion).55
The pancreas should quickly be harvested after the animal’s
death to prevent autolysis. Since the density of endocrine pancreatic islets is not equal across the different pancreatic lobes, it
is recommended to include cross sections of the 3 parts of thepancreas (right and left lobes and body).26 In nonclinical safety

130

Toxicologic Pathology 49(1)studies, usually the left lobe or tail (splenic portion) of thepancreas is sampled for microscopic examination.
For investigative studies,26 3 samples were recommend
including left and right lobes and body of the pancreas (Figure 4.25. Minipig, Pancreas).
Histologically, middle- and small-sized islets arepresent in all lobes of the pancreas in the porcine. The

islets of Langerhans are not as clearly delimited from theadjacent exocrine tissue as in rodents. The islet cells getin the adjacent exocrine tissue and occasionally constitutea part of the acinus. The distribution pattern of a- versus
b-cells in the islet is not distinctive and is basically uniform in all lobes of the pancreas regardless of the size ofthe islet. 26,56

Table 4.8. Microscopic Findings of Digestive System: Pancreas (exocrine); Minipig.
Pancreas (exocrine)
Congenital
Ectopic tissue
Nonproliferative
Accumulation, adipocytesa
Amyloid
Apoptosis
Apoptosis/necrosis, acinar cell
Atrophy, acinar cell
Ectasia, duct
Edema
Eosinophilic globules
Fibrosis
Focus, basophilic
Halos, peri-insular, decreased
Halos, peri-insular, increased
Hemorrhagea
Hypertrophy, acinar cell
Infiltrateb
Inflammation
Inflammation, vessel
Metaplasia, ductule
Metaplasia, hepatocyte
Mineralization
Necrosis
Pigment
Secretion, decreased, acinar cella
Vacuolation, acinar cell
Proliferative (nonneoplastic)
Hyperplasia, acinar cell
Hyperplasia, ductal cellabCommonUncommonNot observed, but potentially relevant

Not applicable
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.

Accumulation, adipocytes, pancreas (exocrine)
Comment
Adipocyte accumulation has not been observed in minipig nonclinical safety studies yet. However, in a minipig obesity
model, adipocyte accumulation was observed.57

studies, possibly due to a restricted diet. This can also be
observed as a secondary finding in moribund and/or anorexic
animals with protein deficiency. Microscopically, the acinar
cells appear shrunken with increased basophilia of thecytoplasm.

Secretion, decreased, acinar cell, pancreas(exocrine)

Hemorrhage, pancreas (exocrine)

Comment

Focal interstitial hemorrhage around the pancreatic islets canoccasionally be observed at minimal level in minipig nonclinical safety studies.7

Decreased zymogen granules in the pancreas are a commonlyrecorded background finding in minipig nonclinical safety

Comment

Skydsgaard et al.

131

Chapter 5. Endocrine System

I. Pituitary GlandFor detailed general considerations on the endocrine system, please refer to the rodent INHAND publication.58 In
this document, the pituitary, pineal, thyroid, parathyroid,
adrenal glands, and endocrine pancreas will be discussed
separately.

The pituitary gland is structurally and functionally complex
and exhibits a typical mammalian morphology in the pig. It
is located immediately posterior to the optic chiasm within thebony sella turcica. The pituitary gland is harvested after
removal of the brain and sagittally cut into 2 halves along its
rostrocaudal axis.26

Table 5.1. Microscopic Findings of Endocrine System: Pituitary gland; Minipig.
Pituitary gland

CommonCongenital
Aberrant craniopharyngeal structures
Aplasia/hypoplasia
Persistent Rathke’s pouch
Nonproliferative
Angiectasis
Atrophy
Cyst
Fibrosis
Gliosis, pars nervosa
Hemorrhage
Hypertrophy, pars distalis
Hypertrophy, pars intermedia
Infiltratea
Inflammationa
Metaplasia, osseous
Mineralizationb
Pigmenta
Pseudocyst(s)Thrombus
Vacuolation
Proliferative (nonneoplastic)
Hyperplasia, pars distalis
Hyperplasia, pars intermediaab

UncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Mineralization, pituitary gland (Figure 5.1)

II. Pineal GlandComment

The pineal gland is not routinely examined in toxicology studies. It is one of the circumventricular organs of the brain andlocated on the posterior wall of the third ventricle, between thehemispheres, rostral to the cranial colliculi. It can be assessed
when the brain is dissected longitudinally and it is longitudinally cut into 2 halves.26 Dilated vessels can be quite prominent
in the pineal gland of the minipig (Figures 5.2 and 5.3), andmelanin pigment (melanosis; Figure 5.4) may be present.

Mineralized cells, generally randomly distributed and notassociated with inflammation, may occasionally occur in thepars distalis.3,7,13,59

Table 5.2. Microscopic Findings of Endocrine System: Pineal Gland; Minipig.
Pineal gland

CommonNonproliferative
Fibrosis
Infiltratea
Mineralization
Striated muscle fiber
Vacuolationa

Terminology addressed in the Systemic Pathology section.

Uncommon
X
X
X
X
X

Not observed, but potentially relevant

Not applicable

132

Toxicologic Pathology 49(1)No minipig-specific terminology, criteria, or comments nor
references are available.
Since the pineal gland is not routinely evaluated, there islimited experience in incidences of these microscopic findingsin the minipig.

III. Thyroid GlandIn the pig, the thyroid is a bilobed organ with fusion of the lobes
along the ventral trachea between trachea and thymus cranial to
the thoracic inlet. The parathyroid glands are not embedded in the
thyroid gland. Either a transverse or longitudinal section of theisolated thyroid gland can be prepared for histological evaluation.26

Table 5.3. Microscopic Findings of Endocrine System: Thyroid Gland; Minipig.
Thyroid gland
Congenital
Cyst, ultimobranchial
Ectopic tissue, thyroida
Ectopic tissue, thymusa
Persistent thyroglossal ducts
Thyroid dysplasia
Nonproliferative
Amyloid
Atrophy
Colloid alteration
Cystic follicle
Dilatation, follicle, diffuse
Fibrosis, capsuleb
Hemorrhageb
Hypertrophy, follicular cell
Infiltratea
Inflammationa,bMetaplasia, squamous cellb
Mineralization
Pigmenta
Proliferative (nonneoplastic)
Hyperplasia, C-cell
Hyperplasia, follicular cell
Neoplastic
Adenoma, follicular cellbabCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Fibrosis, thyroid gland (Figure 5.5)

Metaplasia, squamous cell, thyroid glandSee comment under Inflammation.

Comment
Minimal focal squamous metaplasia of follicular epithelium inthe thyroid gland has been reported as spontaneous lesion.7

Hemorrhage, thyroid gland (Figure 5.5)
See comment under Inflammation.

Adenoma, follicular cell, thyroid gland(Figures 5.6 and 5.7)

Inflammation, thyroid gland (Figure 5.5)

Comment

Comment

Spontaneous thyroid follicular cell adenomas are very rare in
the minipig (Jeppesen, personal communication, 2020). In
other species, there appears to be a continuum from focal
hyperplasia to carcinoma in induced thyroid tumors. As inrodents, the differentiation between focal hyperplasia and adenoma is not well defined. Size is not a reliable criterion. The

The thyroid gland is often injured by accidental mechanicalintervention due to blood sampling via the jugular vein leading
to hemorrhage, inflammation, and fibrosis and, in some cases,
may clinically affect thyroid hormone levels.13,60,61

Skydsgaard et al.

133
are only 2 to 4 mm in diameter, they are not easy to find at
necropsy but can be recognized by their firm texture and reddish color.26,62 Collecting the parathyroid gland can be particularly difficult if there is extensive hemorrhage related to bloodcollection in the ventral neck region. While embryologists suggest that there are 2 pairs, only 1 pair has been observed
grossly.62,63 The parathyroid glands are small oval bodies, surrounded by a thin connective tissue capsule. For histology, they
can be split into 2 halves.26

use of neoplasm modifiers (eg, follicular, cystic, papillary,solid) is optional.58

IV. Parathyroid GlandIn pigs, the parathyroid glands are separate from the thyroid
gland and can be found near the cranial part of the cervicalportion of the thymus opposed to a branch of the carotid arteryat the approximate level of the larynx. The glands have a beanlike shape and are often embedded in thymic tissue. Since they

Table 5.4. Microscopic Findings of Endocrine System: Parathyroid Gland; Minipig.
Parathyroid gland
Congenital
Ectopic tissue, parathyroid
Ectopic tissue, thymusa
Nonproliferative
AmyloidAngiectasis
Atrophy
Cyst
FibrosisHypertrophyb
Infiltratea
Inflammationa
Multinucleated giant cell
Vacuolationc
Proliferative (nonneoplastic)
Hyperplasia

CommonUncommonNot observed, but potentially relevant

Not applicable
X
X

X
X
X
X
X
X
X
X
X
X
X

b

Finding more frequent as an induced change.
Terminology addressed in the Systemic Pathology section.
cTerminology with diagnostic criteria or comments described below.
afasciculata, the largest of the 3 zones of the cortex, are occasionally present directly beneath the capsule (Figure 5.8). For
histology, one horizontal section from the middle portion of
one adrenal and one cross section of the middle portion of theother adrenal including both cortex and medulla are recommended.26 In the medulla, there is some variation in the degreeof vacuolation.

Vacuolation, parathyroid gland
Comment
Cytoplasmic vacuolation of the parathyroid cells has been
described for the Göttingen minipigs.7

V. Adrenal GlandIn the adrenal cortex, the zona glomerulosa lies immediately
beneath the capsule. However, parts of the underlying zona

Table 5.5. Microscopic Findings of Endocrine System: Adrenal Gland; Minipig.
Adrenal gland
Congenital
Ectopic tissue, adrenocorticala
Nonproliferative
AmyloidAngiectasis
Atrophy, cortex
Cyst, cortex
Degeneration, cystic

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
(continued)

134

Toxicologic Pathology 49(1)Table 5.5. (continued)
Adrenal gland
Fibrosis
Hematopoiesis, extramedullary
Hemorrhage
Hypertrophy, cortex, diffuse
Hypertrophy, cortex, focal
Infiltratea
Inflammationa,bMetaplasia, osseous
Mineralization
Necrosis
Pigmenta,bThrombus
Vacuolation cortex, increased, diffuseb
Vacuolation cortex, increased, focal
Vacuolation cortex, decreased, diffuse
Vacuolation cortex, decreased, focal
X-zone persistence
Proliferative (nonneoplastic)
Hyperplasia, cortex, diffuse
Hyperplasia, cortex, focal
Hyperplasia, medulla, diffuse
Hyperplasia, medulla, focal
Hyperplasia, subcapsular cellabCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the systemic pathology section
Terminology with diagnostic criteria or comments described below.

Inflammation, adrenal gland

VI. Endocrine Pancreas: Islets of Langerhans

Comment

The endocrine pancreas consists of discrete aggregates ofcells throughout the exocrine pancreas (islets of Langerhans). The islets are composed of pale staining polygonalcells of variable size and are scattered throughout the exocrine pancreas; however, the density is not equal across thedifferent pancreatic lobes.26 The islets are composed of anumber of different endocrine cell types responsible for the
production of different hormones. Alpha cells secrete glucagon; beta cells secrete insulin; delta cells secrete somatostatin; gamma cells secrete pancreatic polypeptide(thereby also referred as PP cells); and enterochromaffincells secrete substance P. Application of immunohistochemical staining for the different hormones or the use of electron microscopy is necessary to differentiate the structure of
the secretory granules in each cell type. In the pig, beta cells
are found as single cells or grouped together to form thecore of the islets at all ages. Some alpha cells are located in
the center of islets, but most reside in the periphery, alongwith a smaller number of delta cells and an even smaller
number of PP cells. The number of delta cells and PP-cells
in islets decreases as animals’ age. PP cells are more abundant in the head, as opposed to the tail area of the pancreas.
Though the number of larger islets increases with gestational age, the percentage volume density of beta cells doesnot. In pigs at 8 months of age, there are differences in thecellular composition of islets from different regions of thepancreas with islets in the head being rich in PP cells andInflammation of the adrenal has been occasionally been
observed in the minipig in association with systemic diseaseor as an extension of peritonitis.10,64

Pigment, adrenal gland (Figure 5.9)
Comment
Small amounts of lipofuscin are commonly observed in aged
minipigs. However, pigment in young animals may be indicative of increased cellular organelle turnover or impaired cell
metabolism (inhibition of steroid synthesis). Lipofuscin pigmentation may be associated with severe hormone-induced
atrophy. Dietary deficiency of antioxidants such as vitamin E
enhances the production and storage of lipofuscin pigment. Its
severity can be enhanced by the administration of estrogens
and adrenocorticosteroids (personal communication).

Vacuolation, cortex, increased diffuse,adrenal gland (Figure 5.10)
Comment
The amount of vacuolation caused by lipid droplets in all 3
zones can be variable even in normal minipigs.3,7

Skydsgaard et al.

135

poor in alpha cells, while islets in the tail are rich in alphacells and poor in PP cells.65 Since the density of endocrine
pancreatic islets is not equal across the different pancreatic
lobes, it is recommended to include cross sections of the 3parts of the pancreas (right and left lobes and body).26 In
nonclinical safety studies, usually the left lobe or tail (splenic portion) of the pancreas is sampled for microscopic
examination.

Table 5.6. Microscopic Findings of Endocrine System: Endocrine Pancreas; Minipig.
Endocrine pancreas

CommonUncommonNonproliferative
Amyloid, isleta
Angiectasis, islet
Apoptosis, islet cell
Atrophy, isleta,bDegranulation, islet cell
Fibrosis, islet
Hemorrhage, islet
Hypertrophy, islet cell
Infiltratec
Inflammationc
Metaplasia, hepatocyte
Pigment, islet
Single-cell necrosis
Vacuolation, islet cell
Proliferative (nonneoplastic)
Hyperplasia, islet cells

Not observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Finding more frequent as an induced change.
cTerminology addressed in the Systemic Pathology section.
b

Amyloid, islet, pancreas, endocrine
Comment
In pigs, islet amyloid polypeptide (IAPP1) is expressed mostly
in beta cells but also in some alpha and delta cells. The
sequence of IAPP in the amyloidogenic domain is dissimilarin pigs and humans, so that pigs are not prone to formation of
pancreatic amyloid whereas humans are.57 Amyloid deposits
can be observed in humanized IAPP mutant pigs.66

Atrophy, islet, pancreas, endocrine(Figures 5.11 and 5.12)
Comment
Göttingen minipigs are used as type 2 diabetes model induced
by streptozotocin with or without combination with nicotinamide leading to reduction in beta cell mass and atrophy of theislets.37,67 Similarly, the Wuzhishan miniature pigs show islet
atrophy after alloxan administration.68

Chapter 6. Hematopoietic and Lymphoid
SystemThis document follows a similar anatomical approach to thatused in the rodent INHAND document; however, descriptions
will focus on microscopic findings that differ from the rodent in
the minipig. Acknowledging that a tiered approach to findings

is used in the rodent INHAND document, due to the smaller
number of animals in general toxicology studies using minipigs, the authors of this document recommend use of descriptive terms (which in most cases are comparable to enhanced
terminology in the rodent). Where lesions in the minipig androdent are similar they will be tabulated with an indication of
their prevalence or applicability to the minipig, and the reader
is referred to the rodent manuscript for a full description of thelesion.
This article deals with a standardized nomenclature for classifying microscopic findings observed in the hematolymphoidsystem of minipigs, namely bone marrow, thymus, lymph
node, spleen, mucosal-associated lymphoid tissue (MALT),
tertiary lymphoid structures, and a section on general hematolymphoid changes. The minimum recommendation for examination of the hematolymphoid system is to examine thymus,
spleen, draining lymph nodes/lymph nodes local to parenteralor topical administration sites, bone marrow in situ, and any
gross lesions of a lymphoid organ.69

General
As in other toxicology species, focal interstitial infiltrates ofinflammatory cells, mostly comprised of lymphocytes withplasma cells and macrophages, are the most frequently
observed lesion in minipigs. Infiltrating eosinophils can beobserved, particularly in the mesenteric lymph node.3,13

136

Toxicologic Pathology 49(1)Table 6.1. Microscopic Findings of Hematopoietic and Lymphoid System: General; Minipig.
GeneralCommonUncommonCongenital
Aplasia/hypoplasia
Nonproliferative
Abscess
Amyloid
Apoptosis, increased, lymphocyte
Cellularity increased, mast cell
Extramedullary hematopoiesis (EMH)a
Infiltrateb
Inflammationa,bMetaplasia, osseous
Mineralization
Necrosis
Macrophages, vacuolateda
Phospholipidosis (see Vacuolated Macrophages)a,c
Pigment, macrophagea
Tingible body macrophage, increased
Vacuolation, macrophage

Not observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cFinding more frequent as an induced change.
bExtramedullary hematopoiesis

Comment

Comment

Vacuoles positive for LAMP2 or characteristic appearance onelectron microscopy. Similar to the other laboratory species,
the minipig is susceptible to phospholipidosis.15

Extramedullary hematopoiesis is generally greatest at around 14
days postpartum, is decreased or absent by 35 days, and is absent
after 63 days5 or 6 weeks.6,7 In older pigs, extramedullary hematopoiesis may be seen in response to severe anemia. The
INHAND term for rodents is “extramedullary hematopoiesis,
increased”; however, in minipigs over 6 weeks old, as this is notconsidered a normal feature of the spleen, the modifier“increased” has been dropped.

Pigment, macrophage
Comment
Pigment within macrophages can be seen in piglets due to prophylactic postnatal iron injection.3,13

Inflammation
Comment

Bone Marrow

Inflammation can be seen in and around the thymus, thyroid,
trachea, esophagus, and mandibular lymph nodes due to blood
sampling procedures.3,7

Phospholipidosis
Other term(s)Vacuolation, macrophage

Bone marrow is usually examined in formalin-fixed, paraffinembedded, decalcified 5 or 3 mm sections of femur and sternum(and occasionally vertebrae) stained with H&E. It is good practice to prepare contemporaneous bone marrow smears at
necropsy, as unequivocal identification of cell lines on H&Estained sections is difficult. Should changes be detected on
H&E, Romanowsky-stained bone marrow smears can be
examined.70

Table 6.2. Microscopic Findings of Hematopoietic and Lymphoid System: Bone Marrow; Minipig.
Bone marrow
Nonproliferative
Angiectasis
Cellularity, decreased, adipocyte

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X(continued)

Skydsgaard et al.

137

Table 6.2. (continued)
Bone marrow
Cellularity, decreased, bone marrow
Dyshematopoiesis
FibrosisHypersegementation, granulocyte
Inflammationa
Necrosisa
Serous atrophy of adipose tissueb
Proliferative (nonneoplastic)
Cellularity, increased, adipocyte
Cellularity, increased, bone marrow
Cellularity, increased, macrophage
Cellularity, increased, mast cellabCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Serous atrophy of adipose tissue, bonemarrow (Figure 6.1)

ThymusOther term(s)Gelatinous transformation, serous atrophy. Please refer to the
Systemic Chapter for a detailed description.

Diagnostic features

Focal or diffuse depletion of atrophied or degenerated
adipocytes, often starting in the epiphysis. If only the
epiphysis is affected, it is graded as minimal or slight.
With increasing severity, the metaphysis and diaphysis
are affected and the assigned grade would be moderate
or marked.
Reduced cellularity of the hematopoietic cells.
Interstitial accumulation or total replacement of adipose
tissue by homogenous eosinophilic gelatinous tissue(hyaluronic acid, mucopolysaccharides).

Differential diagnoses

Adipocyte atrophy in the absence of eosinophilic gelatinous tissue.
Decreased hemopoietic cells.

The thymus consists of cervical and thoracic lobes, with the pig
having a well-developed cervical lobe with a cranial and caudal
portion. The cervical thymus runs along the jugular grooves up
to the pharyngeal region, while the thoracic portion extends
from the pericardium into the thoracic inlet.71 Keratinizationof Hassall’s corpuscles is prominent in minipigs as in young
dogs and nonhuman primates. The minipig thymus does not
involute to the same extent as that of dogs and nonhumanprimates, at least not in the course of routine toxicology studies.71 In the experience of the authors, the thymus is relatively
resistant to stress-induced atrophy.7 When sampling, both parts
should be weighed and histological analysis performed. Sampling of the thoracic portion may avoid venipuncture artifact.
Albl et al suggest that the caudal cervical portion should be
examined; however, sampling sites vary between laboratories.26,69 The position of the parathyroid gland is variable
and may be found embedded in the thymus or in the connective
tissue adjacent to the thymus. As such, ectopic parathyroid
gland is not considered to be a meaningful diagnosis in thisspecies.3

Table 6.3. Microscopic Findings of Hematopoietic and Lymphoid System: Thymus; Minipig.
ThymusNonproliferative
Apoptosis, increased, lymphocytea
Cellularity, decreased, lymphocyte
Corticomedullary ratio, decreased
Corticomedullary ratio, increased
Cyst, epithelial
Ectopic tissue, parathyroida
Ectopic tissue (specify tissue)

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
(continued)

138

Toxicologic Pathology 49(1)Table 6.3. (continued)
Thymus
Ectopic tissue, thymus
Hemorrhagea
Hypoplasia
Infiltratea,bInflammationb
Involution, age-relateda
Loss of corticomedullary distinction
Necrosis, lymphocytec
Tingible body macrophage, increased
Proliferative (Nonneoplastic)
Cellularity, increased, epithelial cell
Cellularity, increased, lymphocyte
Epithelium-free areas, increased
Thymic corpuscles, increased

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cFinding more frequent as an induced change.
b

Apoptosis, increased, lymphocyte, thymus(Figure 6.2)
Ectopic tissue, parathyroid, thymus
Comment
As the parathyroid gland is not associated with the thyroid
glands in the minipig, and as the pig has a cervical thymus,
parathyroid gland is commonly found within the thymus and is
not considered to be ectopic.

Hemorrhage, thymus (Figure 6.3)
Pathogenesis/cell of origin
Extravasated erythrocytes in the vicinity of thethymus and thyroid. Blood sampling procedures are themost common cause of hemorrhage in and aroundthe thymus.

Diagnostic features
This lesion may vary in appearance from acute to chronic.
When acute, extravasated blood is present around the thymus. As the lesion ages, extravasated blood is graduallyreplaced by inflammatory cells, fibrous tissue, and pigmented macrophages.

Differential diagnoses

Hemorrhagic syndrome

Also see inflammation and/or infiltrate, inflammatory cells in
the “General” section and below.

Infiltrate, inflammatory cell and inflammation,
thymusComment
Due to the proximity of the thymus to the major vessels of theneck used in venipuncture procedures, and the difficulty sometimes experienced while taking blood samples from pigs,
inflammatory cell infiltrate or inflammation, usually in conjunction with hemorrhage, in the vicinity of the thymus is notuncommon.

Involution, age-related, thymus
Thymic involution is thought to begin at puberty, with changes
visible at 180 days72 or 18 months,73 depending on which paper
is considered. Distinguishing between age-related involution
and test article-related thymic lymphocyte depletion can bechallenging. Prominent necrotic and/or apoptotic lymphocytes
would be considered unusual in involution; however, fewer
lymphocytes in cortex and medulla with a lack of definition
between these two compartments, an increase in perivascularspaces, more Hassall’s corpuscles with keratinization anddegeneration, and adipocyte infiltration in capsule and interlobular connective tissue may be seen. Careful comparison
between control and treated animals with reference to the ageof animals should be made.

Comment

SpleenDue to the proximity of the thymus to the major vessels of theneck used in venipuncture procedures, and the difficulty sometimes experienced while taking blood samples from pigs,
hemorrhage in the vicinity of the thymus is not uncommon.

The role of the spleen in humans and rodents is primarily
defensive, and in dogs, it is primarily a storage organ; however, in minipigs, the spleen shares characteristics of both.
The minipig spleen is nonsinusoidal. 71 There is a thickSkydsgaard et al.

139
quite prominent.29,71 Their function is clearance of bloodborne particles. Extramedullary hematopoiesis may be seenin juveniles up to 6 weeks old or as a response to systemic
disease such as hemorrhagic syndrome.7,29 Accessory spleens
may be found in the gastrosplenic ligament.71 A cross-section
through the thickest part of the spleen should be examinedhistologically (Figures 6.4 and 6.5).

capsule of smooth muscle and elastic fibers and similarly
structured trabeculae. Lymphoid follicles are less prominent
than in rats, but the periarteriolar lymphoid sheath is welldeveloped. The red pulp contains pale eosinophilic, contractile, phagocytic structures surrounding splenic capillaries,
which are known as periarteriolar macrophage sheaths, splenic ellipsoids, or Schweigger-Seidel sheaths, which can be

Table 6.4. Microscopic Findings of Hematopoietic and Lymphoid System: Spleen; Minipig.
SpleenCommonUncommonCongenital
Aplasia/hypoplasia (see General section)
Nonproliferative
White pulp (PALS, follicles, germinal centers, marginal zones)
Apoptosis, increased, lymphocyte
Cellularity, decreased, white pulp
Necrosis, lymphocyte
Tingible body macrophages, increased
Red pulp
Angiectasis
Cellularity, decreased, red pulp
CongestionX
Contraction
Ectopic tissue, spleen
Erythrophagocytosis
Fibrosis
Pigment, macrophagea
Vacuolation, macrophagea
White pulp (pals, follicles, germinal centers, marginal zones)
Proliferative (nonneoplastic)
Aggregates, macrophage, increased
Cellularity, increased, plasma cell, white pulp
Cellularity, increased, white pulp
Red pulp
Cellularity, increased, adipocyte
Cellularity, increased, macrophage
Cellularity, increased, mast cell
Cellularity, increased, mesothelial cell
Cellularity, increased, plasma cell, red pulp
Cellularity, increased, stromal cell
Extramedullary hematopoiesisb
Hyperplasia, nodular
Periarteriolar macrophage sheaths (PAMS)bXab

Not observed, but potentially relevant

Not Applicable

X

X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Extramedullary hematopoiesis, spleen(Figure 6.6)
Comment
Extramedullary hematopoiesis is generally greatest ataround 14 days old, is decreased or absent by 35 days,
and is absent after 63 days 5 or 6 weeks.6,7 In older pigs,
extramedullary hematopoiesis may be seen in response
to severe anemia. The INHAND term for rodents is“extramedullary hemopoiesis, increased”; however, inminipigs over 6 weeks old, as this is not considered anormal feature of the spleen, the modifier “increased” hasbeen dropped.

Periarteriolar macrophage sheaths, Spleen
Other term(s)Ellipsoids, Schweigger-Seidel sheaths

Pathogenesis/cell of origin
These are normal structures formed from phagocytic cells andreticular fibers.

140

Toxicologic Pathology 49(1)Diagnostic features
Generally rounded, pale eosinophilic accumulations ofcells around capillaries or small arterioles in the red pulp.

Comment
Periarteriolar lymphoid sheaths are prominent in the Göttingen
minipig. Their prominence varies between animals. Their function is to filter blood, retain circulating particulate matter, andtrap immune complexes.6,71,74

Lymph Node
Histologically, lymph nodes of pigs have a unique structure,with centrally located cortical tissue and germinal centersand peripheral medullary sinuses and cords that appear
inside out when compared to other species. The follicles
are arranged centrally and not peripherally, and the lymph
flow is reversed with lymph entering centrally at the hilus

and leaving through high endothelial veins at the capsularsurface.71 This results in few lymphocytes in the efferent
lymph ducts. These differences in structure do not appear toresult in significant functional changes. The appearance oflymph nodes can vary considerably depending on anatomical
location, region drained, age, and plane of section (Figures 6.7,
6.8). There are varying numbers of eosinophils normally present in the porcine lymph node sinuses.7 The mesenteric lymph
node is large and is located at the root of the mesentery. In
addition, the mesenteric lymph nodes are supplied by a prominent perimesenteric plexus.29 This lymph node should besampled in all studies as it can reflect the inflammatory stateof the gastrointestinal tract.69 It is common practice for themandibular lymph node to be examined as a representative
peripheral lymph node. For enhanced histopathology of thelymph nodes, the most proximal regional lymphoid tissues thatdrain the application/ administration site should be examined
microscopically.75

Table 6.5. Microscopic Findings of Hematopoietic and Lymphoid System: Lymph Node; Minipig.
Lymph node
Congenital
Aplasia/hypoplasia (see General section)
Cortex, paracortex, and medullary cords
Nonproliferative
Apoptosis, increased, lymphocyte
Cellularity, decreased, lymphocyte
Inflammationa
Necrosisa,bPigment, macrophagea
Tingible body macrophage, increaseda
Sinuses and lymphatics
Nonproliferative
Dilatation, sinus
Erythrocytes, intrasinusoidal
FibrosisHemorrhageb
Lymphangiectasis
Pigment, macrophagea
Vacuolation, macrophagea
Cortex, paracortex, and medullary cords
Proliferative (nonneoplastic)
Aggregates, increased, macrophage
Cellularity, increased, interdigitating dendritic cell
Cellularity, increased, lymphocyte
Cellularity, increased, plasma cell
Cellularity, increased, stromal cell
Hyperplasia, angiomatous
Hypertrophy/hyperplasia, high endothelial venule (HEV)
Pigment, macrophagea
Sinuses and lymphatics
Proliferative (nonneoplastic)
Cellularity, increased, macrophage, intrasinusoidal
Cellularity, increased, mast cell
FibrosisabTerminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Common Uncommon Not observed, but potentially relevant Not applicable
X

X
X
X
X
X
X

X
X
X
X
X
X
X

X
X
X
X
X
X
X
X

X
X
X

Skydsgaard et al.

141
samples from minipigs, hemorrhage in the vicinity of thislymph node is not uncommon. Also see inflammation in the“General” section.

Hemorrhage, lymph node
Pathogenesis/cell of origin
Extravasated erythrocytes in and/or around the mandibular lymph node. Blood sampling procedures are themost common cause of hemorrhage in and around thislymph node, although agonal changes may be involved
in some cases.3

Diagnostic features
This lesion may vary in appearance from acute to chronic.
When acute, extravasated blood is present around the lymph
node, and/or within the sinuses. As the lesion ages, blood is
gradually replaced by inflammatory cells, fibrous tissue, andpigmented macrophages.

Differential diagnoses

Hemorrhagic Syndrome

Comment
Due to the proximity of the mandibular lymph node to themajor vessels of the neck used in venipuncture procedures, andthe difficulty sometimes experienced while taking blood

Necrosis, mesenteric lymph node (Figure 6.9)
Mucosa-associated lymphoid tissue
Mucosa-associated lymphoid tissues (MALT) include gutassociated lymphoid tissue, nasopharynx-associated lymphoidtissue (NALT), and bronchus-associated lymphoid tissue(BALT). Gut-associated lymphoid tissue should be examinedin studies where the test article is administered orally and isusually examined in a routine section of jejunum or ileum that
contains Peyer’s patches. Minipigs have 2 types of Peyer’s
patches: discrete nodules in the jejunum and proximal ileum
and linear continuous structures in the distal ileum. Antigenic
stimulation is not required for growth of Peyer’s patches in theminipig, which increase in number and size with age.71
Nasopharynx-associated lymphoid tissue and BALT should
be examined in studies where the test article is administeredintranasally or by inhalation. Nasopharynx-associated lymphoid tissue may be examined in routine nasal cavity sections,and BALT in routine lung sections, generally at bronchial/bronchiolar bifurcations.76,77

Table 6.6. Microscopic Findings of Hematopoietic and Lymphoid System: MALT; Minipig.
MALT

CommonCongenital
Aplasia/hypoplasiaa
Nonproliferative
Apoptosis, increased, lymphocytea
Cellularity, decreased, lymphocyte
Degeneration, follicle-associated epithelium
Hyaline material
Inflammationa
Lymphangiectasis
Mineralizationa
Necrosisa
Pigment, macrophagea
Tingible body macrophage, increaseda
Proliferative (nonneoplastic)
Aggregates, macrophage
Cellularity, increased, lymphocyte
Cellularity, increased, macrophage
Hyperplasia, follicle-associated epithelium
Hyperplasia, goblet cell, follicle-associated epithelium
Hypertrophy/hyperplasia, high endothelial venule (HEV)
Metaplasia, squamous, follicle-associated epitheliumaUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic Pathology section.

Table 6.7. Microscopic Findings of Hematopoietic and Lymphoid System: Other Lymphoid Tissues; Minipig.
Other lymphoid tissues
Tertiary lymphoid structures (TLSs)
Serosa-associated lymphoid clusters (SALCs), increased

CommonUncommonXX

Not observed, but potentially relevant

Not applicable

142

Toxicologic Pathology 49(1)Table 6.8. Microscopic Findings of Hematopoietic and Lymphoid System: Hematolymphoid Neoplasms; Minipig.
Hematolymphoid neoplasms

CommonUncommona,b

Leukemia, myeloid
Lymphoid neoplasms
Lymphomaa,bMast cell proliferations
Leukemia, mast cellaab

Not observed, but potentially relevant

Not applicable

X
X
X

Terminology with diagnostic criteria or comments described below.
Finding more frequent as an induced change.

Leukemia, myeloid
Comment
Myeloid leukemias have been induced in Pitman-Moore miniature pigs by administration of strontium 90.78

Lymphoma (Figures 6.10 and 6.11)
Comment
Lymphoma has not, to our knowledge, been described in theliterature in Göttingen minipigs. However, 2 lymphomas havebeen diagnosed but not reported in Göttingen minipigs. One wasdiagnosed in an 8-month-old male and one in a 3-month-old
female. Neoplastic lymphocytes were present in intestine, liver,and spleen; however, limited tissues were available for examination. Neither pig was on study (McKeag, unpublisheddata).79-81 Lymphoid neoplasms have been induced in miniaturepigs by oral administration of Strontium 90.78

Leukemia, mast cell
Comment
Mast cell leukemia has been diagnosed in a 3-year-old male pet
Göttingen minipig82 and a 9-year-old female Sinclair miniature
pig.83

Chapter 7. Hepatobiliary System
For detailed description of the hepatobiliary system lesions,refer to the rodent publication.84 While the type and classification of pathological findings of the hepatobiliary system are
identical or very similar across species, their frequency inuntreated individuals may differ substantially among various
species.

I. LiverThe minipig liver consists of 6 lobes. It has 4 main lobes (rightlateral, right medial, left medial, and left lateral) and 2 lesser lobes(caudate and quadrate). The right lateral lobe is divided dorsally
to form the caudate lobe, which in turn may be divided by a fissure
into 2 parts. The quadrate lobe is ventral to the portal fissure and to
the left of the gall bladder. In contrast to the other laboratory
species and humans, the porcine liver displays prominent

lobulation that is macroscopically visible. The lobules are demarcated by white stromal septa which become apparent in animals at
approximately 4 weeks of age. The liver has 2 surfaces. The
diaphragmatic surface lies in close proximity to the diaphragmand is markedly convex. The visceral surface is concave andclosely associated with the abdominal viscera. The margins of
the normal liver are sharp and not rounded26,85 (Figure 7.1.).
For nonclinical safety studies, left lateral and right median
lobes are sampled longitudinally in relation to the edge of thelobes, ensuring 2 peripheral surfaces (diaphragmatic and visceral) are available for microscopic examination.
Histologically, unlike rodents, normal minipig liver has
markedly defined lobulation (well-defined acinar structureswith well-delineated portal triads; Figures 7.2a and 7.2b). Vastamounts of interlobular and subcapsular connective tissue cangive disorganized appearance of the surrounding hepatic parenchyma.7,54 Although an increased amount of interlobularconnective tissue is present, there does not appear to be any
increased tendency of the porcine liver to develop cirrhosis
after toxic injury compared with other nonhuman species.44
Spontaneous changes in the porcine liver have been reportedwith very low incidence. They include minimal to slight focal
parenchymal necrosis, minimal focal single cell necrosis, minimal to slight focal infiltration of mononuclear cells or mixed
inflammatory cells, minimal to moderate vacuolation of thehepatocytes, minimal to slight focal extramedullary hematopoiesis, and minimal to moderate focal subcapsular/interlobular fibrosis.7
Toxicologic lesions in the porcine liver are similar to those
noted in other species, including rodents and dogs, and theminipig is considered predictive for human toxic injury.44
All the main metabolic activities typical for human cytochromes P450 (CYPs) are found in liver microsomes of pigs
and minipigs. Unlike other routinely used animals, minipigs
possess a CYP ortholog to the main human liver enzyme of
drug biotransformation, CYP3A, both in similar amounts andactivities, as well as orthologs with similar sequences to human
CYP2A, 2C, 3A. Some porcine CYP enzymes have been characterized including substrate specificity, inhibition, and regulation. Porcine (conventional, minipigs, and micropigs)
CYP1A, 2A, and 3A metabolize the same test substrates ashuman enzymes, whereas porcine 2B, 2D, and 2E are different
from the corresponding human enzymes regarding metabolismof the well-known substrates. Porcine CYPs that metabolize

Skydsgaard et al.

143

human test substrates are approximately 75% identical in
N-terminal cDNA sequences. cDNA sequences of porcine(conventional and minipig) CYP2A, CYP2C, and CYP3A are

similar to human counterparts. Although there is sequencehomology, substrate specificity and activity may be altered incomparison with man.31,86,87

Table 7.1. Microscopic Findings of Hepatobiliary System: Liver; Minipig.
Liver

CommonCongenital
Nonproliferative
Amyloida,bAngiectasis
Atrophy, hepatocytea
Bile plugs, canaliculia
Cholangiofibrosis
Congestiona
Crystals
Cyst, bile duct
Cytoplasmic alteration
Degeneration, cystic
Degeneration, hydropic
Disease, Tyzzer’s (Clostridium piliforme infection)
Extramedullary hematopoiesis
Fatty change
Fibrosisa
Hemorrhagec
Helicobacter sp. hepatitisa
Hepatitis, porcine hepatitis virusc
Hypertrophy, hepatocytea,bHypertrophy/hyperplasia, Kupffer cell
Inclusions, cytoplasmic
Inclusions, intranuclear
Infarctb
Infiltratea,cInfiltration, peribiliary
Intrahepatocellular erythrocytes
Intravascular hepatocytes
Karyocytomegaly and/or multinucleated hepatocytes
Metaplasia, glandular, hepatocyte
Metaplasia, pancreatic acinar cell
Mineralization
Necrosis, focal/multifocala
Necrosis, single cell
Necrosis, zonal
Phospholipidosisb
Pigment, hepatocytesa
Pigment, Kupffer cellsa
Rarefactiona
Single-cell necrosis
Thrombus
Vacuolation, hepatocytea,bProliferative (Nonneoplastic)
Focus of cellular alteration
Hyperplasia, bile duct
Hyperplasia, hemangioendothelial
Hyperplasia, hepatocyte, nonregenerativea,bHyperplasia, hepatocyte, regenerative
Hyperplasia, Kupffer cell
Hyperplasia, oval cell
Hyperplasia, stellate cell
Hepatodiaphragmatic nodule
Neoplastic
LymphomaaaTerminology with diagnostic criteria or comments described below.
Uncommon as a background finding; commonly induced.
cTerminology addressed in Systemic Pathology section.
b

UncommonNot observed, potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

144

Toxicologic Pathology 49(1)Amyloid, liver

Hypertrophy, hepatocyte, liver

Comment

Comment88

Amyloidosis may be induced during acute infections in pigs.
Serum amyloid A genes are both hepatically and extrahepatically upregulated in pigs infected with Actinobacillus pleuropneumoniae or Staphylococcus aureus which is important for theuse of the pig as a model for human inflammatory responses, forexample, sepsis, cancer, and obesity research.88

With cholestasis, bilirubin may be encountered in dilated
canaliculi or hepatocytes and appears as a yellow or olive
green pigment. Bile pigment stains green with Hall’s
method.

Liver enlargement in response to hepatic enzyme induction istypically associated with hepatocellular hypertrophy and can beinitiated by a wide range of stimuli including pregnancy andlactation, hormonal fluctuations, dietary constituents, infections
associated with acute-phase proteins, and responses to exposure
to xenobiotics such as androstane receptor (CAR), the peroxisome proliferator-activated receptor, the aryl hydrocarbon receptor, and the pregnane-X-receptor. The hypertrophy may show
considerable species, strain, and sex differences in susceptibility
and severity.90 Hepatocellular hypertrophy as a result of hepatic
enzyme induction to xenobiotics has also been observed in minipigs (Jeppesen, personal communication).
Phenobarbital, a barbiturate sedative known for its liver
enzyme-inducing effects, has been shown to induce liver
CYP2B, CYP3A, and UGT enzymes in rodents, leading toincreased thyroid hormone clearance followed by increased
thyroid-stimulating hormone and subsequent stimulation ofcell proliferation in the thyroid. This pathway has also beenstudied in minipigs by Forster et al, and similar pattern ofchanges were also observed in the minipig as observed in therodents (ie, increases in the liver weights associated with diffuse hepatocellular hypertrophy, reduction in plasma concentrations of T3 and T4, and increases in enzymatic activities of
CYP3A429, CYP4A24, CYP1A2, and CYP2B22).
Portal vein embolization induces segmental hypertrophy ofthe nonembolized lobe in the liver of the minipig.91 However,portal reperfusion by collateral vessels may limit the occurrence of hypertrophy.

Fibrosis, liver (Figure 7.3)

Hyperplasia, hepatocyte, nonregenerative, liver

Comment

Comment

In normal minipig liver, considerable interlobular and subcapsular fibrosis are present. This is a characteristic histologicalfeature for the minipig and pig compared with other species.7
Therefore, drug-induced fibrosis in the liver should carefully
be assessed in the nonclinical safety studies. Additionally, there
does not appear to be any increased tendency of the porcine
liver to become cirrhotic after toxicologic injury comparedwith other nonhuman species.44 Adhesions and its associatedfibrosis of the hepatic capsule are common in Hanfords rather
than Göttingens and Yucatans.13

Spontaneous intralobular hepatocellular hyperplasia was seenin 5 out of a total of 51 female Yucatan minipigs ranging from 4
to 7 months of age but not observed in males. It was characterized by randomly scattered, discrete, intralobular nodules composed of hepatocytes that lacked lobular organization with anapparent compression of adjacent hepatocytes. Mitotic figures
within these foci were negligible. Liver enzymes were within
expected ranges for these animals.92 Intralobular hepatocellular
hyperplasia have not been reported in other strains of pigs.

Atrophy, hepatocyte, liver
Comment
Diffuse hepatocellular atrophy may occur in starvation, fibrosis,
portosystemic shunts, or congenital hepatic hypoplasia in minipigs. Stores of hepatocyte glycogen, lipid and protein, are utilized in starvation, resulting in a smaller liver.85 Althoughhepatocellular atrophy is uncommon, minimal to mild hepatocyte atrophy can be observed in association with fibrosis during
nonclinical safety studies when xenobiotics/test articles result inpoor clinical condition of a minipig.

Bile plugs, canaliculi, liver
Comment

Infarct, liver
Hepatitis, Helicobacter sp, liver
Comment
Helicobacter pylori and other Helicobacter species have been
isolated from porcine livers89; however, Helicobacter infections have not been observed or demonstrated in minipig nonclinical safety studies.3

Comment
Dearterialization by a combination of distal occlusion of thehepatic artery with Ivalon (polyvinyl alcohol foam) particles of
150 to 250 mm and proximal occlusion of the common hepatic
artery with Gianturco coils can result in necrosis/infarction of
up to 90% of the liver.93

Skydsgaard et al.

145
triglyceridemia.44 Presence of lipid can be demonstrated with
Oil Red O and osmium staining.

Lymphoma, liver
Comment
Swine multicentric lymphomas can occur in young pigs before theonset of maturity. The liver can be enlarged with multifocal, randomly distributed, small to large raised white masses throughout theparenchyma. Histologically densely packed large lymphoid cell
infiltrates can be seen in the liver94 (see Hematopoietic and Lymphoid System chapter, McKeag, unpublished data).

Rarefaction, hepatocyte (glycogen), liver(Figure 7.6)
Sinusoidal congestion, liver (Figure 7.7)
Infiltrate, mixed cell, liver (Figure 7.8)

Pigment, liver (Figure 7.4)
Comment
Pigment is seen frequently in hepatocytes and Kupffer cells injuvenile minipigs and occasionally in 4-week nonclinicalsafety studies due to iron injection. Hemosiderin can be seenin the liver associated with hemorrhages or generalized intraphagocytic hemolysis. With cholestasis, bilirubin may be
encountered in dilated canaliculi or in hepatocytes and appears
as a yellow or olive green pigment.

Vacuolation, liver (Figure 7.5)
Comment
Hepatocyte cytoplasmic vacuolation due to lipid accumulation
can occasionally be observed. These focal or multifocal, clear,sharply demarcated, empty vacuoles usually have no specificzonal distribution.3 These are generally of minimal to slightseverity. Fatty liver is a common finding in the streptozotocin
diabetic pig model, as it is in other porcine models of hyper-

Hepatocyte necrosis and congestion, liver(Figure 7.9)
Hepatocyte necrosis with neutrophilic cell
infiltrate, liver (Figure 7.10)
II. Gall Bladder
The gall bladder is located in a fossa on the visceral surface ofthe right medial lobe and is pear-shaped. Its serosal surface iswhite, and the mucosal surface is light green.85
At necropsy, upon opening the abdominal cavity, the gall
bladder should be examined and sampled immediately to prevent autolysis. A transverse section from the central region of
the gall bladder is examined in nonclinical safety studies.
Some of the commonly observed background changes in theGöttingen minipig are necrotizing cholecystitis, and hypoplasia
or aplasia of the gall bladder which are findings unique to theGöttingen minipig.3,7Table 7.2. Microscopic Findings of Hepatobiliary system: Gall Bladder; Minipig.
Gall bladder
Congenital
Aplasiaa
Hypoplasiaa
Nonproliferative
Calculus
Cholecystitis, porcinea
Edemaa,b
Hyalinosis
Infiltratec
Metaplasia, glandular
Proliferative (Nonneoplastic)
Hyperplasiaa,babcCommonUncommonNot observed, potentially relevant

X
X
X
X

Terminology with diagnostic criteria or comments described below.
Uncommon as a background finding; commonly induced.
Terminology addressed in Systemic Pathology section.

X
X
X
X
X

Not applicable

146

Toxicologic Pathology 49(1)Aplasia, gall bladder

Edema, gall bladder (Figures 7.15 and 7.16)

Pathogenesis

CommentUnknown, possibly congenital.

Diagnostic features

Macroscopic appearance; gall bladder is gray to beige,
small, and difficult to discern at necropsy.
Residual tissue consisting mainly of connective tissueelements observed microscopically.

Differential diagnoses


Atrophy, hypoplasia.

Comment
Aplasia of the gall bladder is occasionally observed in minipig
nonclinical safety studies, particularly in Gottingen minipigs.
There is no trace of bile macroscopically or microscopically in
the gall bladder. In addition, there are no associated clinicalsigns or changes in the clinical pathology parameters routinely
measured.3 Aplasia of the gall bladder has not been reported in
Hanford and Yucatan minipigs.13

Cholecystitis, porcine, gall bladder(Figures 7.11-7.14)
Other term(s)Unknown.

Diagnostic features






Macroscopically, slightly decreased in size with thickened walls.
Bile is absent or if present is inspissated.
Described as acute, subacute, chronic, or chronic activewith a severity ranging from moderate to marked.
Acute cases often characterized as necrotizing.
Microscopically diffuse necrotic, hemorrhagic mucosal
layer associated with granulomatous inflammation
extending into the muscular layer.

Differential diagnoses

Hyperplasia, gall bladder (Figure 7.18)
Comment
In juvenile minipigs, treatment with Gattex (teduglutide, a 33
amino acid recombinant analog of the human GLP-2) can resultin microscopic changes in the gall bladder (cystic mucosal
hyperplasia) and extrahepatic bile duct (cystic mucosal hyperplasia) as observed in mice and monkeys.52

Hypoplasia, gall bladder (Figures 7.19a and7.19b)
Pathogenesis
Hypoplasia.

Unknown, possibly congenital.

Diagnostic features

Necrotizing cholecystitis.

Pathogenesis
Edema in the gall bladder can occasionally be observed in minipig
nonclinical safety studies. Microscopically edema appears as apale eosinophilic amorphous accumulation with minimal or no
inflammatory cells in the mucosa/submucosa.



Dark gray and small at macroscopic examination.
Microscopically flattened or folded mucosal epithelium(disappearance of mucosal crypts), absence of glands,
and underlying minimal to slight amounts of loose connective tissue rich in blood vessels.
Absence of bile macroscopically and microscopically.

Differential diagnoses


Atrophy, aplasia.

Comment
Hypoplasia of the gall bladder is occasionally observed inminipig nonclinical safety studies. There are no associatedclinical signs or changes in the clinical pathology parameters
routinely measured.3 Hypoplasia of the gall bladder has only
been observed in Gottingen minipigs and has not been reported
in Hanfords and Yucatans yet.13

Gallstone in an aged microminipig, gall
bladder (Figure 7.20)

Comment
Cholecystitis is one of the commonly observed backgroundchanges unique to the Göttingen minipig. Chronic necrotizing
cholecystitis is the most common form. No clinical signs orchanges in clinical pathology parameters are observed in animals
diagnosed with cholecystitis in nonclinical safety studies. While
this is a background finding, bile is important in drug metabolism and this entity may affect toxicological outcomes.3,7Chapter 8. Integumentary System
The anatomy of minipig skin is comparable to that of othermammals. Therefore, for detailed general considerations on
the integument, please refer to the INHAND publication on
rodent integument.95 In general, pigs (domestic and minipigs)
have thicker skin with less hair than most animals, especiallycompared to rodents. Although different minipig strains, such

Skydsgaard et al.
as Göttingen, Sinclair, Yucatan, and Hanford, exhibit differences in cutaneous pigmentation, hair coats, and growth patterns of their skin, they share many similarities in terms ofgeneral morphology, epidermal thickness, cellular composition, immunological reactivity, and cellular turnover tohuman skin.96-100 The minipig epidermal thickness varies
only minimally between birth and adulthood, whereas thedermal and especially the subcutaneous thicknesses increases
over time.101 Although the epidermis generally consists of 5
to 7 cell layers with only minor regional variations, the thickness of porcine epidermis varies from 30 to 140 mm (compared to 50-120 mm in human skin) and is thinner in theabdominal region and thicker at the neck, particularly inmales. Similarly, the dermis and subcutis are thicker on thedorsum and thinner at the abdomen and inner thighs. Similar
to humans, pig skin has well-developed rete ridges and pars
papillaris. Both pig and human skin have abundant subcutaneous fat tissue, which is divided in a superficial and a profound layer. This may lead to differences in compound
diffusion depending on the depth of the injection. In general,
the fat tissue layer is thicker in pigs and the skin is less vascular compared to humans. Pigs have extensive apocrine
sweat glands, which play a limited role in thermoregulationand sweating. Porcine eccrine glands are only located on thesnout and in the carpal area.102 Seasonal change from apocrine to eccrine glands occurs in some pig strains. The pH ofminipig skin depends slightly on the body region and is
between 6 and 7, which is slightly higher than in human skin(approximately pH 5).
The similarities of porcine and human skin makes the minipig an attractive animal model in dermatotoxicology, in particular for investigating wound healing.103,104 Regional variationsin the porcine skin such as thickness of epidermis, dermis, andsubcutis, as well as occurrence of apocrine and eccrine glands,
and differences in the hair coat have to be considered during theplanning of cutaneous toxicology studies.
This chapter does not cover hooves. Lesions in the mammary gland are covered in Chapter 9. Dermal lesions, which arecovered in the soft tissue, nervous system, or cardiovascularsystem INHAND manuscripts, are not discussed here.
Nonproliferative skin lesions are commonly encountered asspontaneous background findings in young minipigs used in
nonclinical toxicity studies. In contrast, proliferative skinlesions are observed rarely as background findings due to theyoung age of animals at study initiation and the short durationof toxicity studies. In domestic pigs, squamous cell papillomas
and carcinomas have been reported to occur rarely in whiteskinned strains.105-107 Melanomas have been described indarkly colored pigs such as Duroc and Hampshire breeds.108
There are certain specific minipig strains, namely Sinclair,109
MeLiM (Melanoma-bearing Libechov Minipigs) also known as
Libechov strains110 and Munchener Miniaturschwein (Troll)minipig strains111 that frequently develop regressing cutaneous
melanomas at a very young age.
Minipigs are susceptible to many of the same skin diseases
that affect domestic pigs. The term “Dippity Pig Syndrome” is147commonly used to describe a skin condition in various strainsof domestic pigs, as well as in minipigs.12 The condition isrecognized in Göttingen minipigs in all age groups. It is acutaneous neurological syndrome characterized by skin sores(“weeping blisters”) and sensitivity to touch. Other clinicalmanifestations include assuming “hunkered-down” stancewhen touched, pain, weakness and/or inability to use the hindlegs, falling down, and other signs of distress. The etiology is
unknown, but the condition may be precipitated by stress. The
cutaneous manifestations are consistent with erythema multiforme. The lesions are mainly seen in the lumbar region. Histopathologic appearance includes increased dermal congestion,
edema, and perivascular lymphocytic infiltration (Figure 8.1).
Although this syndrome is self-limiting, with spontaneousrecovery typically within 1 to 2 weeks, the occurrence withina toxicity study in minipigs may confound interpretation offindings, particularly in the safety evaluation of topical agents.
There is no known treatment; however, palliative veterinary
treatment for pain (eg, oral meloxicam) may be beneficial.
Intermittent withdraw of topical application of the test article
may be needed in a toxicity study to allow healing of the
cutaneous lesions associated with “Dippity Pig Syndrome”
before reinitiating application of the test article.
Other skin diseases such as porcine juvenile pustular psoriasiform dermatitis (Pityriasis rosea), which occurs in young
domestic white pig breeds, has also been found in young white
minipigs. However, there are some skin diseases that arereported to occur specifically in minipigs. One such example
is a thrombocytopenic purpura-like syndrome also called thehemorrhagic syndrome described in mature male and femaleGöttingen minipigs, characterized by extensive subcutaneous
hemorrhage accompanied by marked thrombocytopenia, anemia, and membranoproliferative glomerular lesions,18 which is
discussed in the systemic pathology chapter. Another example
is bullous pemphigoid reported in Yucatan minipigs characterized by subepidermal vesicles with many intact and degranulated eosinophils and exhibiting linear immunoglobulin(specifically IgGs) deposits at the dermoepidermal junction.29,112 With advances in animal husbandry in minipig facilities, infectious skin diseases are likely to be a rare occurrence.

Trimming
For organ sampling and trimming in rats and mice, the revised
guides113 recommend the collection of skin samples from theinguinal region. Sampling of minipig skin differs between
laboratories with some using the abdomen and others the dorsum. For standardization and comparison of the samples, it isimportant to collect the skin samples consistently. To represent
the large skin surface of pigs with different morphology at
different body locations, at least 2 samples from different locations may be necessary.
In dermal studies, 3 to 4 treated skin samples are routinely
examined in addition to an untreated sample from a similar
anatomical location. The treated skin samples should be subjected to a pooled evaluation.

148

Toxicologic Pathology 49(1)Nomenclature
The incidence data are based on Göttingen, Hanford, Sinclair, and Yucatan minipigs routinely used in toxicity

studies. In general, inflammatory lesions and associatedreactions were the most commonly observed findings in
the skin. 13

Table 8.1. Microscopic Lesions of the Integument: Skin; Minipig.
Integument

CommonNonproliferative
Abscessa
Adnexal dysplasia
Atrophy, adnexa
Atrophy, dermis
Atrophy, epidermis
CrustbCyst, squamous
Edema, dermis
Edema, intercellular, epidermis
Edema, intracellular, epidermis
Elastosis
Erosion/ulcer
Hyperkeratosis, adnexa
Hyperkeratosis, epidermis
Infiltrate, epidermisa
Inflammation, adnexaa
Necrosis, adnexa
Necrosis, epidermis
PustuleVesicleProliferative (nonneoplastic)
Hyperplasia, adnexa
Hyperplasia, epidermis
Hyperplasia, melanocyte
Neoplastic
Carcinoma, squamous cell
Papilloma, squamous cell
Melanoma, benignb,cMelanoma, malignantb,cabcUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.
Melanomas have been described in Sinclair, MeLiM also known as Libechov strains and Munchener Miniaturschwein (Troll) minipig strains but have not been
described in white skinned minipigs such as Göttingen.

Crust, Skin (Figures 8.2 and 8.3)
Sublocation
Epidermis.

Other term(s)Crust, serocellular; exudate, serocellular.

Modifier(s)within the stratum corneum of the epidermis. Crusts canbe formed through aging of pustules.

Diagnostic features
Desiccated accumulation of inflammatory cells, pustular debris,
erythrocytes, epithelial squames, clotted plasma proteins, or microorganisms on or within the stratum corneum of the epidermis.

Inflammation

Serous, hemorrhagic, cellular, serocellular.

Inflammation of the skin is not confined to the stratum corneum
level of the epidermis.

Pathogenesis/cell of origin

Comment

Crusts are indicative of a previous exudative process.
They are formed by inflammatory cells and cellular debris

Crust are by far the most common microscopic finding in theskin of minipigs.7 The crusts are usually focal in nature and of aSkydsgaard et al.
low severity. The crusts commonly have a traumatic etiology(eg, due to rubbing against fixed items) but might be test
article-induced in toxicology studies, especially when the dermal route of administration is used. In general, crusts are composed of a serocellular exudate but might also include othercomponents such as dressing material in dermal studies. In
wound healing studies, crusts covering the surface of thewound should be carefully characterized with respect to size/
severity as well as the individual constituents, since these parameters change when the wound healing process proceeds
toward complete healing.

Melanoma, benign, skin
Comment
The Sinclair, Libechov, and Munchener Miniaturschwein(Troll) minipig strains show a high incidence of spontaneous
cutaneous melanomas in newborns, which continue to develop
after birth. The benign melanomas can further develop in
malignant melanomas and can also undergo regression. Histologically, tumor regression is characterized by melanomacrophages, infiltrates of lymphocytes, fibrosis, and free melanin
replacing the tumor.114

Melanoma, malignant, skin
Comment
The Sinclair, Libechov, and Munchener Miniaturschwein(Troll) minipig strains show a high incidence of spontaneous
cutaneous malignant melanomas in newborns, which continue
to develop after birth. The tumors may invade muscle fascia
and have high metastatic activity. Metastatic spread to thedraining lymph nodes is frequently observed, whereas metastases to other organs is less frequent but not uncommon.115
Spontaneous regression of primary and metastatic lesions has
been observed in Sinclair minipigs within the first year of life.
Tumor regression is accompanied by depigmentation, which is
not limited to the tumor itself but may range from a localized
depigmentation of adjacent hair and skin to a generalized
depigmentation including the iris of the eye.109 Histologically,tumor regression is characterized by melanomacrophages,

149
infiltrates of lymphocytes, fibrosis, and free melanin replacing
the tumor.114. Though many melanomas in these minipig
strains have been classified as malignant, they may regress,
which should be considered during evaluation of these tumors.

Chapter 9. Mammary Gland
Standard INHAND nomenclature for nonproliferative and proliferative mammary gland findings in rats and mice has previously been published116 and will be used for the minipig as
appropriate following the anatomical approach used for rodent
species, with a focus on those findings that are likely to be
encountered frequently and those that might be consideredunique to the minipig. The mammary gland is a milkproducing exocrine gland characteristic of all female mammals
and present in a rudimentary and generally nonfunctional form
in males. Mammary glands are regulated by the endocrine
system. Minipigs have 6 to 7 pairs of mammary glands on theventral abdomen. At necropsy, usually one of the inguinal
mammary glands is sampled for histology (Figures 9.1-9.6).
In immature female minipigs, there is very little mammary
gland development with no secretion. There are few ducts
which are small and scattered. Alveoli, where present, are lined
by epithelial cells that are either quiescent (small and single
layered) or show early development (larger, multilayered). In
mature female minipigs, mammary glandular tissue is moderately to markedly developed, with numerous alveoli. Hyperplastic glandular cells are visible in about one-third of the caseswith markedly developed mammary glands, and alveoli andcanals are in many cases filled with eosinophilic material.117
The mammary gland of female minipigs does not reflect thestage of the estrus cycle as it does for example in dogs.118
Pathologic changes in the mammary gland of minipigs arerare in preclinical toxicity studies.7 Proliferative lesions inlaboratory-maintained minipigs may theoretically arise fromgenotoxic test items, infectious agents, and changes in hormonal homeostasis (eg, as part of the aging process). However, thereports on proliferative changes in the mammary gland of minipigs are sparse and is most likely due to the young age of theanimals used for routine preclinical toxicity testing.

Table 9.1. Microscopic Findings of Mammary Gland; Minipig.
Mammary gland
Nonproliferative
AmyloidAngiectasis
Atrophy
Basophilia
Congestion
Corpora amylacea
Degeneration

CommonUncommonNot observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X
(continued)

150

Toxicologic Pathology 49(1)Table 9.1. (continued)
Mammary gland

CommonDilation
EdemaaFibrosisa
Hemorrhagea
Infiltratea
Inflammationa
Mineralizationa
Necrosisa
Pigmenta
Single cell necrosis
Thrombus
Proliferative (nonneoplastic)
Hypertrophy/hyperplasia, alveolar, and/or ductal epithelial cell
Hyperplasia, lobuloalveolar
Proliferative, neoplastic
Adenocarcinomabab

UncommonNot observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Adenocarcinoma, Mammary Gland, Acinus
Comment
A case of a mammary gland adenocarcinoma has been reported
in a 10-year-old female Göttingen minipig.119

Chapter 10. Nervous System
For detailed general considerations on the nervous system,
please refer to the INHAND publication on rodent nervous
system.120,121The cellular composition of minipig nervous system is comparable to that of other mammals.
Minipigs are susceptible to many of the same nervous system diseases that affect domestic pigs. However, with advancesin animal husbandry in minipig facilities, infectious diseases
are likely to be a rare occurrence.

Trimming
The brain is typically trimmed for examination of 7 sections as recommended by Bolon et al, but a more extended

trimming procedure of the minipig brain has beenpresented, with 14 sections examined.122,123 Sections from
3 divisions of the spinal cord are collected including cervical, thoracic, and lumbar sections.26 For general toxicity
testing, the sciatic nerve is representative of the peripheral
nervous system and is trimmed in cross and longitudinal
section.26 For studies in which neurotoxicity is expectedor known, sampling of ganglia and nerves should be more
extensive.123

Nomenclature
The incidence data in the following tables are based onGöttingen, Hanford, Sinclair, and Yucatan minipigs routinely used in toxicity studies. In general, inflammatory
cell infiltration and mineralization of the meninges werethe most commonly observed findings in the nervous
system.13

Table 10.1. Microscopic Lesions of the Central Nervous System: Brain and Spinal Cord; Minipig.
Brain and spinal cord
Congenital
Cyst, squamous
Hamartoma, lipomatousa
Neuronal heterotopia
Nonproliferative
Artifact, dark neuronb
Astrocyte swelling
Astrocyte swelling/vacuolation
Astrocytosis
Atrophy, axon

CommonUncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

151

Table 10.1. (continued)
Brain and spinal cord

Commonb

Bubbles, myelin
Chromatolysis
Cholesterol clefts
Decreased cellularity, neuron
Degeneration, axon
Degeneration, nerve fiber
Demyelination
Dystrophy, axon
Ectopic tissue
Edema, intramyelinic
Gliosis, not otherwise specified (NOS)
Hemorrhage
Hydrocephalus
Hydromyelia
Infarct
Infiltratea,cInflammationc
Necrosis/Inflammation, vascular/perivasculara,cMicrogliosis
Mineralizationa,dNecrosis, neuron
Neuronophagia
Pigment, lipofuscin
Satellitosis
Syringomyelia/hydromyelia
Syringomyelia
Thrombus
Type II astrocytes
Vacuolation, choroid plexus
Vacuolation, neuron
Vacuolation, white matterb
Neoplastic
Medulloblastomaa

UncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
As artifact.
Terminology addressed in the Systemic/General Pathology section.
dSpecifically observed in the meninges.
bcHamartoma, lipomatous, brain and spinal cordComment
Though they have not yet been reported in minipigs,
lipomatous hamartomas have been described indomestic pigs. 124 These are occasionally described as
lipomas.

Necrosis/Inflammation, media or wall, artery,
Brain and Spinal Cord

Infiltrate, eosinophilic, brain and spinal cord
(Figure 10.1)
Comment
Meningeal and perivascular cortical infiltration of eosinophilic
granulocytes has been described in pigs in several disease states,
including leukomalacia of vitamin E/selenium deficiency andvarious encephalitides but is specifically seen in salt toxicity(inadequate water intake followed by adequate or excess water
intake). Combined with the laminar loss of cortical neurons, thisinfiltrate is pathognomonic for salt toxicity.124,125

Comment

Medulloblastoma, brain and spinal cord

A generalized arteritis or periarteritis in the minipig may
also affect the nervous system. For detailed information
about this condition please refer to the cardiovascular
chapter.

Comment
Blastomas of the nervous system such as medulloblastomas
have been reported to occur rarely in young domestic
pigs124,126 and might therefore also be found in the minipig.

152

Toxicologic Pathology 49(1)Table 10.2. Microscopic Lesions of the Central Nervous System: Meninges; Minipig.
Meninges

CommonCongenital
Aggregate, melanocytea,bCyst, squamous
Hamartoma, lipomatousc
Meningioangiomatosis
Nonproliferative
Cholesterol clefts
Hemorrhage
Infarct
Infiltratec,dInflammationd
Necrosis/inflammation, vascular/perivascularc,dPigment, lipofuscin
Mineralizationa,eThrombus

UncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
In pigmented animals.
Terminology with diagnostic criteria or comments described above (microscopic lesions of the brain and spinal cord).
dTerminology addressed in the Systemic/General Pathology section.
eSpecifically observed in the meninges.
bcAggregates, melanocyte

Comment

Other term(s)The leptomeninges of pigmented pigs can have melanin with ahigh variation from animal to animal concerning the extent anddegree of pigmented deposition. Congenital meningeal melanosis produces no clinical impairment in affected animals.126

Meningeal melanosis; melanosis.

Pathogenesis/cell of origin
Neuroectodermal origin.

Mineralization (Figure 10.2)
Diagnostic features

Comment

Aggregates of polygonal melanocytes filled with pigment.
No cellular atypia.
No displacement of normal structures.

Mineralization as a focal minimal lesion is often observed in
the meninges of minipigs.7,13,41

Table 10.3. Microscopic Lesions of Nervous System: Peripheral Nervous System; Minipig.
Peripheral nervous system
Congenital
Neuronal heterotopia
Nonproliferative
Accumulation, laminar, Schwann cell
Accumulation, matrix
Atrophy, axon
Autophagy, neuron, ganglion
Chromatolysis
Decreased cellularity, neuron
Degeneration, axon
Degeneration, nerve fiber
Demyelination
Dystrophy, axon
Infiltratea

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

153

Table 10.3. (continued)
Peripheral nervous system

CommonUncommona

Inflammation
Necrosis/inflammation, vascular/perivasculara,bIntramyelinic edema
Necrosis, neuron
Neuronophagia
Renaut body
Vacuolation, neuronab

Not observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X

Terminology addressed in the Systemic/General Pathology section.
Terminology with diagnostic criteria or comments described above (microscopic lesions of the brain and spinal cord).

Chapter 11. Reproductive System—Female
Standard INHAND nomenclature for nonproliferative and proliferative female reproductive system findings in rats and micehas previously been published127 and will be used for the minipig as appropriate following the anatomical approach used forrodent species, with a focus on those findings that are likely to
be encountered frequently and those that might be consideredunique to the minipig. Generally very few spontaneously or
treatment induced microscopic changes are seen in the female
reproductive tract of minipigs. If published references werelocated for experimentally induced changes, they have been
indicated in the reference section and included in the comments
for the changes.
Discussion of histological features of the female reproductive system with regard to sexual maturity status as well as
estrus cycle specific changes is included to enable familiarization with normal variations and proper recording of alterations
from normal. Discussion of maturity status in this chapter isstrictly related to the degree of maturity of the reproductive
tract organs and does not include consideration of skeletal
maturity.

that opens to the outside through the common urogenital orifice. Ovaries are generally bean-shaped and may weigh as little
as 2 g, even in the adult. The ovarian stroma is composed oftightly packed spindle-shaped fibroblast-like cells, often
arranged in a whirling pattern, and may contain lipid droplets.
Under hormonal influence, the stromal cells may accumulate
more lipid (luteinization).

Tissue Collection, Fixation, and Trimming
Albl et al have thoroughly detailed the proper collection, fixation, and trimming schemes for the female reproductive tract.26
Briefly, for routine general toxicology studies, the sections
examined include one cross section of the right and left oviduct(uterine tube), one mid-sagittal section of each ovary, one crosssection of the right and left uterine horns (from the middle ofthe organ), one cross section of the cervix, and one cross section of the vagina. For advanced studies, trimming guidelines
for the ovary, oviduct, and uterus described in26 may befollowed.26

Sexual Maturity in the Female Minipig
Female Minipig Reproductive Anatomy
The female reproductive tract in the minipig is similar todomestic swine, consisting of a bicornuate uterus with tortuous
fallopian tubes. In adult domestic pigs, the fallopian tubes are
similar in diameter to the human but longer.128 The uterus
consists of 3 functional zones comparable to the macaqueendometrium including the luminal epithelium (zone I), glandular layer subjacent to the luminal epithelium (zone II), and thebasal glandular layer above the myometrium (zone III). The
minipig is similar to humans with regard to uterine histology
and estrus cyclicity but unlike humans and cynomolgus monkeys does not menstruate. A characteristic feature of the porcine cervix not observed in humans is the pulvini cervicales,129
which consists of numerous interdigitating prominent solid
mucosal folds and protrusions throughout the length of thecervix. In pigs, more than 90% of the cervix may have a vaginal
type of epithelium with stratified squamous epithelium that
undergoes cyclic alterations. In minipigs, the urethra opens
on the ventral surface of the vagina, creating a urogenital sinus

Repeat-dose toxicity studies conducted using sexually mature
animals provide important information on potential effects ofthe test article on the reproductive tract. The International
Council for Harmonization Safety Guidance Document S5R3 lists histopathology of reproductive tract organs fromrepeat-dose toxicity studies as a sensitive method for detecting
effects on male and female fertility, provided animals are sexually mature at the termination of the study.130 However, reliable interpretation of changes in the female reproductive tract
in routine toxicology studies is often confounded by the wide
window of sexual maturity of female minipigs. Female minpigs, based on available reports103,117,131 as well as personal
experience of the authors, generally mature between 4 and 8months of age. Younger age (<5-6 months) at study initiation,
small group size, and a wide window of sexual maturity can
result in uneven distribution of immature minipigs in various
treatment groups, particularly in studies of 3 months durationor less. It is therefore recommended that studies be initiatedusing minipigs of appropriate age, taking into consideration the154
study duration and the wide maturation window of this species,
to ensure that adequate numbers of animals would be sexuallymature in each treatment group at the end of study.
While reproductive tract organs exhibiting typical immature phenotype histologically should be considered andrecorded by pathologists as within normal limits, it is prudent to capture information regarding the sexual maturity
status for the study animals. Pathologists should therefore
be familiar with the normal variation in the histologicalfeatures of the reproductive tract related to sexual maturity
status. Sexual maturity status in female minipigs can bedivided into 2 major categories, immature and mature, withan intervening transitional period when the histologic
appearance of the reproductive tract organs can be categorized as partially mature or peripubertal.
At the onset of sexual maturity, the female should havesuccessfully ovulated, with progression of the ruptured follicle to formation of a corpus luteum. Progesterone levels
increase following ovulation, and therefore, analysis of progesterone levels is considered a reliable method to detect
the onset of sexual maturity. The first cycle of progesterone
release can be considered as an indicator of the onset of
sexual maturity. There have been conflicting reports on thecorrelation of body weight with age of onset of sexual
maturity. Tortereau et al reported significant correlation
between body weight and uterine weight but not ovarian
weight with maturity status from an evaluation of 39
females.117 However, de Rijk et al (2014) reported thatbody weight did not correlate with age of sexual maturity.118 The histological appearance of the reproductive
tract, especially changes in the ovary, enables identificationof the sexual maturity status117,131,132 and is briefly summarized below. Histological features of the uterus andvagina correlate well with ovarian changes.117

Immature StageThe ovary contains primordial and small to medium-sizedsecondary follicles but lacks tertiary follicles and corpus
lutea. In the uterus, the endometrial glands are few, simple and tubular without luminal secretions. The uterine
lumen tends to be narrow. The vaginal epithelium is generally regular and uniform, varying from 4 to 18 cells inthickness, with no evidence of estrus cycle activity(Figure 11.1)

Partially Mature Stage (Peripubertal Stage)
Female minipigs are considered to be partially mature or in aperipubertal stage when the ovaries contain primordial, secondary, and tertiary follicles but lack corpora lutea (CL). The
uterus appears similar to the immature stage, with endometrial
glands moderately developed, but may contain frequent apoptotic bodies in the glandular and superficial epithelial cells(Figure 11.2).

Toxicologic Pathology 49(1)Mature Stage
Ovaries contain primordial, secondary, and tertiary follicles andone or more CL. The presence of a corpus luteum in any stage isconsidered confirmation of sexual maturity. The uterus contains
well-developed, often branched and coiled, endometrial glands,
often with mucinous secretions. The uterine lumen is also moderately developed with papillary surface endometrium. Histologyof the vaginal epithelium in mature females generally varies
according to the stage of estrus cycle (Figure 11.3).

Estrus Cycle
Recommendations for evaluation of the female reproductive
system and estrus cycle staging are similar to that forrodents.127 While it is not necessary to record the stage of
estrus cycle in routine toxicity studies,127 it is critical that thepathologist be aware of the normal estrus cycle phase-specifichistologic characteristics of the reproductive tract. Generally,a high degree of synchronicity is seen in the histologicchanges in the various segments of the reproductive tract(ovaries, uterus, and vagina) during the various phases of the
estrus cycle. Familiarity with the normal estrus cycle associated histology will therefore permit identification of changes,
which could be direct or indirect effects of xenobiotic
administration.
Unlike rodents, which have a very short estrus cycle of 4 to 5days, the duration of estrus cycle in the mature minipig is longer,
ranging from 20 to 22 days, and is more comparable to theduration of the menstrual cycle in the cynomolgus macaque(approximately 30 days). Estrus cycle-related systemic hormonal fluctuations have been well characterized and include a preovulatory luteinizing hormone surge followed by ovulation withsubsequent increase in progesterone levels.118 Progesterone levels increase for about 5 to 6 days following ovulation (estrus),
stay high for another 6 to 7 days (metestrus), and finally fall
during the last 3 to 4 days of the estrus cycle (end of diestrus).
Staging of the estrus cycle and identification of perturbations in
the cycle, based on morphologic features of the reproductive
tract, is best achieved when the histologic features of the ovary,uterus, and vagina are taken into consideration together rather
than relying on any one of them individually. The histologic
distinction between phases, however, is not as clear as in rodents,
rabbits, dogs, or monkeys.117 The estrus cycle and the associatedhistological changes in the reproductive tract in the minipig havebeen described in detail117,118 and are briefly described below.
Estrus cycle-associated changes seen in the reproductive tract
have been categorized as “common” in the tables below. Thesechanges are, however, not routinely recorded as separate
diagnoses.

Proestrus (Follicular/Proliferative Phase)
Ovary: The primary feature includes the presence of large
tertiary (Graafian) follicles. Regressing CLs from previous cycle/s may also be seen (Figure 11.4).

Skydsgaard et al.

155

Uterus: The uterus generally has a small lumen with few
invaginations of the surface epithelium. Surface epithelium is generally cuboidal and contains many apoptotic
and mitotic cells. The luminal surface is lined by a thinlayer of periodic acid–Schiff (PAS)-positive material.
Endometrial glands in zones 2 and 3 are small and contain few apoptotic cells (Figures 11.5 and 11.6).

central cavities may be present which are considered torepresent either CLs that have not yet completely filled orGraafian follicles that have failed to rupture and haveundergone luteinization (luteinized unruptured follicles[LUFs]).
Uterus: The luminal epithelium of the uterus is tallest(columnar) at this stage (Figure 11.13), and the epithelium has a maximal number of invaginations. Luminal
epithelial cells no longer have a pseudostratified appearance. Endometrial glands in zones 2 and 3 are developedwith abundant luminal secretion. Glandular stroma may
contain lymphocytic infiltration. Few apoptotic bodies
and mitotic cells may be noted during early metestrus
but are generally not present during late metestrus.

Vagina: The vaginal epithelium is characterized by the presence of a layer of stratum germinativum, up to 10 intermediary layers, and a superficial layer of cuboidal to
ovoid cells with pale cytoplasm (Figure 11.7).

Estrus (Ovulatory Phase)
Ovary: The ovary contains a collapsed Graafian follicle
indicative of ovulation (Figure 11.8). Regressing CLs
from pervious cycles may be present.
Uterus: In the uterus, very few distinct changes from theproestrus stage are noted. Apoptotic bodies tend to disappear toward the later part of estrus and a pseudostratified appearance of nuclei is seen in the luminal
epithelium (Figure 11.9).

Vagina: Vaginal epithelium is characterized by 4 to 8 layersof cuboidal to ovoid cells with polymorphonuclear cell
infiltration of the epithelium and lamina propria(Figure 11.14).

Diestrus (Late Luteal/Late Secretory Phase)

Vagina: Unlike rodents, cornification of the superficial
epithelium is not a feature in the vagina. A layer ofbasal stratum germinativum is overlain by intermediate layers and several layers of superficial flattened
cells (Figure 11.10).

Metestrus (Early Secretory/Mid-Secretory Phase)
Ovary: The ovary contains large, newly formed CLs with acentral space during the early phase, which progresses tofully mature CLs filled with luteinized cells (Figures 11.11 and 11.12). Tertiary follicles may be present
at this stage. Hyalinized regressed CLs from previous
cycles may be present. Other CL-like structures with

Ovary: Degeneration of CLs is noted with cells losing eosinophilic character and medium-sized to large tertiary
follicles are present (Figure 11.15).
Uterus: Endometrial glands in zone 3 are lined by flattenedepithelium and contain inspissated secretions. Luminal
epithelium is comparable to proestrus with many apoptotic cells. Mitotic activity is present in the luminal
epithelium but not in zone 2 and 3 glandular epithelium.
Periodic acid–Schiff-positive material is present on theluminal surface (Figure 11.16).
Vagina: Epithelium generally is similar to that of metestrus
with the exception that polymorphonuclear cell infiltration is minimal or absent (Figure 11.17).

Table 11.1. Microscopic Findings of the Female Reproductive System: Ovary; Minipig.
Ovary
Nonproliferative
AmyloidAngiectasis
Atretic follicles, increased number
Atrophy
Atrophy, corpora lutea
Cyst, bursal
Cyst, epithelial
Cyst, follicular
Cyst, luteal
Cyst, NOS
Cyst, rete ovarii
Cyst, paraovarian
Decreased number/absent corpora lutea

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

156

Toxicologic Pathology 49(1)Table 11.1. (continued)
Ovary
Decreased number/absent follicles
Degeneration, corpora luteaa
Degeneration, oocyte
Edema
Follicle, luteinizeda
Follicle, polyovular
Immaturity
Infiltrateb
Mineralizationc
Ovotestisc
Pigment
Ectopic tissue
Vacuolation, corpora luteaa
Vacuolation, granulosa cell
Vacuolation, interstitial cell
Vacuolation, theca cell
Proliferative (nonneoplastic)
Hyperplasia, cystic/papillary
Hyperplasia, granulosa cell
Hyperplasia, rete ovarii
Hyperplasia, Sertoli cell
Hyperplasia, sex cord stromal, mixed
Hyperplasia, smooth muscle, mesovarial
Hyperplasia, theca cell
Hyperplasia, tubulostromal
Hypertrophy, corpora lutea
Hypertrophy, interstitial cell
Increased number, corpora lutea
Hyperplasia, interstitial cell
Neoplastic
Teratoma, benignc

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Indicates changes commonly seen as part of normal estrus cycle and not generally recorded as an individual finding.
Terminology addressed in Systemic Pathology section.
cTerminology with diagnostic criteria or comments described below.
b

Follicle, Luteinized

Ovotestis

Comment

Comment

Graafian follicles that fail to rupture undergo atresia resulting in
atretic follicles. Occasionally CL-like structures with prominentcentral cavities lined by luteinized epithelium can be seen in theovaries and may represent unovulated Graafian follicles thathave luteinized.118 Luteinized unruptured follicles should be
differentiated from normal nonclosed corpus lutea. Presence ofoocyte is confirmative of LUF. Similar LUFs have been
described as a background finding in beagle dogs.133 Hormonal
manipulation can induce LUFs in rats and monkeys.134

Ovotestis has been seen rarely (unpublished data) and, similar
to rodents, is characterized by gonads containing both ovarianand testicular tissue.

Mineralization (Figure 11.18)
Comment
Mineralization can be seen in the ovaries and is often associated with atretic follicles.7

Teratoma
Comment
Neoplastic changes in the ovary are rare. One instance ofa benign teratoma was noted in a female Göttingen minipig.7 Benign teratomas are generally encapsulated, expansile masses composed of varying proportion of tissues
derived from the endoderm, mesoderm, and ectoderm.

Skydsgaard et al.

157

Table 11.2. Microscopic Findings of the Female Reproductive System: Oviduct; Minipig.
Oviduct
Nonproliferative
Atrophy
Infiltratea
Inflammation
Metaplasia, squamous cellb
Proliferative (nonneoplastic)
Hyperplasia, epithelial cellabCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Metaplasia, squamous cell (Figure 11.19)
Comment
Squamous cell metaplasia can be commonly found in the oviduct7 and is characterized by the presence of keratinizing or nonkeratinizing squamous cells replacing normal cuboidal or columnar epithelium of the oviduct.
Table 11.3. Microscopic Findings of the Female Reproductive System: Uterus; Minipig.
UterusCongenital
Aplasia, segmental
Nonproliferative
Abscess(es)AmyloidAngiectasis
Apoptosisa
Atrophy
Decidual reaction
Decidualization, focal
Dilatation, glandular, cystic
Dilatation, luminala
Erosion/ulcer
Fibrosis
Granuloma
Hemorrhageb
Hypertrophy, epithelial
Hypertrophy, myometrial
Hypoplasia
Infiltratea
Inflammation, endometrium
Inflammation, myometrium
Mesonephric duct remnants
Metaplasia, squamous cellb,cNecrosisMesonephric duct remnants
Pigment
Prolapse
Pyometrab
Vacuolation, epithelial cell
Proliferative (nonneoplastic)
Adenosisb
Hyperplasia, angiomatous
Hyperplasia, endometrial stromal
Hyperplasia, endometrial, diffuse
Hyperplasia, glandular, cysticb,cCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

158

Toxicologic Pathology 49(1)Table 11.3. (continued)
UterusCommonHyperplasia, glandular, focal
Hyperplasia, granular cell
Hyperplasia, myometrial
Polyp, endometrial stromal
Polyp, glandular
Neoplastic
Adenocarcinoma, endometrialb
Adenoma, endometrial
Leiomyomab
Leiomyosarcomab

UncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X

.

a

Indicates changes commonly seen as part of normal estrus cycle and not generally recorded as an individual finding.
Terminology with diagnostic criteria or comments described below.
cUncommon as a background finding; can be induced.
b

Hemorrhage

Adenosis

Comment

Comment

Focal hemorrhage of minimal severity has been noted with avery low incidence in the uterus of the minipig.7

Adenosis is an uncommon change reported to occur in theuterus of female miniature pigs135 and may be observed macroscopically as nodular lesions. Diagnostic features are similar to
those reported for rodents and include the presence of endometrial glandular and stromal elements that do not exhibit atypicalfeatures within the myometrium. Spread of endometrial elements (glands and stroma) to the peritoneum has not beenreported, and therefore, unlike human and nonhuman primates,
adenomyosis in pigs should not be equated with endometriosis.
Adenosis should be differentiated from endometrial adenoma
and adenocarcinoma.

Hyperplasia, glandular, cystic
Comment
Cystic glandular hyperplasia, characterized by dilated and cystic
glands, has been reported in female miniature pigs as a background finding.135 Cystic glandular hyperplasia has also been
induced by exposure to zearalenone136 and methoxychlor.137

Metaplasia, squamous cell
Comment

Neoplastic changes

Squamous metaplasia, characterized by focal or multifocal
replacement of the surface and/or glandular columnar epithelium by keratinizing or nonkeratinizing epithelium, is an
uncommon finding. However, cystic dilatation of endometrial
gland with squamous metaplasia has been reported following
experimental administration of zearalenone to pigs.136

PyometraComment
Pyometra characterized by suppurative (purulent) luminal exudate, which can be a sequel to inflammation of the endometrium,
has been reported infrequently in female miniature pigs.135

Comment
Neoplastic changes are generally rarely observed in the female
reproductive system of minipigs. A few sporadic reports ofneoplastic changes include endometrial adenocarcinoma in an
8-year-old mixed breed (Spotted Poland China  Chapel Hill)
gilt138 and adenocarcinoma, leiomyoma, and leiomyosarcoma
in female potbellied pigs and miniature pet pigs.135,139 Most ofthese cases involved older pigs, and therefore, the low incidence of neoplasms observed could potentially be related to
the fact that the vast majority of minipigs used in safety assessment programs are relatively young. Studies involving pigs oflonger durations (12 months) are rare.

Table 11.4. Microscopic Findings of the Female Reproductive System: Uterine Cervix; Minipig.
Uterine cervix
Nonproliferative
Aggregate, granular cell
Atrophy
Atrophy, epithelial
Cyst, NOS

CommonUncommonNot observed, but potentially relevant

Not applicable
X

X
X
X(continued)

Skydsgaard et al.

159

Table 11.4. (continued)
Uterine cervix
Degeneration, epithelial
Erosion/ulcer
Hypertrophy, stroma
Increased keratinization
Increased mucification
Infiltratea
Inflammation
Necrosis, epithelial
Prolapse
Vacuolation, epithelial
Proliferative (nonneoplastic)
AdenosisHyperplasia, granular cell
Hyperplasia, stromal
Hyperplasia, epithelial
Neoplastic
Leiomyoma
LeiomyosarcomaaCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Indicates change commonly seen as part of normal estrus cycle and not generally recorded as an individual finding.

Table 11.5. Microscopic Findings of the Female Reproductive System: Vagina; Minipig.
VaginaCongenital
Imperforate vagina
Prostatic rudiment
Nonproliferative
Adenosis
Aggregate, granular cell
Atrophy
Atrophy, epithelial
Cyst, NOS
Degeneration, epitheliala
Erosion/ulcer
Increased keratinization
Increased mucification
Infiltratea
Inflammation
Necrosis, epitheliala
Prolapse
Vacuolation, epithelial
Proliferative (nonneoplastic)
AdenosisHyperplasia, epithelial
Hyperplasia, granular cellaCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Indicates change commonly seen as part of normal estrus cycle and not generally recorded as an individual finding.

Chapter 12. Reproductive System—Male
Standard INHAND nomenclature for proliferative and nonproliferative male reproductive tract findings in rats and mice has
previously been published140 and will be followed for the minipig as appropriate. There are some specific anatomical featuresof the porcine male reproductive tract that will be addressed

here, with the remainder of the document following the anatomical approach used for rodent species, with a focus on thosefindings unique or applicable to the minipig. Specifics of anatomical and histological differences of the minipig from therodent as well as the suggested examination, fixation, and sampling procedures will be addressed.

160This chapter deals with a standardized nomenclature forclassifying microscopic lesions observed in the reproductive
system of male minipigs, including the testes, epididymides,
seminal vesicles (or vesicular glands), prostate, efferent ducts,
spermatic cord, bulbourethral glands, penis, prepuce, and dorsal preputial diverticulum. Spontaneous lesions, as well as
lesions induced by exposure to test article, are included.
The reproductive system is susceptible to injury or change
caused by a wide range of xenobiotics/test articles and should
be routinely assessed in general toxicity studies. However, acomprehensive evaluation of the male reproductive system isbest performed on fully mature animals or animals of known
maturation status as a specialized study emphasizing reproductive tract collection, preservation, sampling, and proper staining for enhanced evaluation of any microscopic changes. As
minipigs are more widely used in general toxicity studies andas dermal administration of systemically acting compounds
becomes more common, an increased emphasis on proper evaluation of reproductive tract changes may be expected.
Nonproliferative lesions in general may be associated withexperimental perturbation or are a result of degenerativechanges frequently associated with aging. Modern laboratory
animal management practices within minipig facilities are suchthat spontaneous infectious processes should be infrequently
encountered; thus, the lesions related to infectious reproductive
tract diseases are not described in detail in this document.
Proliferative lesions in the male reproductive tract are rarely
seen as background findings but may be due to toxicity, infectious agents, or background lesions. This may be seen, forexample, in instances of experimental perturbation of the normal hormonal balance. Minipigs are not used in long-term
carcinogenicity studies; thus, there is a paucity of data on neoplastic lesions.

Male Minipig Reproductive Anatomy, Histology,
and Examination (Figures 12.1-12.12)
The anatomy of the male minipig reproductive tract is the sameas that of domestic swine but differs from the rodent anatomy
in several features. Notably, coagulating glands and preputial
glands are not prominent or easily sampled and the bulbourethral glands are more prominent and readily accessible in swine
as compared to rodents. The suggested set of tissues for routine
evaluation in the boar includes testes, epididymides, seminal
vesicles (or vesicular glands), and prostate.26 Sections of testis
for routine histopathological evaluation should include the capsule, parenchyma, and the rete testis. It is recommended thatthe head, tail, and body of the epididymides are examined.
Additional tissues may be added for specific studies but are
not generally sampled in routine toxicity studies. These secondary tissues would include efferent ducts, spermatic cord,bulbourethral glands, penis, prepuce, and dorsal preputial
diverticulum. Albl et al have thoroughly detailed a collection
and trimming scheme for the male reproductive tract, often
significantly more in depth than needed for routine toxicologic
pathology assessment.26 Representative photomicrographs of

Toxicologic Pathology 49(1)the normal microscopic anatomy of these nonstandard tissues
are presented for reference in this article (Figures 12.1-12.12).
Testes in the minipig are characterized by much the samefeatures as those seen in the rat or mouse. Leydig (interstitial)
cell density, size, and numbers may vary widely in the minipig,
both as a function of maturation status and between individual
animals (Figures 12.11 and 12.12 demonstrate age-matchedmature adult male control animals). Leydig cells sustain intratesticular androgen levels and may be affected by direct toxic
effects on steroidogenesis as well as indirectly by toxic effects
on gonadotropin release. However, in light of the extensive
histological variation in Leydig cell numbers, size, and appearance in normal mature control boars, evaluation of toxic effects
on Leydig cells must be carefully considered.
Stage-aware evaluation of the minipig testis: At the present
time, there are limited published guidelines available for stageaware evaluation of the Göttingen minipig testis. Extant methods
for other swine breeds often use periodic acid–Schiff (PAS) stain
for increased resolution and improved evaluation of spermatogonia substructures rather than a more general H&E stain. An 8stage method (PAS and H&E) to assess spermatogenesis in theGöttingen minipig has been proposed,141,142 and Kangawa et al
have published a staging method in the microminipig using an
11-stage method (PAS).143 Some further guidelines may beinferred from literature detailing reproductive maturation of thetestes of various breeds of domestic swine, but at this time,stage-aware evaluation is not routinely performed for this species and there are no clear, comprehensive published guidelines
for performance of this evaluation in the Göttingen minipig.

Sexual Maturation in the Male Minipig
Particularly for the porcine testis, but also for other reproductive
tract tissues, a comprehensive knowledge of normal cellularmorphology, spermatogenesis, and maturation processes isimportant for detecting any abnormalities. In many short-term
studies in minipigs, the male reproductive system is undergoing
maturation over the course of the study. Thus, there may benotable differences in histoanatomical features within the reproductive tract as a result of normal maturation variance in the
population. These differences must be separated from histopathologic findings of either background pathology or toxicity.
Taberner et al reported onset of histological puberty at 2
months of age in a group of 6 Göttingen minipigs, although
there was no information regarding the onset of full reproductive maturation.144 There are reports in the literature asserting
histological reproductive maturation in the Yucatan minipig
and microminipig at *4.5 months of age.145,146 However,these studies have small sample sizes and may or may not
represent a consistent level of maturation suitable for histological evaluation of potential reproductive toxicity in large-scale
research facilities. Reports from breeding facilities suggest thatmature sexual behavior begins at 3 to 4 months of age in maleGöttingen minipigs (Ellegaard), although this may not directly
correlate with complete histomorphological reproductive
maturity. In the experience of the INHAND Minipig group,Skydsgaard et al.

161some seminiferous tubules, with variable numbers of
seminiferous tubules containing intraluminal spermatids
and/or occasionally germ cell debris and multinucleatedcells. Small amounts of mature sperm and/or germ celldebris may be seen in the ductular lumen of the epididymides, with cell population consisting primarily of giant
cells and spermatids, with rare to no mature spermatozoa.
Secondary sex organs development may be variable.
Mature: Mature spermatozoa predominate in all/most seminiferous tubules and epididymides. Seminiferous tubules
are fully developed with mature spermatids/spermatozoa
and all 4 generations of germ cells (spermatogonia, spermatocytes, round spermatids, and elongating spermatids).
Epididymides contain abundant spermatozoa in the ductular lumen with rare to absent rounded germ cells. Secondary sex organs development may be variable.

groups of animals less than 4 to 5 months of age will have somenumber of males that are immature and it is not until animals
are of 5 to 6 months of age that we may be assured of theiroverall reproductive histomorphological maturity and full progression past pubertal status. While this is not always vital inindividual toxicity studies, ability to assess effects of test compounds on the reproductive tract of mature individuals is
important, particularly in instances where there is a potential
reproductive effect, which cannot always be predicted. Thus,
we recommend routinely using animals of 5 to 6 months of age
at the start of the study period to mitigate potential confounding
effects. A more definitive study of maturation status at various
ages should be undertaken, as full histomorphological maturation of the reproductive organs is important for accurate assessment of toxic effects.
As in many nonrodent species, maturation in the male minipig may occur over a range of age, so individual animals of thesame age may be at very different points in their reproductive
maturation. It is suggested that sexual maturity be routinely
recorded, using the immature, peripubertal, and mature categories. This will allow for more rigorous evaluation of potential
toxicity affecting maturation status or sperm development.
Brief definitions of the 3 separate stages of sexual maturation
are given below:

Neoplasia
Although there are no published reports of neoplasia withinGöttingen minipigs or other minipig breeds used in toxicologic pathology, a single incidence of Leydig cell adenoma
has been noted (Figures 12.13 and 12.14)147 and should beconsidered as an uncommon finding in the Göttingen minipig. Additionally, tumors of the male reproductive tract
have been rarely reported in the literature in domestic
swine. These include Leydig cell (interstitial cell) tumorsof the testis,148-150 Sertoli tumors of the testis,149,151 hemangiomas and hemangiosarcomas of the scrotum and testis,148,151 intratubular germ cell tumors of the testis,151,152
and papillomas of the penis.106 These neoplasms may beseen at a very low incidence in the minipig and should beconsidered as possible findings, although they have not yet
been reported.

Immature: Undeveloped seminiferous tubules lacking spermatozoa. Undistended seminiferous tubules are lined
only by Sertoli cells and gonocytes (lacking mature spermatids) with rare luminal germ cell debris and multinucleated cells. No mature spermatids and rare to absent
germ cell debris in epididymides. Secondary sex organs
are undeveloped.
Peripubertal: Seminiferous tubules in varying stages ofdevelopment. Mature spermatozoa may be present inNomenclature
Table 12.1. Microscopic Findings of the Male Reproductive System: Testis; Minipig.
TestisCongenital
Aplasiaa
Cryptorchidism
Hypoplasiaa
Nonproliferative
Amyloida
Angiectasis
Atrophy, Leydig cell
Atrophy, tubular
Degeneration, germ cell
Degeneration, tubular
Degeneration/atrophy, tubularb
Depletion, germ cell
Dilation, rete testis

CommonUncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
(continued)

162

Toxicologic Pathology 49(1)Table 12.1. (continued)
TestisCommonDilation, tubular
EdemaaExfoliation, germ cell
Fibrosisa
Hemorrhagea
Hypoplasia/atrophy, tubularc
Infiltratea
Infiltrate, vascular/perivasculard
Inflammationa
Mineralizationa
Multinucleated giant cells
Necrosis, Leydig cell
Necrosis
Necrosis, tubular
Necrosis, vasculard
Pigmenta
Residual bodies, atypical
Retention, spermatid
Sperm granuloma
Spermatocele
Stasis, sperm
Vacuolation, Leydig cell
Vacuolation, macrophageb
Vacuolation, tubular
Proliferative (nonneoplastic)
Hyperplasia, Leydig cellc
Hyperplasia, mesothelium
Hyperplasia, rete testis

UncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology addressed in the Systemic/General Pathology section.
Finding more frequently observed as an induced change.
Terminology with diagnostic criteria or comments described below.
dTerminology addressed in the Cardiovascular Section.
bcHypoplasia/Atrophy, Tubular, Testis,
Epididymis (Figure 12.15)
Other term(s)Hypospermatogenesis, hypoplasia

Diagnostic features

A continuum of microscopic changes, including one or moreof the following, multifocally affecting a number of tubules:
 Decreased numbers of spermatocytes and/or spermatids (hypospermatogenesis) to a complete
absence of germ cells, leaving only Sertoli cells lining the affected tubules.
 Vacuolation of seminiferous tubule epithelium withdecreased spermatogonia.
 Increased numbers of multinucleated or rounded
germ cells in the lumen of seminiferous tubules.
Changes seen in tubules immediately surrounding therete (tubuli recti) are considered normal and do not represent tubular hypoplasia/atrophy.

Affected tubules are clumped multifocally and adjacent
to normal tubules.
This finding may be accompanied by decreased luminal
sperm in the epididymides, along with increased numbers of round and multinucleated germ cells.
Number of Leydig cells might seem more prominent
when the severity of hypoplasia/atrophy is high.

Differential diagnoses
Tubular degeneration/atrophy.
The presence of degenerate and apoptotic germ cells is
one potential differentiator, with apoptotic cells being afeature of this finding and not noted in the more common background finding of tubular hypoplasia/atrophy.
Tubular degeneration/atrophy may be seen in alcohol
intoxication or folate deficiency, with tubular epithelial
vacuolation and germ cell degeneration.153

Comments
Tubular hypoplasia/atrophy is a testicular finding that canbe present with variable severity in a majority of GöttingenSkydsgaard et al.

163

minipig males. It may be a developmental lesion but does
not seem to have an age-associated variance in incidenceor severity. This finding is commonly seen in Göttingen
minipig males, with reported incidences ranging from70%154 to 25%,7,13 and can affect over 50% of the testis
in some animals.154 There does not appear to be an age-

related progression or regression of the lesion. 7 , 1 5 4
Hypospermatogenesis is one facet of this continuum offindings, as is tubular atrophy, and care should be taken
in differentiating this finding from possible test articlerelated decreased spermatogenesis or tubular degeneration/atrophy.

Table 12.2. Microscopic Findings of the Male Reproductive System: Efferent Ducts, Epididymis, and Spermatic Cord; Minipig.
Efferent Ducts, Epididymis, and Spermatic Cord
Congenital
Aplasiaa
Hypoplasiaa
Nonproliferative
Adenosis
Amyloida
Angiectasisa
Atrophy, ductal
Cell debris, luminal
Cribriform change
Cysta
Degeneration, epithelial
Dilation, ductal
EdemaaFibrosisa
Hemorrhagea
Infiltrate
Infiltrate, vascular/perivascularb
Inflammation
Karyomegaly
Metaplasia, squamous cell
Necrosis, vascularb
Sperm decreased, lumen
Single-cell necrosisa
Sperm granuloma
Sperm stasis
Spermatocele
Vacuolation, epithelialabCommonUncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in the Systemic/General Pathology section.
Terminology addressed in the Cardiovascular section.

Table 12.3. Microscopic Findings of the Male Reproductive System: Bulbourethral Gland, Seminal Vesicles, and Prostate; Minipig.
Bulbourethral gland, seminal vesicles, and prostate
Congenital
Aplasiaa
Hypoplasiaa
Nonproliferative
Amyloida
Angiectasis
AtrophyConcretions
Dilation, acinar/vesicle
Fibrosis, stromala
Hemorrhagea
Infiltrate
Infiltrate, vascular/perivascularb

CommonUncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
(continued)

164

Toxicologic Pathology 49(1)Table 12.3. (continued)
Bulbourethral gland, seminal vesicles, and prostate

CommonInflammation
Metaplasia
Necrosisa
Necrosis, vascularb
Single-cell necrosisa
Vacuolation, epithelial
Proliferative (nonneoplastic)
Hyperplasia, atypicalHyperplasia, diffusec
Hyperplasia, reactive
Mesenchymal proliferative lesion

UncommonNot observed, but relevant

Not applicable

X
X
X
X
X
X
X
X
X
X

a

Terminology addressed in the Systemic/General Pathology section.
Terminology addressed in the Cardiovascular section.
cTerminology with diagnostic criteria and/or comments described below.
bHyperplasia, diffuse, prostate, seminal
vesicles (Figures 12.16 and 12.17)
Other terms
Hyperplasia, functional; hyperplasia, adaptive

Pathogenesis
Specific pathogenesis is not always known in the swine but
may, as in other mammals, represent effects of increased steroidal compounds or steroidogenic compounds producing
increased testosterone, stimulating growth of glandular epithelium of the accessory sex organs.

Diagnostic featuresThickening of the vesicle epithelium, with increased
glandular epithelial cell numbers.
May include formation of small papillary fronds of proliferating cells.

Differential diagnoses

N/A.

Comments
Functional hyperplasia of accessory sex organs is steroid
dependent in other species, such as the canine, and may be
so in the minipig as well.

Chapter 13. Respiratory System
The respiratory system is susceptible to injury caused byinhaled and blood-borne (including dermally absorbed) xenobiotics/test articles and thus should be routinely examined in all
toxicity studies. This is especially important since inhaledxenobiotics are the most frequent cause of toxicity and theminipig is beginning to be used for short-term inhalation studies.
In addition, the minipig is being used to generate animal modelsof respiratory disease, for example, inhaled lipopolysaccharide(LPS) has been used as a model for chronic obstructive pulmonary disease,155-157 one of the leading causes of human mortality
and morbidity worldwide. More porcine inhalation studies andmodels of disease may be anticipated given the similarity of the
porcine respiratory system to that of humans.
Lung toxicity in the pig may be mediated by similar
mechanisms as in other species including biotransformation,
via cytochrome P450 and phase II enzymes.158 Metabolizing
enzymes, several of which are inducible, have been identified
in the porcine lung.159 In addition to injury of pulmonary
epithelial and endothelial cells, toxicological insults in the pig
may cause injury to resident pulmonary intravascular macrophages (PIMs).158,160 Unlike rodents and humans, PIMs thatadhere to endothelium are a significant resident cell populationin the pig lung. Pulmonary intravascular macrophages increasein number with age and function to remove particulate matterincluding bacteria from the blood.161,162 Injection of microspheres has been reported to lead to severe pulmonary vasoconstriction in the pig due to release of thromboxane A2 from
PIMs.160 In rodents and humans, PIMs are not normally present
but may be induced in cases of liver dysfunction.163
Respiratory tract lesions can be treatment-related, spontaneous background changes or due to infection, environmental
factors, or aging. Modern laboratory animal management practices within minipig facilities are such that spontaneous infectious processes should be infrequently encountered; thus,
lesions caused by infectious respiratory tract diseases are notdescribed in this document. Excellent reviews have been published on the effects of infectious disease, diet, and environmental factors on the pig respiratory tract.164-166 It has recently
been proposed that the microbiome plays a role in the pulmonary response to infection and by extrapolation may have effects
on toxicological responses.164

Skydsgaard et al.
While the focus is on the minipig, it should be noted thatmost lesions described in the domestic pig could occur in theminipig since both are Sus scrofa. Reports of neoplastic lesions
in the respiratory tract of pigs are rare, and to the best of ourknowledge, none have been reported in minipigs.
Systematic sampling and careful fixation by airway or intravascular perfusion are prerequisites to identifying lung lesions;however, this is not as easily done in larger animals as it is inrodents. The entire larynx, trachea, or lung lobe cannot be examined histologically. Thus, a uniform method must be developed
within laboratories which includes consistent sections from eachof these tissues (see Albl et al for recommendations).26 Larynx
should be sampled, although there is variation in sampling procedures among laboratories. Trachea should be sampled at 1 to 2
sites; either longitudinal or transverse sections are acceptable,
and again, consistency within laboratories is important. Both left
and right lung lobes should be sampled, with sections containing
main stem bronchi, secondary bronchi, and lung margins. The
primary goal is to assess all anatomical features. Lung sections
should be carefully inflated with fixative,26 but again there isvariation in techniques used among laboratories.

I. Nasal Cavity
Swine sinonasal anatomy is largely similar to human as demonstrated by computed tomography of domestic and Yucatan pig
heads.167 The middle and inferior turbinates arise from asingle uniturbinate and the superior turbinates containlarge concha bullosa. Unlike human, swine nasal septum
consists of bone anteriorly and cartilage posteriorly. 167
The nasal cavity is structurally complex, reflecting thediverse physiological functions associated with this anatomical site. Kuper et al168 provide an overview of normal
nasal passages of Gottingen minipigs from the neonatal
period to young adult. Several additional articles describe
the anatomy and histology of porcine nasal cavity

165
tissues.167,169 In pigs, a caudal diverticulum of the pharynx lies immediately dorsal to the esophagus. In young
pigs, foreign materials occasionally lodge in this diverticulum, causing pharyngitis and cellulitis that may lead todysphagia and death from starvation.
There are few reports of inhalation studies or chemicallyinduced upper respiratory lesions in pigs or minipigs.170-173
The majority of reported inhalation studies do not include morphologic evaluation of the nasal cavity and the nasal cavity is
not routinely examined in most noninhalation toxicology studies. Therefore, the frequency of individual microscopic findings, as provided in the Tables 13.1 and 13.2, may needrevision in the future.
Albl et al26 recommend that in routine noninhalation studies, one sample each of the nasal septum, turbinates (frontal
section at level of first premolar), and ethmoidal labyrinth(parasagittal section) be taken to examine the transitional,
respiratory, and olfactory epithelia. For inhalation studies,
Albl et al recommend 2 additional frontal sections of thenasal septum and turbinates (caudal to the first incisors andcaudal end of turbinates) to examine squamous and transitional epithelium. Kuper et al168 provide additional guidance for a standard method to sample the minipig nasal
passages, including a section of the nasopharynx to examine
NALT. The importance of consistent sampling cannot be
overemphasized in evaluation of the nasal cavity since normal changes in epithelial type with location may be mistaken for xenobiotic-induced lesions. It is recommended thatthe nasal cavity be first flushed with 10% neutral-bufferedformalin. Appropriate samples of the turbinates and ethmoidal labyrinth should be fixed in formalin and then decalcified before embedding in paraffin. A standardized approachfor recording histologic lesion distribution is essential andnasal diagrams, such as provided by Yang et al,169 canprovide assistance in mapping lesions.168

Table 13.1. Microscopic Findings of Respiratory System: Nasal Cavity; Minipig.
Nasal cavity
Congenital
Cleft palatea,bDeviation, nasal septuma,bNonproliferative
Amyloidc
Angiectasisa
Atrophy
Congestionc
Corpora amylacea
Cellularity, decreased, nasal-associated lymphoid tissue (NALT)dCellularity, increased, NALTd
Degeneration
EdemacEosinophilic globules

CommonUncommonNot observed, but
potentially relevant

Not Applicable

X
X
X
X
X
X
X
X
X
X
X
X
(continued)

166

Toxicologic Pathology 49(1)Table 13.1. (continued)
Nasal cavity

CommonErosion/ulcer
Hemorrhagea,cHypertrophy, media or wall, arterya
Infiltratec
Infiltrate, vascular/perivasculara,cInflammationa,cInflammation, vascular/perivasculara,cIntimal thickening, acellular, arterya
Metaplasia, respiratory, olfactory/glandular epithelium
Metaplasia, squamous cella,b,c
Necrosisc
Perforation, septum
Pigment/foreign materiala,cRegenerationc
Thrombusa
Proliferative (nonneoplastic)
Hamartomaa
Hyperplasia, atypicalHyperplasia, basal cell
Hyperplasia/metaplasia, mucous cellaHyperplasia, neuroendocrine cell
Hyperplasia, olfactory epithelium
Hyperplasia, respiratory epithelium
Hyperplasia, squamous cell
Hyperplasia, transitional epithelium

Not observed, but
potentially relevant

UncommonNot Applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Induced lesion.
c
Terminology addressed in the Systemic Pathology section.
dTerminology addressed in the Hematolymphoid section.
b

Table 13.2. Microscopic Findings of Respiratory System: Nasopharynx, Paranasal Sinuses; Minipig.
Nasopharynx, paranasal sinuses
Nonproliferative
Infiltratea
Inflammationa
Inflammation, vascular/perivasculara
Metaplasia, squamous cella,b,c
Necrosisa
Regenerationa
Thrombus
Proliferative (nonneoplastic)
Hyperplasia, atypicalHyperplasia, basal cell
Hyperplasia/metaplasia, mucous cellb
Hyperplasia, neuroendocrine cell
Hyperplasia, respiratory epitheliumaCommonNot observed, but potentially relevant

X

Terminology addressed in the Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.
cInduced lesion.
b

Uncommon

X
X
X
X
X
X
X
X
X

X

X
X

X

Not applicableSkydsgaard et al.

167

Comment

via the retroorbital plexus in the medial canthus of the eye is
not done in pigs, and therefore, this is not a cause of hemorrhage in the minipig.

Unlike rodents, mononuclear cell leukemia has not beenreported in pigs, so angiectasis of the nasal cavity is not associated with this lesion.

Hypertrophy, media or wall, artery, nasal cavity

Angiectasis: Nasal cavity

Cleft palate: Nasal cavity
Comment
Cleft palate (palatoschisis) has been reported as a backgroundfinding in the Gottingen minipig174 and induced in domestic
pigs by feeding poison hemlock (Conium maculatum),
tobacco plants (Nicotiana glauca and N tabacum), and Crotalaria retusa seed during pregnancy.175-177 The toxic principle in poison hemlock is a piperidine alkaloid. The
mechanism of cleft palate formation by poison hemlock andtobacco is believed to be the lack of fetal movement during
the critical stage of gestation with arthrogryposis also frequently present. Feeding vitamin A depleted domestic sowsa vitamin A-free ration during the first month of pregnancy
also resulted in cleft palate formation.178

Deviation, nasal septum: Nasal cavity
Comment
Deviation of the nasal septum as a congenital lesion should be
differentiated from that due to atrophic rhinitis.

Hamartoma: Nasal cavity
Pathogenesis/cell of origin
Defective development of blood vessels resulting in disordered
proliferation of mature blood vessels.

Differential Diagnosis
Hemangioma or hemangiosarcoma, however these have notbeen reported.

Diagnostic features
Tumor-like mass consisting of primitive to well-formed bloodvessels.

Comment
A vascular hamartoma has been reported in the minipig nasal
cavity.13

Comment
See cardiovascular section. Medial hypertrophy of the vessels in
the nasal cavity has been reported in the minipig.13

Hyperplasia/metaplasia, mucous cell: nasal
cavity, nasopharynx, paranasal sinus
Comment
Mucous cell hyperplasia has been reported in domestic pigs
following chronic aerogenous exposure to ammonia.179

Infiltrate, vascular/perivascular: Nasal cavity
Comment
See systemic section. Perivascular mononuclear cell infiltrates
in the nasal cavity have been reported in the minipig.13

Inflammation (granulomatous): Nasal cavity
Comment
Foci of granulomatous inflammation with or without mineralization have been reported in the propria/submucosa of the nasal
cavity and trachea in pigs. They are believed to be due toimpacted particles of inhaled calcium salts from the feed.180

Inflammation, vascular/ perivascular: Nasal
cavityOther terms
Arteritis, periarteritis.

Comment
See systemic and cardiovascular sections. Inflammation of thevasculature (arteritis) has been reported in the minipig nasal
cavity.10

Hemorrhage: Nasal cavity

Intimal thickening, acellular, artery: Nasal cavity

Comment

Comment

Hemorrhage in the nasal cavity is most likely due to trauma or
foreign material. Unlike rodents, antemortem blood collection

See cardiovascular section. Intimal proliferation of the vessels
in the nasal cavity has been reported in the minipig.13

168

Toxicologic Pathology 49(1)Metaplasia, squamous cell: Nasal cavity,
nasopharynx, paranasal sinus

Thrombus: Nasal cavity

Comment

Unlike rodents, mononuclear cell leukemia has not beenreported in pigs so thrombosis of the nasal cavity is not associated with this lesion.13 See Cardiovascular section for additional information.

Comment

In the domestic pig, squamous metaplasia has been observed
following chronic exposure to corn dust and SO2181 as well aswith experimental infection with porcine reproductive andrespiratory syndrome virus.182

II. Larynx, Trachea, Bronchi, and Bronchioles
Pigment/foreign material: Nasal cavity
Other terms
Foreign body

Pathogenesis/cell of origin
Foreign material may be seen in the respiratory system of theminipig and may be associated with porcine rooting behavior.59

Comment
This term includes foreign body. Although not reported specifically in the nasal passages, this is likely due to the fact that
there is limited information available on this portion of therespiratory tract.

The mucosal epithelium of the airway conducting system in the
pig larynx, trachea, and bronchi is similar to other species inthat it is less complex than that lining the nasal airways. The pig
has a large larynx. The supraglottis and glottis are lined bystratified squamous epithelium and the subglottis by pseudostratified columnar epithelium.183,184 Sampling of the rightaryepiglottic fold is recommended by Albl et al.26
Unlike other species, there is a bronchial branch emanatingfrom the trachea cranial to the carina that supplies the rightcranial lung lobe.185 Similarly to humans, but unlike therodent, the pig has cartilage and submucosal glands in intrapulmonary bronchi. Bronchial glands are similar in size andsecretion to those in humans, which increase in number as theanimal ages.186 Cartilage and submucosal glands are not present at the level of the bronchioles.187 Bronchial-associatedlymphoid tissue (BALT) only develops when animals are
antigenically stimulated and therefore is not present ingerm-free pigs.188,189 When BALT is present, it manifests
as a single follicle found at bronchial bifurcations and protrudes into the lumen.190

Table 13.3. Microscopic Findings of Respiratory System: Larynx; Minipig.
LarynxNonproliferative
Degenerationa
Ectasia, submucosal gland
Erosion/ulcera
Exudatea
Fibrosisa,bHemorrhageb
Hypertrophy, media or wall, arteryc
Infiltratea,bInflammationa,bInflammation, vascular/perivascularb
Intimal thickening, acellular, arteryc
Metaplasia, osseousb
Metaplasia, squamous cella,bMineralizationa,bNecrosisb
Pigment/foreign materiala,bRegenerationb
Thrombusc
Proliferative (nonneoplastic)
Epithelial alteration
Hyperplasia, atypical

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

169

Table 13.3. (continued)
LarynxCommonUncommonHyperplasia, mucous cell
Hyperplasia, neuroendocrine cell
Hyperplasia, respiratory epithelium

X

Hyperplasia, squamous cellX

Not observed, but potentially relevant

Not applicable

X
X

aTerminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Table 13.4. Microscopic Findings of Respiratory System: Trachea: Minipig.
Trachea
Nonproliferative
Degenerationa
Ectasia, submucosal gland
Erosion/ulcera
Exudatea
Fibrosisa,bHemorrhageb
Hypertrophy, media or wall, artery
Infiltratea,bInflammationa,bInflammation, vascular/perivasculara,bIntimal thickening, acellular, arteryc
Metaplasia, osseousb
Metaplasia, squamous cella,bMineralizationa,bNecrosisb
Pigment/foreign materiala,bRegenerationb
Thrombusc
Proliferative (nonneoplastic)
Hyperplasia, atypicalHyperplasia, mucous cell
Hyperplasia, neuroendocrine cell
Hyperplasia, respiratory epithelium

CommonUncommonNot observed, but potentially relevant

Not applicableNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Hyperplasia, squamous cellXaTerminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Table 13.5. Microscopic Findings of Respiratory System: Bronchi: Minipig.
BronchiNonproliferative
Bronchiectasis
Cellularity, increased (BALT)aDegenerationa
Ectasia, submucosal gland
Erosion/ulcera
Exudatea
Fibrosisa,bHemorrhageb

CommonUncommon
X
X
X
X
X
X
X

X
(continued)

170

Toxicologic Pathology 49(1)Table 13.5. (continued)
BronchiCommonUncommoncHypertrophy, media or wall, artery
Infiltratea,bInflammationa,bInflammation, vascular/perivascularb
Intimal thickening, acellular, arteryc
Metaplasia, osseousb
Metaplasia, squamous cella,bMineralizationa,bNecrosisb
Pigment/foreign materiala,bRegenerationb
Thrombusc
Proliferative (nonneoplastic)
Hyperplasia, atypicalHyperplasia, mucous cell
Hyperplasia, neuroendocrine cell
Hyperplasia, respiratory epithelium

Not observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Hyperplasia, squamous cellXaTerminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Table 13.6. Microscopic Findings of Respiratory System: Lung: Bronchioles: Minipig.
Lung: bronchioles

Common Uncommon Not observed, but potentially relevant Not applicable

Nonproliferative
Bronchiectasis
Cellularity, increased (BALT)aDegenerationa
Erosion/ulcera
Exudatea
Fibrosisa,bHemorrhageb
Hypertrophy, media or wall, arteryc
Infiltratea,bInflammationa,bInflammation, vascular/perivasculara,bIntimal thickening, acellular, arteryc proliferation, vessels
Material, extracellular [insert morphology/color]
Metaplasia, osseousb
Metaplasia, squamous cella,bMineralizationa,bNecrosisb
Pigment/foreign materiala,bRegenerationb
Thrombusc
Proliferative (nonneoplastic)
Hyperplasia, atypicalHyperplasia, mucous cell
Hyperplasia, neuroendocrine cell
Hyperplasia, respiratory epithelium
Hyperplasia, squamous cellaTerminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Skydsgaard et al.

171

Cellularity, increased; BALT: Bronchi,
bronchioles

Fibrosis: Trachea, bronchi, bronchioles

Pathogenesis/cell of origin

Blood sampling in the pig is often performed in the cervical region
and can result in lesions within surrounding tissues. If the trachea
or surrounding tissue is damaged during sampling, peritracheal
hemorrhage/inflammation and fibrosis may result.13 Mild peritracheal fibrosis can also be found in the Hanford minipig.191

BALT is only present in pigs that are antigenically stimulated. It
normally consists of a single follicle-like structure at the bifurcation of bronchi.188-190 When antigenic stimulation is excessive, itcan expand in both size and location to encompass other regionsof the bronchi or be multifollicular.

Pathogenesis/cell of origin

Infiltrate: Larynx, trachea, bronchi, bronchioles
Differential diagnosis
Inflammation, acute, chronic, or granulomatous.

Comment
Cell infiltrates are common in the pig in all tissues and should
not be confused with inflammation.7,13,59 See Systemic section.

Comment
See hematolymphoid system for additional information.

Inflammation (acute, chronic, granulomatous):
Larynx, trachea, bronchi, bronchioles

Degeneration: Larynx, trachea, bronchi,
bronchioles

Comment

Comment
This lesion has been reported in the minipig.13

Erosion/ulcer: Larynx, trachea, bronchi,
bronchioles
Comment
Erosions/ulcerations of the vocal folds are hypothesized to berelated to excessive vocalizations. Erosion and ulceration in the
trachea may be a spontaneous lesion13 but may also be due toinjury from an endotracheal tube if the animal was anesthetized(as in device studies).

Exudate: Larynx, trachea, bronchi, bronchioles
Pathogenesis/cell of origin
Cell accumulations are typically primarily neutrophils andmacrophages but can be composed of any inflammatory cell
type (lymphocytes, neutrophils, eosinophils, and macrophages). Exudate may be related to inhalation of foreignmaterial.13

Diagnostic features
The cells accumulate within the lumen of the tissue but without
damage to surrounding tissue which is typical of inflammation.
This may be due to translocation by the mucociliary apparatus
from inflammation in the lower respiratory tract which may or
may not be associated with foreign particles.

Acute inflammation is often seen in pigs as a background finding secondary to inhaled particles. Inflammation secondary toforeign particles needs to be carefully differentiated frominflammation due to an inhalant or test article.10,13,59 Whenforeign material becomes trapped in respiratory tissues, it canlead to chronic inflammation.10,13,59 Many instances of granulomatous inflammation in the pig respiratory system are due tothe propensity for rooting behavior leading to inhaled foreignmaterial. This foreign material often gets trapped in respiratory
tissues leading to granulomatous inflammation.10,13,59 Inflammation of glands in larynx, trachea, and bronchi has beenreported in the minipig.13

Metaplasia, squamous cell: Larynx, trachea,
bronchi, bronchioles
Comment
Tiny islands of squamous metaplasia within bronchi have beenreported in domestic pigs.192 Squamous metaplasia has also
been noted to occur in the ducts of the bronchial glands ofdomestic pigs.193

Mineralization: Larynx, trachea, bronchi,
bronchioles
Pathogenesis/cell of origin
It is believed that multifocal mineralization in the lamina propria of respiratory mucosa is due in part to inhaled calcium salts
from dry and pelleted feed.180

Diagnostic features
Differential diagnosis
Inflammation, acute, chronic, or granulomatous.

If related to inhalation of particles, the mineralization will be
primarily along the dorsal wall of the trachea.180

172

Comment
This change has also been reported as an incidental finding.13

Pigment/foreign material: Larynx, trachea,
bronchi, bronchioles

Toxicologic Pathology 49(1)dominated by macrophages, with varying numbers of other cell
types; fibrosis may also be present. Foreign material may beseen in the respiratory system of the minipig and is often associated with porcine rooting behavior.59

Other term

III. Terminal Bronchioles, Alveoli, and Pleura

Foreign body.

Unlike rodents, the pig lung is lobulated meaning that there isdivision of the lung into lobules by connective tissue septa.194
Humans have incomplete lobulation. Lobulation limits movement and transport of cells, molecules, and potentially toxic
substances between adjacent lobules.194 Interlobular septa and a
thickened pleura are prominent in the pig. Similarly to rodents,
but unlike humans, pigs have few to no respiratory bronchioles.195,196 It is difficult to impossible to distinguish between
respiratory bronchi and alveolar ducts in the porcine lung.197
Similarly to humans and cattle, the visceral pleura and interstitialconnective tissue of porcine lungs are relatively thick as compared to other domestic and laboratory species.195,198,199

Pathogenesis/cell of origin
Foreign bodies, generally either food particles or bedding
material, may be found in the respiratory tract. Normal porcine
behavior, such as rooting, can result in foreign bodies and an
associated inflammatory response being encountered in minipigs.7,13 Foreign bodies initially elicit a suppurative inflammatory response, accompanied or followed by influx ofmacrophages. If the foreign body persists, the inflammatory
response will gradually evolve to chronic inflammation

Table 13.7. Microscopic Findings of Respiratory System: Lung: Terminal Bronchioles; Minipig.
Lung: Terminal bronchioles
Congenital
Cyst, congenitala
Hypoplasia
Nonproliferative
Alveolar lipoproteinosis
Atelectasisa
Bacteriaa,bCongestiona,bDegeneration
Edemaa,b
Embolusa
Exudatea
Fibrosisa,bGranuloma, foreign materiala
Hemorrhagea,bHypertrophy, media or wall, arterya,cInfiltratea,bInfiltrate, vascular/perivascularb
Inflammation (acute alveolar/interstitial, chronic interstitial, acute bronchoalveolar,
chronic bronchoalveolar, granulomatous)a,b
Inflammation, vascular/perivasculara,bIntimal thickening, acellular, arterya,cMacrophages, increaseda
Material, extracellular [insert morphology/color]
Metaplasia, mucous cell
Metaplasia, osseousa,bMetaplasia, squamous cellb
Mineralization a,b
Necrosisa,bPigment/foreign materialb
Regenerationb
Thrombusa

Not observed, but
Not
Common Uncommon potentially relevant applicable
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

173

Table 13.7. (continued)
Lung: Terminal bronchioles

Not observed, but
Not
Common Uncommon potentially relevant applicable

Proliferative (nonneoplastic)
Hyperplasia, bronchioloalveolar
Syncytial cells, (alveolar epithelial cells or macrophages)aX
Xa

Terminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Table 13.8. Microscopic Findings of Respiratory System: Lung: Alveoli: Minipig.
Lung: Alveoli
Congenital
Cyst, congenitala
Hypoplasiab
Nonproliferative
Alveolar emphysemaa
Alveolar lipoproteinosis
Atelectasisa
Bacteriaa,bCongestiona,bDegeneration
Ectasia, acinus
Edemaa,b
Embolusa
Exudatea
Fibrosisa,bGranuloma, foreign materiala
Hemorrhagea,bHypertrophy, media or wall, arterya,cInfiltratea,bInfiltrate, vascular/perivascularb
Inflammation, (acute alveolar/interstitial, chronic interstitial, acute bronchoalveolar,
chronic bronchoalveolar, granulomatous)a,b
Inflammation, vascular/perivasculara,bIntimal thickening, acellular, arterya,cMacrophages, increaseda
Material, extracellular [insert morphology/color]
Metaplasia, mucous cell
Metaplasia, osseousa,bMetaplasia, squamous cellb
Mineralizationa,bNecrosisa,bPigment/foreign materiala,bRegenerationb
Thrombusa,cProliferative (nonneoplastic)
Cyst, keratinizing
Hyperplasia, bronchiolo-alveolar
Syncytial cells (alveolar or macrophages)aaTerminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Not observed, but
Not
Common Uncommon potentially relevant applicable
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

174

Toxicologic Pathology 49(1)Table 13.8. Microscopic Findings of Respiratory System: Pleura: Minipig.
PleuraCommonCongenital
Cyst, congenitala
Nonproliferative
Adhesiona
Bacteriaa,bEffusion noninflammatory
ExudateFibrosisa,bGranuloma, foreign materiala
Hemorrhageb
Infiltratea,bInflammationa,bInflammation, vascular/perivascularb
Intimal thickening, acellular, arteryc
Metaplasia, osseousa,bMineralizationa,bPigment/foreign materiala,bPyothorax
Proliferative (nonneoplastic)
Hyperplasia, mesotheliuma

UncommonNot observed, potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

a

Terminology with diagnostic criteria or comments described below.
Terminology addressed in the Systemic Pathology section.
cTerminology addressed in the Cardiovascular section.
b

Adhesion: Pleura (Figure 13.1)

Alveolar emphysema: Alveoli

Other terms

Comment

None.

This lesion can be induced by mechanical ventilation, which
may be relevant in medical device studies. Emphysema may
also be induced in minipigs using inhaled LPS.157

Pathogenesis/Cell of origin
Adhesions can follow damage or inflammation of the visceralor thoracic pleura. These are frequently encountered in animals
that have had parasites or respiratory infections. They also
occur in SPF animals and have been infrequently observed in
Hanford minipigs.191

Diagnostic features
This lesion is typically diagnosed grossly, but irregularities of thepulmonary margin can be identified histologically. The adhesions
present on the surface of the lung may contain abundant collagen(fibrosis) or inflammatory cells.

Atelectasis: Terminal bronchioles, alveoli
Comment
This lesion has been reported in the minipig.13

Bacteria: Terminal bronchioles, alveoli, pleura
Comment
The rooting behavior of the pig results in inhalation of foreignmaterials, some of which may contain bacteria.13

Cyst, congenital: Bronchioles, alveoli, pleura
Comment
Adhesions are commonly seen following thoracic surgery(device models).

Comment
This lesion has been reported in the minipig.13

Skydsgaard et al.

Congestion: Terminal bronchioles, alveoli
Comment
Congestion is common in alveoli, uncommon in terminal
bronchioles and pleura, but may be found in all locations.3,10,13,59 See Systemic section.

Edema: Terminal bronchioles, alveoli, pleura(Figure 13.2)
Comment
In rodents, edema is primarily a finding in bronchioles andalveoli, but in the pig, it also occurs in pleura and interlobular
septa.13,29,59 Lymphatics in interlobular septa and pleura can be
widely dilated leading to misinterpretation of the lesion.

175

Hemorrhage: Terminal Bronchioles, Alveoli,
Pleura (Figure 13.8)
Comment
Pulmonary hemorrhage is common in alveoli but uncommon interminal bronchioles and pleura.3,10,13,59 Hemorrhage in thelung may also be seen with thrombocytopenic purpura. See
Systemic section.

Hypertrophy, media or wall, artery: Terminal
bronchioles, alveoli
Comment
See Cardiovascular section. This lesion has been reported in theminipig.7,13

Hyperplasia, mesothelium: pleura
Embolus: Terminal Bronchioles, Alveoli(Figures 13.3a and 13.3b)

Comment
This lesion has been reported in the minipig.7,59

Comment
Several types of emboli may be found in the porcine lung:lipid,40 thromboembolus, or bacterial. These are described
more fully in the Cardiovascular section. Lipid may be used
as a modifier for embolus in the pig. Embolus has been reported
in the minipig.7,13,59

Exudate: Terminal Bronchioles, Alveoli, Pleura
Comment

Infiltrate: Terminal bronchioles, alveoli,
pleura (Figure 13.9)
Comment
This finding is commonly reported in the minipig.7,13,59

Inflammation, acute bronchioloalveolar:
Terminal bronchioles, alveoli, pleura(Figure 13.10)

Small foci of exudate have been reported in the smaller airwaysof the minipig.13,59

Comment

Fibrosis: Terminal Bronchioles, Alveoli,
Pleura (Figures 13.4-13.6)

Inflammation, acute alveolar/interstitial: Alveoli

Comment

This lesion has been reported in the minipig.3,7,13

Fibrosis is uncommon in both the terminal bronchioles andalveoli but is common in pleura.3,7,13,59 Fibrosis of the interlobular septa has been induced with radiation exposure in theminipig.200

Inflammation, chronic bronchioloalveolar:
Terminal bronchioles, alveoli, pleura

This lesion has been reported in the minipig.7,13,59

Comment

Comment
This lesion has been reported in the minipig.7,13

Granuloma, foreign material: Terminal
bronchioles, alveoli (Figure 13.7)
Pathogenesis/cell of origin
As above in section II. Often associated with inhaled foreignmaterial or potentially parasites.7,13,59

Inflammation, chronic interstitial: Terminal
bronchioles, alveoli, pleura
Comment
This lesion has been reported in the minipig.3,7,13,59

176

Toxicologic Pathology 49(1)Inflammation, granulomatous: Terminal
bronchioles, alveoli, pleura

Pigment/foreign material: Terminal bronchioles,
alveoli, pleura

Comment

Comment
3,7,10,13,59

This lesion has been reported in the minipig.

Inflammation, vascular/perivascular: Terminal
bronchioles, alveoli, pleura

Pigment is common in alveoli but uncommon in terminal
bronchioles and pleura. Pigs often root and inhale dusts andinert materials.3,13,59,201

Comment

Syncytial cells (alveolar epithelial cells ormacrophages): Alveoli

Other terms are arteritis/periarteritis, see Systemic and Cardiovascular sections.10

Other terms
Giant cells, multinucleate cells.

Intimal thickening, acellular, artery: Terminal
bronchioles, alveoli, pleura
Comment
See Cardiovascular section. This lesion has been reported in theminipig.13

Pathogenesis/cell of origin
Syncytial cells in the porcine lung may be formed from alveolar epithelial cells (typically viral in origin) but are more commonly formed from macrophages. Multiple activatedmacrophages may fuse to form syncytia.

Diagnostic features
Macrophages, increased: Terminal
bronchioles, alveoli (Figures 13.11 and 13.12)
Comment
Accumulations of alveolar macrophages have been reported to
be associated with inhaled feedstuffs.201 This is commonly
seen as an incidental finding in pigs.3,7,10,13,29,59,64 Accumulation of alveolar macrophages is also common in Hanford, Sinclair, and Yucatan breeds. Pigmented breeds such as theYucatan contain pigment within some of their cells, and therefore, accumulated macrophages may be pigmented.13 Occasionally, the macrophages are vacuolated and may be usedwith the modifier (vacuolated).

Metaplasia, osseous: Terminal bronchioles,
alveoli, pleura
Comment
This lesion has been reported in the minipig.7,13

Mineralization: Terminal bronchioles, alveoli,
pleuraComment
This lesion has been reported in the minipig.3,7,10,13,59 Also see
Systemic section.

Necrosis: Terminal bronchioles, alveoli
Comment
This lesion has been reported in the minipig.13

Within alveoli, a single cell outline can be identified withmultiple nuclei. Occasionally, foreign material may be foundwithin or in close proximity to the cells.

Comments
This lesion has been reported in the minipig.13

Thrombus: Terminal bronchioles, alveoli
Comments
This is uncommon as a spontaneous lesion but often seen following intravenous continuous infusion studies using vascularaccess ports.7 See also Cardiovascular section.

Chapter 14. Skeletal System
Standard INHAND nomenclature for nonproliferative and proliferative skeletal system findings in rats and mice has previously been published202 and will be used for the minipig as
appropriate following the anatomical approach used for rodent
species, with a focus on those findings that are likely to be
encountered frequently and those that might be consideredunique to the minipig. While the type and classification of boneand joint lesions is identical or very similar across species, theirfrequency in untreated individuals may differ substantially inthe various species. The discussion herein is focused on spontaneous lesions observed in young minipigs (up to 2 years ofage) used in nonclinical toxicity studies.
Skeletal tissues covered in this chapter are bones and joints.
Teeth are not routinely examined in nonclinical toxicity studies
and have thus been excluded from this chapter.

Skydsgaard et al.

177

The standard bone samples examined during a toxicity study
are distal femur and proximal tibia including the femorotibial
joint. Additionally, the sternum is examined with the main
purpose of evaluating the bone marrow. The preparation of thebones for optimal H&E sections is a time-consuming procedurein the nonrodent species. Although several attempts have been
made to optimize the decalcification procedure, the traditional
method often provides the best results.
Fractures of the bones are very rarely observed and joint lesions,
often seen in conventional pigs, are uncommon in minipigs.

Serous atrophy of the bone marrow does not specifically affectthe bones but affects adipocytes and the hematopoietic tissue
located in the bone marrow. This change is described in greater
detail in the section on systemic conditions of minipigs. Serous
atrophy of the adipocytes in the bone marrow occurs with a variable incidence and geographical variation, being more common
in Europe than in North America.13 Although the reason for thisdifference is uncertain, it may be related to different feeding
regimes. Serous atrophy of the adipocytes in the bone marrow
has not been described in Hanford or Yucatan minipigs.13
Unlike the rodent, the physis of minipigs closes when the
animal reaches maturity and growth ceases. The recording ofphysis closure may become necessary if a treatment causes or isconsidered to possibly cause a delay in physis closure or premature physis closure. In the untreated, healthy female Göttingen
minipig, growth plate closure of femur starts at the age of 25months and is complete by 42 months.55 Another paper describes
closure of the physis to be complete by 16 to 24 months.203

I. BoneLesions in the bones are uncommon in minipigs and only 2
lesions not described for rodents will be described herein:(1) serous atrophy of the adipose tissue in the bone marrowin Göttingen minipigs and (2) closed/partially closed/open
growth plate.

Table 14.1. Microscopic Findings of Skeletal System: Bone; Minipig.
Bone
Nonproliferative
Bone, decreased, trabeculae, and/or cortex
Bone, fibrosisa
Bone, increased, trabeculae, and/or cortex
Cyst, bone
Eroded surface, increased
Fibrous osteodystrophy (FOD)b
Fibro-osseous lesion (FOL)
Fracture/callusb
Growth plate closed/partially closed/openb
Hemorrhagea
Infiltratea
Inflammationa
Necrosis
Osteoblastic surface, increased
Osteoclasts, increased
Osteoid, increased
Physis, decreased thickness
Physeal dysplasia
Physis, increased thickness
Serous atrophy, adipose tissuea,bProliferative (nonneoplastic)
Hyperplasia, chrondrocyte
Hyperplasia, osteoblast, focalabCommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

X
X

178

Toxicologic Pathology 49(1)Fibrous osteodystrophy; bone
(Figures 14.2-14.6)

Comment

Other term(s)Renal osteodystrophy

Comment
A case of renal osteodystrophy in sternum and femur of an 8month-old female Göttingen minipig has been described.204
The bones exhibited high bone turnover associated withincreased cortical porosity, higher trabecular thickness, andabnormal collagen texture (woven versus lamellar osteoid andbone). The pathogenesis in humans is described in the following reference.205

Fracture/CallusFractures in minipigs are rare and most often associated withcallus formation which can be used as a modifier.

Gelatinous bone marrow transformation.

Comment
The spontaneous lesion most commonly observed in the femur
and tibia of control Göttingen minipigs is minimal to marked
serous atrophy of the adipose tissue of the bone marrow which
can occur as a physiological response to a normal degradationof adipose tissue under conditions where the minipig needs
additional energy and the diet is insufficient to meet thoseneeds.3,7,13,31,41
Serous atrophy of the adipose tissue in the bone marrow is
described in greater detail in the section on systemic conditionsof minipigs.

Growth plate closed, partially closed, open;
bone
Other term(s)Physis closed, partially closed, open

Pathogenesis/cell of origin
Serous atrophy of adipose tissue (bonemarrow) (Figure 14.1)
Other term(s)Comment

Unlike the rodent, the growth plate (physis) of minipigs closes
when the animal reaches maturity and growth ceases. The
recording of growth plate closure may become necessary if atreatment causes or is considered to possibly cause a delay ingrowth plate closure or premature growth plate closure. In theuntreated, healthy female Göttingen minipig, growth plate closure of femur starts at the age of 25 months and is complete by
42 months.55 Another paper describes closure of the physis tobe complete by 16 to 24 months.203

Ossification of the growth plate

Diagnostic features

Thickness of the growth plate (physis) depends on therate of growth.
When growth slows, the layers of the growth plate
become narrower.
At the end of the process of bone growth, the cartilage ofthe growth plate is replaced by a bony scar, which is
remodeled into trabecular bone.

II. Joint
Spontaneous lesions of the joints and synovium are rare inminipigs used in toxicity studies and just one change, degenerative joint disease (DJD), is described herein. The relativelack of changes in the joints of the minipigs is most likely
related to the young age of the animals used in nonclinical
toxicity studies.

Table 14.2. Microscopic Findings of Skeletal System: Joint and Synovium; Minipig.
Joint

CommonNonproliferative
Degeneration, chondromucinous
Degenerative joint disease (DJD)aHemorrhageb
Infiltrateb
Inflammationb
Osteophyte
Proliferative (nonneoplastic)
Hyperplasia, synovial cellabTerminology with diagnostic criteria or comments described below
Terminology addressed in Systemic Pathology section.

Uncommon
X
X
X
X
X
X
X

Not observed, but potentially relevant

Not applicableSkydsgaard et al.

179




Degenerative joint disease; joint
Other term(s)Osteochondrosis (OC); Osteochondritis dissecans,osteoarthritis

Pathogenesis/cell of origin
Degenerative joint disease is a noninfectious, degenerative condition involving the major weight bearing joints of the limbs,
characterized by a gradual loss of articular cartilage leading tosecondary bone changes. Degenerative joint disease is an
uncommon observation in the minipig, and when it occurs, it
is usually monoarticular. Osteoarthritis is the preferred term inhumans, but as this term incorrectly implies an inflammatory
origin, DJD is the more appropriate term based on the putative
noninflammatory pathogenesis. Degenerative joint disease may
be classified as primary, where no predisposing factors are present (mainly occurring in older animals) or secondary, associatedwith an underlying abnormality (malformation or deformation)in the joint or supporting structures leading to premature degeneration of the joint cartilage.200,206-208
Chondrocyte dysfunction leads to aberrant maintenance ofthe matrix in articular cartilage. The resulting alterations in the
shape and continuity of cartilage surfaces permits the release of
pro-inflammatory factors and promotes joint instability,
thereby leading to secondary reactions in other joint components. A case of osteochondritis in the tarsal joint of a 3-monthold male minipig exhibiting lameness has recently been
described in the literature.29 The histopathology of this lesion
included cartilage necrosis and cartilage flap formation.

Diagnostic features








Macroscopic findings may be absent.
Irregular contours of articular surfaces.
Thickening of articular cartilage.
Tinctorial changes in articular cartilage matrix (eg, focal
loss of basophilia in H&E-stained sections or metachromasia in toluidine blue-stained sections).
Fibrillation of cartilage matrix; erosion/ulceration of
cartilage.
Chondrocytes arranged in closely packed clusters/nests
or “clones” (ie, proliferative response).

Chondrocyte degeneration and necrosis.
Microfissures in articular cartilage.
Gradual progression of the primary cartilage lesion
through the entire articular surface leading to involvement of other joint structures and the following secondary morphologic changes:






Subchondral bone resorption.
Cyst/pseudocyst formation.
Hypertrophy/hyperplasia of synovial lining cells.
Fragments of degenerate cartilage lodged within the
synovium often eliciting an inflammatory reaction.
 Thickening and ossification of the joint capsule.

Differential diagnosesChondromucinous degeneration: An incidental, typically focal change affecting articular cartilage that does
not progress to DJD.

Chapter 15. Soft TissueStandard INHAND nomenclature for nonproliferative and proliferative soft tissue findings in rats and mice has previously
been published209 and will be used for the minipig as appropriate following the anatomical approach used for rodent species, with a focus on those findings that are likely to be
encountered frequently and those that might be consideredunique to the minipig. The discussion herein is focused on
spontaneous lesions observed in young minipigs (up to 2 yearsof age) used in nonclinical toxicity studies.
This chapter includes lesions of the soft tissues, adipose
tissue, mesothelium, skeletal and smooth muscle, and mesothelium. Lesions of the synovium are described in the chapter on
the skeletal system of minipigs. Proliferative neoplastic lesions
are rarely observed as spontaneous background or test articlerelated findings in routine toxicity studies in minipigs due tothe young age of animals and the short duration of toxicity
studies relative to the minipig’s natural life span.
Soft tissue lesions observed in minipigs are generally identical to those observed in rodents and other domestic animals,
as well as in humans.

I. Soft Tissue
Table 15.1. Microscopic Findings of Soft Tissue, Adipose Tissue, and Mesothelium; Minipig.
Soft tissue
Nonproliferative
Amyloid
Atrophy
EdemaaFibroplasia

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X(continued)

180

Toxicologic Pathology 49(1)Table 15.1. (continued)
Soft tissue

CommonFibrosisHemorrhagea
Infiltratea
Inflammationa
Inflammation, lipogranulomatous
Metaplasia
Mineralization
Necrosis
Proliferative (nonneoplastic)
Hyperplasia, adipose tissue
Hyperplasia, mesotheliumbab

UncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Hyperplasia, mesothelium; mesothelium(Figures 15.1 and 15.2)
Other term(s)Little or no cellular atypia.
May possess a small fibrovascular core or stalk.
In the minipig, fibrosis and inflammation rarely accompany the changes.

Proliferation, mesothelial; proliferation, serosal

Differential diagnoses
Pathogenesis/cell of originHyperplasia of mesothelial cells of the pleura, pericardium,
peritoneum, or tunica vaginalis. In the Göttingen minipig, thefinding is most often minimal and focal, primarily observed inrelation to the arch of the aorta or the atria of theheart.3,7,13,31,41

Mesothelioma, malignant: Is highly cellular and pleomorphic and shows extensive spread within the body
cavities or infiltrates adjacent tissues.
Epithelioid mesothelioma, malignant: Exhibits poorly
formed glandular structures or ill-formed glands. Sarcomatoid malignant mesothelioma consists of spindleshaped cells.

Diagnostic featuresUsually localized but may be diffuse.
Focal thickening or villous projections covered by relatively uniform cuboidal cells with little or no stratification, usually limited to 1 to 2 cells in thickness.
Lacks evidence of significant mitotic activity.

Comment
In the Göttingen minipig, focal mesothelial proliferation is acommon incidental finding thought to arise due to friction at
the predilection sites (mainly the aortic arch and the atria of theheart).

II. Skeletal and Smooth Muscle
Table 15.2. Microscopic Findings of Skeletal and Smooth Muscle; Minipig.
Skeletal and smooth muscle
Nonproliferative
AtrophyDegeneration
EdemaaFibrosisa
Hemorrhagea
Infiltratea
Inflammationa
Mineralization
Myopathy, porcineb

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

181

Table 15.2. (continued)
Skeletal and smooth muscle

CommonNecrosis
Regeneration, skeletal musclea
Vacuolation
Proliferative (nonneoplastic)
Hypertrophy
Hyperplasia, smooth muscleab

UncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Myopathy, porcine; skeletal muscle(Figures 15.3 and 15.4)

discussions in the working group considered the mostappropriate.3,7,13,31,41,210

Other term(s)Chapter 16. Special Senses

Myositis; degeneration/regeneration/necrosis/inflammation of myofibers; myopathic changes; cytopathic
changes.

Pathogenesis/cell of origin
Myofibers of the skeletal muscle

Diagnostic features







Signs of degeneration, including vacuolation of fibers,
split fibers, and hyaline changes.
Myofiber necrosis may be present.
Hemorrhage and edema may be observed.
Inflammatory cell infiltrates are often present. Macrophages are often a prominent feature of infiltrates.
Regenerating basophilic myofibers might occur.
Mineralization may be present.

Differential diagnoses


A variety of different pathological processes areembraced by this term. Clear distinction often requires
special staining techniques to clarify the precise natureof the degenerative alterations.

Comment
In the minipig, “porcine myopathy” occurs as a common incidental background finding. The finding ranges from a focal
minimal lesion to a multifocal severe lesion leading to theprescheduled euthanasia of the affected animal due to adverse
clinical signs. The term covers the entire span of diagnostic
features ranging from acute lesions dominated by necrosis,
hemorrhage, edema, and mixed inflammatory cell infiltrates
to the more chronic lesions characterized by basophilic regenerating myofibers, mineralization, and occasionally fibrosis.
Some prefer to use the term “myositis” to describe this entity;
however, as the inflammatory component is not always a prominent feature, the term “myopathy, porcine” is after longStandard INHAND nomenclature for nonproliferative and proliferative special sense findings in rats and mice has previously
been published211 and will be used for the minipig as appropriate following the anatomical approach used for rodent species, with a focus on those findings that are likely to be
encountered frequently and those that might be consideredunique to the minipig. This chapter covers the ocular system.
The otic (ear) and olfactory systems are not routinely evaluatedin toxicity studies in the minipig. The ocular system is subdivided into the eye and the surrounding glands. At necropsy,
Davidson’s fixative is usually the fixative of choice for theeyes.
For a thorough examination of the ocular system, H&Estained slides of samples from the eyes, eyelids, lacrimal
glands (glandulae lacrimales), third eyelids/nictitating membranes with superficial accessory lacrimal glands (glandulae
superficiales, nictitating glands), Harderian glands (glandulae
profundae palpebrarum tertiarum), and optic nerves should be
prepared.26 However, for most routine toxicity purposes, it issufficient to evaluate sagittal sections of the eyes as well as the
optic nerves and not examine the glands. The lacrimal gland islocated dorsal and caudal to the eye in the orbital cavity. The
nictitating gland is closely associated with the third eyelid.
Both the lacrimal and nictitating glands are thin, pale structuresthat are difficult to distinguish from the surrounding connective
tissue. The Harderian gland is located medio-rostro-ventral tothe eye in the orbital cavity, is pale brown in color, and has alobulated structure.
The recommended eye section for a routine toxicity study isa superior–inferior sagittal section, passing through the optic
nerve head, with proper orientation and free of artifacts. The
cornea should be free of clefts or folds, and the corneal
endothelial cells should not be vacuolated. Shattering or vacuolation of the lens should be avoided, and the lens should becorrectly oriented in the globe, with the epithelium facing thecornea. Artifactual retinal separation or vacuolation is a common problem, and evaluation of photoreceptors demands sections no greater than 5 mm in thickness. Specialized ocular

182

Toxicologic Pathology 49(1)studies may require a different sectioning protocol, dependingon the route of administration, the nature of the test article(aqueous solution, viscous depot, slow-release capsule, stem
cells, subretinal device), or as a result of unusual ophthalmoscopic findings. Pathologists should be involved in determining
the best protocol for a particular study.
The genesis of a good ocular section begins at necropsy.
Rough handling of the eye at enucleation can induce retinal
separation and optic nerve artifacts. The optic nerve should betransected at the level of the orbit to maximize the available
nerve tissue. Extraocular tissues, including glands, should betrimmed off the globe prior to fixation to optimize the fixationof the retina and avoid separation; this also allows better visualization of the landmarks for subsequent trimming. Incision of
the globe prior to fixation will compromise the architecture of
the retina due to the reduced pressure inside the globe. Similarly, injection of fixative into the globe is not recommended. If
orientation is critical, consider using tissue marking fluid or asuture to identify landmarks or the 12-o’clock position at thetime of collection, as landmarks are more difficult to see in a
fixed globe. Left and right eyes should be clearly differentiatedto allow correlation with clinical findings.
A variety of fixation methods may be used. Since perfusion
fixation frequently results in artifactual spaces in the retina,
immersion fixation is probably the best option. Ensure that theeye is immersed in a sufficiently large volume of fixative (atleast 10 the volume of the eye) as rapidly as possible toprevent autolytic change in the retina. Submersion in 10% formalin is frequently used in toxicology studies, but retinal preservation is often compromised.
Davidson’s solution gives better retinal fixation than 10%
formalin, but prolonged exposure will result in artifacts associated with hardening of the lens and clefting and pseudoedematous changes in the cornea. For best results, the eyes shouldbe transferred directly to ethanol in the tissue processor; consider washing and transferring to ethanol if a short delay (up to

10 days) is anticipated, but longer term archival of eyes warrants transfer to 10% formalin. Since Davidson’s fixation isassociated with artifactual vacuolation in the optic nerve dueto the ethanol content, a small section of optic nerve should becollected for fixation in 10% formalin for cross-section examination. Davidson’s fixation is compatible with immunohistochemistry techniques for many antigens and morphology issuperior to that obtained with formalin fixation, but it is notsuitable for electron microscopy evaluation.
Fixation with solutions containing glutaraldehyde (eg, Karnovsky’s solution) is suitable if electron microscopy is planned.
To improve results, submerge the globe in the fixative for 2
hours to allow initial firming of the globe and then cut a small
window in one side of the globe before continuing submersion
for another 2 days. This fixative tends to cause distortion of the
globe due to osmolality effects but generally gives good corneal, lens, and retinal morphology. Possible artifacts include
fissuring of the lens, clefting of the cornea, and vacuolation ofphotoreceptors.
Trimming of globes requires a very sharp blade and shouldbe performed with a single cut; a sawing action will result in
retinal separation.

I. EyeIn general, spontaneous or treatment-related changes in theocular system of the minipig are rare. The most commonly
observed, albeit rare, finding of degeneration of the lens fibers
is presented below.
Apart from degeneration of lens fibers, inflammatory cell
infiltrates or inflammation are the most common (but with a
low incidence) findings in the eyes of the minipigs. These aremost commonly observed in the uvea, conjunctiva, and cornea.
The diagnostic features, pathogenesis, and differential diagnosis for the minipig are similar to those described in the
INHAND rodent manuscript for the special senses.

Table 16.1. Microscopic Findings of Special Senses: Eye; Minipig.
EyeConjunctiva/eyelid
Congenital
Dermoid, ocular
Nonproliferative
Atrophy, epitheliuma
Atrophy, meibomian gland
Congestiona
Cyst, inclusion
Edema
Erosion/ulcer
Fibrosisa
Hemorrhagea
Infiltrate, conjunctivaa
Infiltrate, eyelida

CommonUncommonNot observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

183

Table 16.1. (continued)
Eye
Infiltrate, meibomian glanda
Inflammationa
Inflammation, eyelida
Inflammation, meibomian glanda
Pigment
Proliferative (nonneoplastic)
Hyperplasia, squamous cell
Hyperplasia/hypertrophy, endothelium
CorneaCongenital
Dermoid, ocular
Nonproliferative
Atrophy, epithelium
Attenuation, endothelium
Cyst, inclusion
Edema
Erosion/ulcer
Fibroplasiaa
Fibrosis, stroma
Hemorrhagea
Hypertrophy, Descemet’s membrane
Infiltratea
Inflammationa
Keratinization
Mineralizationa
Neovascularization
Pigmenta
Vacuolation, epithelium, or endothelium
Proliferative, nonneoplastic
Hyperplasia, squamous cell
Hyperplasia/hypertrophy, endothelium
Anterior chamber/aqueous humor
Nonproliferative
Proteinaceous fluid
Inflammationa
Filtration angle/trabecular meshwork
Congenital
Filtration angle, narrowed
Malformation, filtration angle
Proliferative (nonneoplastic)
Proliferation, trabecular meshwork (TM)
Uvea
Proliferative (nonneoplastic)
Hyperplasia, melanocyte
Iris (Uvea)
Congenital
Malformation, iris
Persistent pupillary membrane
Nonproliferative
Adhesion, iris
Atrophy, iris
Congestiona
EdemaaFibrosisa
Hemorrhagea
Infiltratea
Inflammationa
Pigment, increased/decreased, iris

CommonUncommonNot observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X

X
X
X

X

X
X
X
X
X
X
X
X
X
X
X
(continued)

184

Toxicologic Pathology 49(1)Table 16.1. (continued)
Eye
Ciliary body (uvea)
Nonproliferative
Atrophy, ciliary body
EdemaaHemorrhagea
Hypoplasia, ciliary body
Infiltratea
Inflammationa
Vacuolation, cytoplasmic, epithelium
Choroid (uvea)
Nonproliferative
EdemaaHemorrhagea
Infiltratea
Inflammationa
Neovascularization
Lens
Nonproliferative
Degeneration, lens fiberb
Dislocation, lens, anterior or posteriorFibroplasia, lens epithelium
Hypertrophy, lens capsule
Hypertrophy, lens epithelium
Hypertrophy, lens fiber
Infiltratea
Inflammation, lensMineralization, lens fiber
Necrosis, lens epithelium
Rupture, lens capsule
Vacuolation, lens epithelium or lens fiber
Proliferative, nonneoplastic
Hyperplasia, lens epithelium
Vitreous
Congenital
Aplasia, vitreous
Persistent hyaloid vessels
Persistent hyperplastic primary vitreous
Nonproliferative
Fibroplasia: vitreous fibrosis
Hemorrhage
Infiltratea
Inflammationa
Metaplasia, bone or cartilage
Mineralization, vitreous
Pigment, macrophage, hemosiderin
RetinaCongenital
Arteriolar loop, preretinal
Nonproliferative
Atrophy, inner retina
Atrophy, outer retina
Atrophy, retina, global
Detachment, retina
Displacement, photoreceptor nuclei
Fibroplasia, retinal, or epiretinal
Hemorrhage
Glial cells, increased number
Infiltratea
Inflammationa

CommonUncommonNot observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X

X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
(continued)

Skydsgaard et al.

185

Table 16.1. (continued)
Eye

CommonUncommonMineralizationa
Myelin, increased
Single-cell necrosis
Pigment, increased
Retinal folds
Retinal rosettes
Vacuolation, cytoplasmic
Vacuolation, extracellular
Proliferative, nonneoplastic
Neovascularization
RPE (retinal pigmented epithelium)
Atrophy, RPE
Deposits, extracellular matrix, subretina
Fibroplasia, subretinal
Hypertrophy, RPE
Inclusions (intracytoplasmic accumulation), RPE
Necrosis
Pigment, increased
Pigment, decreased
Polarity, loss, RPE
Single-cell necrosis
Proliferative, nonneoplastic
Hyperplasia, RPE
ScleraNonproliferative
Atrophy
Hemorrhagea
Infiltratea
Inflammationa
Metaplasia, bone or cartilage
Optic nerve
Nonproliferative
AtrophyDegeneration, axon
Demyelination
Glial cell, increased number
Hemorrhagea
Infiltratea
Inflammationa
Vacuolationab

Not observed, but
potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X

X
X
X
X
X
X
X
X

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

Degeneration, fiber (lens) (Figures 16.1 and16.2).
In the minipig, the lesion is most commonly observed in the posterior segment of the lens.3,7,13,41
Table 16.2. Microscopic Findings of Special Senses: Glands of the Eye; Minipig.
Glands of the eye
Harderian gland
Nonproliferative
Apoptosis
Atrophy
Cytoplasmic alteration, acinar cell
Cyst

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X(continued)

186

Toxicologic Pathology 49(1)Table 16.2. (continued)
Glands of the eye

CommonDegeneration
Dilation
Hemorrhage
Hypertrophy
Infiltratea
Inflammationa
Inflammation, granulomatous
Porphyrin, increased
Regeneration
Single-cell necrosis
Proliferative, nonneoplastic
Hyperplasia, acinar cell
Lacrimal gland
Nonproliferative
Cytoplasmic alteration, acinar
Apoptosis
Atrophy
Cyst
Degeneration
Dilation
Hemorrhage
Infiltratea
Inflammationa
Karyomegaly
Regeneration
Single-cell necrosis
Proliferative, nonneoplastic
Hyperplasia, acinar cell
Nasolacrimal duct
Proliferative (nonneoplastic)
Hyperplasia, epitheliumaUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X

X

Terminology addressed in Systemic Pathology section.

At necropsy, the kidneys are removed from the abdominal
cavity after the adrenal glands and the gastrointestinal tract. The
renal capsule should be removed and both kidneys weighed andmeasured. The renal cortex, outer zone of medulla, and innerzone of medulla (including the papilla) should be sampled fromboth kidneys. The urinary bladder should be incised and a tissue
block of bladder wall that includes mucosa, muscle layer, andserosa should be prepared. If possible, include ureters or proximal urethra in the tissue block with bladder.26

Chapter 17. Urinary System
For detailed general considerations on the urinary system,
please refer to the rodent publication.212 While the type andclassification of kidney lesions is identical or very similar
across species in the renal pelvis, ureter, urethra, and urinary
bladder, the frequency may differ substantially.
Porcine kidneys have anatomical (multireniculate and multipapillate) and physiological characteristics similar tohumans.3

I. Kidney
Table 17.1. Microscopic Findings of Urinary System: Kidneys; Minipig.
KidneysCongenital
Aplasia
Ectopic tissue, adrenal
Hypoplasia
Renal dysplasia

CommonUncommonNot observed, but potentially relevant

Not applicable

X
X
X
X(continued)

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187

Table 17.1. (continued)
KidneysNonproliferative
Accumulation, glycogenAccumulation, hyaline droplets
Accumulation, adipocytes, interstitiala,bAlpha2u-globulin nephropathy
Amyloid, glomerular
Amyloid, interstitial
Atrophy, glomerulus
Atrophy, tubule
Basophilia, tubulea
Basophilic granulesa,bCast
Chronic progressive nephropathy (CPN)
Congestionc
Crystals
Cyst
Degeneration, tubule
Dilatation, Bowman’s space
Dilatation, tubule
Edema, interstitial
Extramedullary hematopoiesis
Fibrosis, interstitiala
Glomerulonephritisa
Glomerulopathy, hyaline
Glomerulopathy, mesangioproliferative
Glomerulosclerosisa
Hemorrhage
Hypertrophy, tubule
Inclusion bodies
Infarct
Infiltrate, interstitiala
Inflammation, interstitialc
Inflammation, vascularc
Interstitial nephritisa
Karyomegaly
Mesangiolysis
Metaplasia, osseous
Metaplasia/hyperplasia, Bowman’s capsule
Microabscess
Mineralizationa
Necrosis
Necrosis, papilla
Single-cell necrosis
Nephropathy, obstructive
Nephropathy, retrograde
Pigmentc
Pyelonephritis
Regeneration, tubule
Vacuolation
Proliferative (nonneoplastic)
Hyperplasia, juxtaglomerular
Hyperplasia, mesangium
Hyperplasia, oncocytic
Hyperplasia, tubule
NephroblastematosisaCommonNot observed, but potentially relevant

Not applicable

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Terminology with diagnostic criteria or comments described below.
Finding more frequent as an induced change.
cTerminology addressed in Systemic Pathology section.
b

Uncommon

X
X
X
X
X

188

Accumulation, adipocytes, interstitial, kidney(Figure 17.1)

Toxicologic Pathology 49(1)Diagnostic featuresSpecies
Minipig.

Comment
Interstitial accumulation of adipocytes was observed in obese
ovariectomized Göttingen minipigs.213

Basophilia, tubule, kidney (Figure 17.2)
Species
Minipig.

Interstitial connective tissue is more abundant in theminipig than in the other species used for routine toxicity testing.
Focal mild interstitial inflammation associated withfibrosis (interstitial nephritis) can occasionally be seen,usually located in the renal cortex.3,7,13,41,216

Comment
These focal and mild lesions can be attributed to inflammation or tubular defects of resorption or secretion. To see
these changes at moderate or marked levels is quite rare.3
The lesion may be observed with hemorrhagic syndrome,
which is further described in Chapter 2, Systemic
Pathology.19

Comment
Tubule basophilia is an extremely common manifestationof mild nephrotoxicity in the minipig kidney and often
occurs without vacuolation, single cell necrosis, or cellsloughing which are prominent with degeneration in otherlaboratory species having a similar degree of tubule
injury. 13 Tubule basophilia may be accompanied bymitoses or cellular hypertrophy, which in other species
would suggest regeneration. However, in minipigs, thisis a stereotypic response to tubule injury and “basophilia,
tubule” should be the preferred terminology with a diagnosis of “regeneration, tubule” reserved for lesions associated in off-dosing phases where resolution is inferred.
With further renal injury, basophilia often progresses totubule dilation and/or necrosis. Tubule basophilia can also
occur as a spontaneous aging lesion in minipigs, which
may involve thickened basement membranes or peritubule
fibrosis, akin to chronic progressive nephropathy in rodent
kidneys. This lesion is most commonly encountered in thecorticomedullary junction, but its pathogenesis remains
uncertain.214

Basophilic granules, kidney (Figure 17.3)
Species
Minipig.

Comment
Basophilic granules may be present in the proximal tubularcells of minipigs exposed to or treated with antisense
oligonucleotides.215

Fibrosis, interstitial, kidney (Figure 17.4)
Species
Minipig.

Glomerulonephritis, Kidney (Figure 17.5)
Species
Minipig.

Diagnostic features
In spontaneous cases of glomerulonephritis, macroscopic
enlargement, hyperemia, and adhesion may be observed
grossly. On microscopic examination, the reported types ofglomerulonephritis in minipigs are commonly membranousor membranoproliferative/mesangioproliferative. Tubular
basophilia, hyaline casts, and interstitial hemorrhage, fibrosis,
and cell infiltration have also been observed in association withglomerulonephritis.7,13,216

Comment
In the streptozotocin (STZ)-induced diabetic model in minipigs, the glomeruli showed a widening of the mesangium withcell proliferation and increased matrix. In some glomeruli, hyaline droplets were observed in the parietal layer of Bowman’s
capsule. Arterioles with hyaline thickening of the walls were
present at the vascular pole.57
The lesion may be observed with hemorrhagic syndrome, which
is further described in Chapter 2, Systemic Pathology.13,18,19,29,39

Glomerulosclerosis, kidney (Figures 17.6 and17.7)
Species
Minipig.

Skydsgaard et al.

189

Diagnostic features


Glomeruli undergo sclerotic changes, with loss of capillaries substituted by mesangial matrix.

of eosinophilic granulocytes has been observed in the pelvic
region in a few cases. Lymphoplasmacytic and/or neutrophilic
infiltrate was also reported in a Göttingen minipig with spontaneous glomerulonephritis.3,7,10,13,31,41,216

Comment
Glomerulosclerosis in minipigs can occasionally be seen in a few
glomeruli at the corticomedullary junction.3,7,216
The lesion may be observed with hemorrhagic syndrome,
which is further described in Chapter 2, Systemic Pathology.13

Interstitial nephritis, kidney (Figure 17.9)

Infiltrate, interstitial, kidney (Figure 17.8)

Comment

Species

Focal mild interstitial inflammation associated with fibrosis(interstitial nephritis), usually located in the renal cortex, canbe seen occasionally.3,7,13,31,216
These spontaneous changes are rarely reported at moderate
or marked levels.3
The lesion may be observed with hemorrhagic syndrome,
which is further described in Chapter 2, Systemic
Pathology.3,19

Minipig.

Other term(s)Infiltration interstitium; infiltrate, inflammatory cell, interstitium; cell infiltration, interstitium.

Modifier(s)Type of inflammatory cell that represents the predominant cell
type in the infiltrate: lymphocyte; plasma cell; mast cell; monocyte/macrophage; mononuclear; neutrophil; eosinophil; basophil; mixed.

Diagnostic featuresSpecies
Minipig.

Mineralization, kidney (Figures 17.10 and
17.11)
Species
Minipig.

Special techniques for diagnostics

Minimal to mild multifocal interstitial mononuclear cell
infiltrates (predominantly lymphocytes) have been observed.

Mineralization can be demonstrated with Alizarin Red S or
Von Kossa stains.

Comment
This lesion has been observed in Göttingen, Hanford, and Yucatan minipigs and is more commonly observed in females than inmales in all breeds. Mononuclear cell and/or mixed inflammatory cell infiltrates are quite common findings. Focal infiltration

Comment
Small foci of mineral deposits observed as amorphous deposits
in H&E-stained sections, mostly in the papilla, are commonly
seen in Göttingen minipig.3,7,10,13,31,41

II. Renal Pelvis
Table 17.2. Microscopic Findings of Urinary System: Renal Pelvis; Minipig.
Renal pelvis
Nonproliferative
Calculus
Crystals
Dilatation, pelvis
ErosionInfiltratea
Inflammation, vascularb
Metaplasia, squamous cell
Mineralization

CommonUncommonNot observed, but potentially relevant

Not applicable

x
x
x
x
x
x
x
x
(continued)

190

Toxicologic Pathology 49(1)Table 17.2. (continued)
Renal pelvis
Pyelonephritis
Ulcer
Vacuolation, urotheliuma
Proliferative (nonneoplastic)
Hyperplasia, urotheliumabCommonUncommonNot observed, but potentially relevant

Not applicable

x
x
x
x

Terminology with diagnostic criteria or comments described below.
Terminology addressed in Systemic Pathology section.

Infiltrate, Renal pelvis, (Figures 17.12 and17.13)
Species
Minipig.

Comment
Focal infiltration of eosinophilic granulocytes has beenreported in the pelvic region of Göttingen minipigs in a few
spontaneous cases.7

Other term(s)Vacuolation, urothelium, renal pelvis(Figures 17.14 and 17.15)

Infiltration; infiltrate, inflammatory cell; cell infiltration.

Species
Minipig.

Modifier(s)Type of inflammatory cell that represents the predominant cell
type in the infiltrate: lymphocyte; plasma cell; mast cell; monocyte/macrophage; mononuclear; neutrophil; eosinophil; basophil; mixed.

Comment
Vacuolation of the urothelium in the kidney has been reported
in Göttingen minipigs. This finding is very common and mostoften not recorded.13

III. Ureter
Table 17.3. Microscopic Findings of Urinary System: Ureter; Minipig.
UreterCongenital
Aplasia, ureter
Nonproliferative
Calculus
Crystals
Dilatation
Infiltratea
Inflammation, vasculara
Metaplasia, glandular
Metaplasia, squamous cell
Vacuolation, urotheliumb
Proliferative (nonneoplastic)
Hyperplasia, urotheliumabCommonUncommonNot observed, but potentially relevant

x
x
x
x
x
x
x
x
x

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

x

Not applicableSkydsgaard et al.

191

Vacuolation, Urothelium, Ureter(Figure 17.16)

Comment
Vacuolation of the urothelium of the ureter has been reported inGöttingen minipigs. This finding is very common and mostoften not recorded.13

Species
Minipig.

IV. Urethra
Table 17.4. Microscopic Findings of Urinary System: Urethra; Minipig.
UrethraCommonUncommonNonproliferative
Infiltratea
Metaplasia, glandular
Metaplasia, squamous cell
Obstruction
Proteinaceous plug
Proliferative (Nonneoplastic)
Hyperplasia, urothelium
Mesenchymal proliferative lesiona

Not observed, but potentially relevant

Not applicable

x
x
x
x
x
x
x

Terminology addressed in Systemic Pathology section.

V. Urinary Bladder
Table 17.5. Microscopic Findings of Urinary System: Urinary Bladder; Minipig.
Urinary bladder
Congenital
Diverticulum
Nonproliferative
Angiectasis
Calculus
Congestiona
Crystals
Dilatation
Edema
ErosionHemorrhageb
Hypertrophy, urothelium
Inclusions, urothelium
Infiltrateb
Inflammation, vascularb
Metaplasia, glandular
Metaplasia, squamous cell
Mineralization
Necrosis
Parasite, nematodeProteinaceous plug
Uropathy, obstructive
Ulcer
Vacuolation, urothelium
Proliferative (nonneoplastic)
Hyperplasia, urothelium
Mesenchymal proliferative lesionabCommonUncommonNot observed, but potentially relevant

Not applicable

x
x
x
x
x
x
x
x
x
x
x
x

Terminology addressed in Systemic Pathology section.
Terminology with diagnostic criteria or comments described below.

x
x
x
x
x
x
x
x
x
x
x
x

192

Hemorrhage, Urinary bladder (Figures 17.17
and 17.18)
Species
Minipig.

Comment
The lesion may be observed with hemorrhagic syndrome,
which is further described in Chapter 2, Systemic
Pathology.3,13,18,19,29

Infiltrate, urinary bladder (Figure 17.17)

Toxicologic Pathology 49(1)monocyte/macrophage; mononuclear; neutrophil; eosinophil;basophil; mixed.

Comment
Small foci of lymphoid aggregates may occasionally beobserved in the mucosa and submucosa, often in association
with minor hemorrhage, which is often described as reddenedmucosa at necropsy.3,7,13,31
Inflammation of the urinary bladder can be observed on
occasion, secondary to kidney pathology. Spontaneous infiltration of inflammatory cells has been reported in Göttingen and
Yucatan minipigs.13,31

Species
Minipig.

Other term(s)Infiltration; infiltrate, inflammatory cell; cell infiltration.

Inflammation, vascular, Urinary bladderSpecies
Minipig.

Modifier(s)Comment

Type of inflammatory cell that represents the predominant cell
type in the infiltrate: lymphocyte; plasma cell; mast cell;

The lesion may be observed with hemorrhagic syndrome,
which is further described in Chapter 2, Systemic Pathology.3,7

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193

Figure 2.1. Minipig; Mesenteric lymph node; Infiltrate, eosinophil; H&E 20. Figure 2.2.—Minipig; Systemic, Kidney; Infiltrate, eosinophil in
renal pelvis; H&E 5. Figure 2.3.—Minipig; Systemic, Kidney; Infiltrate, eosinophil in renal pelvis; H&E x40. Figure 2.4.—Minipig; systemic lungs;
vacuolated alveolar macrophages due to phospholipidosis; H&E 20 (Courtesy of Louise Otzen, H. Lundbeck A/S). Figure 3.1.—Heart: Normal
Purkinje fibers (arrow); H&E x10. Figure 3.2.—Heart: Degeneration/necrosis, cardiomyocyte. H&E x40.

194

Toxicologic Pathology 49(1)Figure 3.3. Heart: Hemorrhage (arrow), infiltrate (arrowhead), and degeneration/necrosis in cardiomyocyte (asterisk). H&E x20. Figure 3.4.—
Heart: Inflammation, endocardium (arrow). Observed in the intravenous infusion study. H&E. Figure 3.5.—Heart: Vacuolation, cardiomyocyte.
Spontaneous. H&E x20. Figure 3.6.—Blood vessels: Atherosclerosis (induced with high-cholesterol diet). Mineralization (arrow). H&E x2.
Figure 3.7.—Blood vessels: Atherosclerosis (induced with high-cholesterol diet). Mineralization (arrow). H&E x 10. Figure 3.8.—Blood vessels:
Atherosclerosis (induced with high cholesterol diet). a-smooth muscle actin. IHC x2.

Skydsgaard et al.

195

Figure 3.9. Blood vessels (induced with high cholesterol diet). a-smooth muscle actin. IHC x10. FIGURE 3.10.—Blood vessels: Atherosclerosis(induced with high cholesterol diet). Oil red O stain x20. FIGURE 3.11.—Blood vessels: Lipid embolus in lung. H&E x40 (Courtesy of Birgitte
Martine Viuff, Novo Nordisk A/S). FIGURE 3.12.—Blood vessels: Lipid embolus of glomerulus. H&E x40 (Courtesy of Birgitte Martine Viuff, Novo
Nordisk A/S). FIGURE 3.13.—Blood vessels: Lipid embolus of glomerulus. Oil red O stain x40 (Courtesy of Birgitte Martine Viuff, Novo
Nordisk A/S). FIGURE 3.14.—Blood vessels: Infiltrate, perivascular. Fibrinoid necrosis (arrow). H&E x20.

196

Toxicologic Pathology 49(1)Figure 3.15. Blood vessels: Infiltrate, perivascular in intestine serosa. H&E x10. Figure 3.16.—Blood vessels: Necrosis/inflammation, media orwall, artery. Fibrinoid necrosis (arrow) and infiltrate, perivascular (arrowhead). H&E x10. Figure 3.17.—Blood vessels: Necrosis/inflammation,
media or wall, artery. Fibrinoid necrosis (arrow). Mesentery of small intestine. H&E x20. Figure 4.1.—Tongue, erosion/ulcer of the squamous
epithelium, 5 (From “Spontaneous background pathology in Göttingen minipigs”. Toxicol Pathol. 2015;43(2):257-266, with permission).
Figure 4.2.—Tongue, ulcer with inflammation due to biting, 2. Figure 4.3.—Tongue, hyperplasia, squamous cell, 5.

Skydsgaard et al.

197

Figure 4.4. Tongue, necrosis and vacuolation of the squamous epithelium, 20. Figure 4.5.—Tongue, vacuolation of the squamous epithelium,20. Figure 4.6.—Pharynx, macroscopic appearance, normal. Figure 4.7.—Esophagus, squamous cell hyperplasia, 2. Figure 4.8.—Stomach,normal macroscopic appearance, opened. Figure 4.9.—Stomach, normal macroscopic appearance, closed.

198

Toxicologic Pathology 49(1)Figure 4.10. Stomach, nonglandular, erosion, 10. Figure 4.11.—Stomach, nonglandular, mucosal hyperplasia, and hyperkeratosis, 5.
Figure 4.12.—Stomach, glandular, pyloric region, erosion, 5 (From “Spontaneous background pathology in Göttingen minipigs”. Toxicol Pathol.
2015;43(2):257-266, with permission). Figure 4.13.—Stomach, glandular, pyloric region, erosion, 20. Figure 4.14.—Stomach, glandular,
pyloric region, erosion, 20. Figure 4.15.—Small intestine, duodenum, erosion, 5.

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199

Figure 4.16. Small intestine, ileum, lymphoma, 2. Figure 4.17.—Small intestine, ileum, lymphoma, 10. Figure 4.18.—Small intestine,
jejunum, basophilic parasite particles, 63. Figure 4.19.—Small intestine, jejunum, basophilic parasite particles in the lumen, 63.
Figure 4.20.—Small intestine, ileum, villous hypertrophy, 2. Figure 4.21.—Small intestine, ileum, control, 2.

200

Toxicologic Pathology 49(1)Figure 4.22. Small intestine, ileum, inflammation, 2. Figure 4.23.—Large intestine, colon, erosion, 20. Figure 4.24.—Sublingual gland,mineralization, 20. Figure 4.25.—Pancreas, normal macroscopic appearance: right and left lobes and body (From “Tissue sampling guides for
porcine biomedical models”. Toxicol Pathol. 2016;44(3):414-420, with permission). Figure 5.1.—Minipig, pituitary gland, mineralization, H&E(From “Background pathological changes in minipigs: a comparison of the incidence and nature among different breeds and populations ofminipigs”. Toxicol Pathol. 2016;44(3):325-337, with permission). Figures 5.2—Minipig, pineal gland, range of normal vascular dilation, H&E.

Skydsgaard et al.

201

Figure 5.3. Minipig, pineal gland, range of normal vascular dilation, H&E. Figures 5.4. Minipig, pineal gland, melanin pigment (melanosis), H&E.
Figure 5.5.—Minipig, thyroid gland, hemorrhage, inflammation, and fibrosis due to blood sampling via the jugular vein, H&E. Figures 5.6 and
5.7.—Minipig, thyroid gland, adenoma, H&E. Figure 5.8.—Minipig, adrenal gland, zona fasciculata (stained brown for CYP17a by immunohistochemistry) extends into the zona glomerulosa.

202

Toxicologic Pathology 49(1)Figure 5.9. —Minipig, adrenal gland, pigment, H&E. Figure 5.10.—Minipig, adrenal gland, diffuse cortical vacuolation, H&E. Figure 5.11.
Minipig, endocrine pancreas, normal islets, H&E. Figure 5.12.—Minipig, endocrine pancreas, islets atrophy induced by streptozotocin, H&E.
Figure 6.1.—Marrow (femur): serous atrophy of adipose tissue, H&E x20. Figure 6.2.—Thymus apoptosis, increased, lymphocyte, H&E x2(Courtesy of Paul Howroyd).

Skydsgaard et al.

203

Figure 6.3. Thymus hemorrhage, H&E, subgross Iatrogenic hemorrhage due to bleeding procedures and the structure of the blood vessels of theneck and cranial thorax. Figure 6.4.—Spleen: normal H&E 10. Note the thick muscular/elastic trabeculae, periarteriolar macrophage sheaths(PAMS), well-developed periarteriolar lymphoid sheath, and poorly developed lymphoid follicles. Figure 6.5.—Spleen: Normal silver, 10 silver
stain of the previous image highlighting the PAMS and trabeculae. Figure 6.6.—Spleen: extramedullary hematopoiesis, H&E 12. Unusual in pigs
over 6 weeks of age. Figure 6.7.—Mesenteric lymph node: Normal structure: H&E: subgross image illustrating the normal “inside-out”
appearance of a porcine lymph node, with central cortex (with germinal centres) and paracortex, with subcapsular medulla filled with histiocytes.
Figure 6.8.—Axillary lymph node: Normal structure: H&E: subgross Illustrating numerous histiocytes and fat infiltration.

204

Toxicologic Pathology 49(1)Figure 6.9. Mesenteric lymph node: Necrosis, focal, lymphocyte: H&E 20. Showing pyknotic nuclei and cell debris. Figure 6.10.—Ileum
lymphoma H&E x2. Diffuse neoplastic lymphocyte infiltration into the mucosa of the ileum of a young pig. Figure 6.11.—Liver lymphoma H&E x2.
Infiltrate of neoplastic lymphocytes into the portal tracts and sinusoids of the liver of a young pig. Figure 7.1.—Liver, macroscopic appearance,
normal. Figure 7.2a.—Liver, microscopic appearance, normal, 5. Figure 7.2b.—Liver, microscopic appearance, normal, 10.

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Figure 7.3. Liver, fibrosis, 4. Figure 7.4.—Liver, pigment in Kupffer cells. Figure 7.5.—Liver, hepatocyte vacuolation, 20. Figure 7.6.—
Liver, hepatocyte rarefaction (glycogen), 10. Figure 7.7.—Liver, sinusoidal congestion, 10. Figure 7.8.—Liver, mixed cell infiltration, 20.

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Toxicologic Pathology 49(1)Figure 7.9. Liver, hepatocyte necrosis with congestion, 10. Figure 7.10.—Liver, hepatocyte necrosis with neutrophilic cell infiltrate, 40.
Figure 7.11.—Gall bladder, porcine cholecystitis, 2. Figure 7.12.—Gall bladder, porcine necrotizing cholecystitis, 1.4. Figure 7.13.—Gall
bladder, porcine necrotizing cholecystitis, 10. Figure 7.14.—Gall bladder, porcine necrotizing cholecystitis, 40.

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Figure 7.15. Gall bladder, mucosal edema, 4. Figure 7.16.—Gall bladder, mucosal edema, 10. Figure 7.17.—Gall bladder, microscopic
appearance, normal, 10. Figure 7.18.—Gall bladder, induced mucosal hyperplasia, 10. Figure 7.19a.—Gall bladder, hypoplasia, 2.
Figure 7.19b.—Gall bladder, hypoplasia, 2.

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Toxicologic Pathology 49(1)Figure 7.20. Gall bladder, gallstone in an aged microminipig. Figure 8.1.—Skin: Histologic lesions of the Dippity Pig syndrome. H&E 20.
Figure 8.2.—Skin: Multifocal crusts within the stratum corneum of the epidermis. H&E x5. Figure 8.3.—Skin: Focal crust within the stratum
corneum of the epidermis. H&E x10. Figure 9.1.—Minipig, mammary gland, normal tissue, right below the nipple, male, magnification, H&E 20.
Figure 9.2.—Minipig, mammary gland, normal tissue, right below the nipple, male, H&E 40.

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Figure 9.3. Minipig, mammary gland, normal tissue, right below the nipple, male, H&E 100. Figure 9.4.—Minipig, mammary gland, normal
tissue, right below the nipple, female, H&E 20. Figure 9.5.—Minipig, mammary gland, normal tissue, right below the nipple, female, H&E 40.
Figure 9.6.—Minipig, mammary gland, normal tissue, right below the nipple, female, H&E 100. Figure 10.1.—Hypo-osmotic edema in the
cerebrum of a pig with sodium chloride toxicity. Note the region of vacuolation (spongy change) in the neurophil. This lesion is often laminar(middle to deep gray lamina) and accompanied by neuronal necrosis (eosinophilic neurons) (arrows) and astrocytic swelling (evident as vacuolation). Unique to pigs is a perivascular infiltrate of eosinophils and, with longer survival, an influx of macrophages (gitter cells). Inset: Note theeosinophils in the perivascular space of this post-capillary venule. H&E stain (From Haschek WM, Rousseaux CG, Wallig MA, eds. Fundamentals of
Toxicologic Pathology. 2nd ed. Elsevier; 2010. Figure 13.19, p 402, with permission). Figure 10.2.—Cerebellar meninx: Focal minimal mineralizationin the meninx of the cerebellum (arrow). H&E 10 (From McAnulthy PA, Dayan AD, Ganderup NC, Hastings KL, eds. The Minipig in Biomedical
Research. CRC Press; 2012. Reproduced by permission of Taylor and Francis Group.

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Toxicologic Pathology 49(1)Figure 11.1. Immature ovary lacking tertiary follicles and corpus luteum. Subgross H&E (From “Onset of puberty and normal histologicalappearances of the reproductive organs in peripubertal female Gottingen minipigs”. Toxicol Pathol. 2013;41(8):1116-1125, with permission).
Figure 11.2.—Partially mature ovary with large tertiary (Graaffian) follicles (F) (From “Onset of puberty and normal histological appearancesof the reproductive organs in peripubertal female Gottingen minipigs”. Toxicol Pathol. 2013;41(8):1116-1125, with permission). Figure 11.3.—
Mature ovary with large corpus luteum. Figure 11.4.—Proestrus ovary with large tertiary (Graaffian) follicles. Figure 11.5.—Proestrus uterus
with small lumen. Figure 11.6.—Proestrus uterus with apoptotic cells in surface epithelium (Courtesy de Rijk, Eveline).

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Figure 11.7. Proestrus vagina. Figure 11.8.—Estrus ovary with collapsed Graaffian follicle. Figure 11.9.—Estrus uterus (Courtesy de Rijk,
Eveline). Figure 11.10.—Estrus vagina. Figure 11.11.—Metestrus ovary. Figure 11.12.—Metestrus ovary with newly formed CL (Courtesy deRijk, Eveline).

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Toxicologic Pathology 49(1)Figure 11.13. Metestrus uterus with tall columnar luminal epithelium (Courtesy de Rijk, Eveline). Figure 11.14.—Metestrus vagina. Figure 11.15.—Diestrus ovary (Courtesy de Rijk, Eveline). Figure 11.16.—Diestrus uterus (Courtesy de Rijk, Eveline). Figure 11.17.—Diestrus
vagina. Figure 11.18.—Mineralization ovary (Courtesy de Rijk, Eveline).

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Figure 11.19. Oviduct squamous cell metaplasia (Courtesy de Rijk, Eveline). Figure 12.1.—Normal minipig efferent ducts. Testis and epididymis are also present in image. H&E 20. Figure 12.2.—Normal minipig efferent ducts. H&E x100. Figure 12.3.—Normal minipig spermaticcord. H&E x100. Figure 12.4.—Normal minipig bulbourethral glands. H&E x40. Figure 12.5.—Normal minipig bulbourethral glands. H&E x100.

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Toxicologic Pathology 49(1)Figure 12.6. Normal minipig penis, cross-section. H&E x6. Figure 12.7.—Normal minipig penis, cross-section. Urethra and underlying glands.
H&E x40. Figure 12.8.—Normal minipig prepuce, sectioned longitudinally. H&E x10. Figure 12.9.—Normal minipig dorsal preputial diverticulum. Penis is also present. H&E x5. Figure 12.10.—Normal minipig dorsal preputial diverticulum. H&E x40. Figure 12.11.—Testis: Control
mature male (11 month old) Göttingen minipig testis with low numbers of Leydig cells. H&E x100.

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Figure 12.12. Testis: Control mature male (11 month old) Göttingen minipig testis with high numbers of Leydig cells. H&E x100.
Figure 12.13.—Testis: Control male Göttingen minipig testis with Leydig cell adenoma. H&E x25. Figure 12.14.—Testis: Control maleGöttingen minipig testis with Leydig cell adenoma. H&E x100. Figure 12.15.—Testis: Tubular hypoplasia/atrophy in the testis of a male Göttingen
minipig. Vacuolation of affected tubules, tubules with decreased numbers of spermatids, and round intraluminal germ cells are also present.
Abundant Leydig cells are present, but are within the range of normal. H&E x100. Figure 12.16.—Section of normal seminal vesicle. Note thesmooth appearance of the vesicle epithelial lining H&E x2. Figure 12.17.—Section of seminal vesicle with diffuse hyperplasia of the epithelium.
Epithelium is thickened, with increased numbers of cells, and forms frond-like protrusions into the lumen of the vesicle H&E x2.

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Toxicologic Pathology 49(1)Figure 13.1. Pleura, adhesion. Pleural adhesion with subpleural inflammation (H&E). Figure 13.2.—Pleura, lung, edema. Interlobular and pleuraledema with dilated lymphatics (arrows). Domestic pig with fumonisin toxicosis (H&E). Figure 13.3.—Lung, alveoli, embolus. Lipid embolus inpulmonary capillaries. A, Alveolar capillaries are markedly distended by discrete nonstaining material appearing as clear spaces (*) (H&E, x20). B,
Capillaries are distended with material that stains red with Oil Red O, consistent with lipid (Oil Red O, x20) (Courtesy Birgitte Martine Viuff,
Novo Nordisk A/S). Figure 13.4.—Lung, bronchiole, exudate. The bronchiole is filled with inflammatory and cell debris. Inflammation is alsopresent in adjacent alveoli (H&E) (Courtesy Shih-Hsuan Hsiao). Figure 13.5.—Lung, alveoli, exudate. Alveoli are filled by fibrinous exudate
consisting of proteinaceous edema and fibrin (arrows). Alveolar walls are thickened due to inflammatory cells (H&E) (Courtesy Shih-Hsuan
Hsiao). Figure 13.6.—Pleura, exudate. The pleura is thickened and covered by exudate consisting of inflammatory cells and fibrin. The lymphatics(arrow) are distended with inflammatory cells (H&E) (Courtesy Shih-Hsuan Hsiao).

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Figure 13.7. Lung, alveoli, granuloma, foreign material. There is foreign material surrounded by macrophages including multinucleated giant cells.
Figure 13.8.—Lung, alveoli, hemorrhage. Red blood cells are free within alveoli. This animal also had thrombocytopenic purpura (see the
Systemic section) (H&E x40). Figure 13.9.—Lung, alveoli, infiltrate. Numerous inflammatory cells fill alveoli with no tissue damage or signs ofinflammation (H&E). Figure 13.10.—Lung, alveoli, macrophages, increased. Alveoli contain numerous alveolar macrophages (H&E, x40) (From“Background pathological changes in minipigs: a comparison of the incidence and nature among different breeds and populations of minipigs”.
Toxicol Pathol. 2016;44(3):325-337, with permission). Figure 13.11.—Lung, alveoli, macrophages, increased (vacuolated). Vacuolated macrophages focally fill many alveolar spaces (H&E, x20). (Courtesy Louise Otzen, H. Lundbeck A/S). Figure 13.12.—Lung, bronchiole, pigment or
foreign material. Foreign material is present within the lumen of the bronchiole and is surrounded by inflammatory cells (H&E).

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Toxicologic Pathology 49(1)Figure 14.1. Minipig, bone marrow, serous atrophy of adipose tissue, H&E 40. Figure 14.2.—Minipig, sternum, osteodystrophy, H&E 1(Courtesy Rosa Anna Manno, ERBC srl). Figure 14.3.—Minipig, sternum, osteodystrophy, H&E 5 (Courtesy Rosa Anna Manno, ERBC srl).
Figure 14.4.—Minipig, kidney, renal changes in minipig with osteodystrophy, H&E 1 (Courtesy Rosa Anna Manno, ERBC srl). Figure 14.5.—
Minipig, Kidney, degeneration, tubular; interstitial fibrosis, regeneration, tubular, H&E 10 (Courtesy Rosa Anna Manno, ERBC srl).

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Figure 14.6. Minipig, kidney, mineralization, tubular, H&E 10 (Courtesy Rosa Anna Manno, ERBC srl). Figure 15.1. Minipig, mesothelium,
hyperplasia, H&E x20. Figure 15.2.—Minipig, mesothelium, hyperplasia, H&E x100. Figure 15.3.—Minipig, skeletal muscle, myopathy porcine,
H&E x100 (From “Spontaneous background pathology in Göttingen minipigs”. Toxicol Pathol. 2015;43(2):257-266, with permission). Figure 15.4.—Minipig, skeletal muscle, myopathy porcine, H&E 40. Figure 16.1.—Minipig: Eye, degeneration, fiber (lens), H&E 20 (FromHaschek and Rousseaux’s Handbook of Toxicologic Pathology. Haschek WM, Rousseaux CG, and Wallig MA, eds. 3rd ed. Elsevier; 2013. Figure 13.19,
p 473, with permission).

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Toxicologic Pathology 49(1)Figure 16.2. —Minipig: Eye, degeneration, fiber (lens), H&E 40 (From Haschek and Rousseaux’s Handbook of Toxicologic Pathology. Haschek WM,
Rousseaux CG, and Wallig MA, eds. 3rd ed. Elsevier; 2013. Figure 13.19, p 473, with permission). Figure 17.1. Kidney: Accumulation, adipocytes(arrows), interstitial. H&E 10. Figure 17.2.—Kidney: Basophilia, tubule (arrows). H&E 10. Figure 17.3.—Kidney: Basophilic granules(arrows). H&E. Figure 17.4.—Kidney: Fibrosis, interstitial. Basophilic granules (arrows). H&E 20. Figure 17.5.—Kidney: Glomerulonephritis.
H&E 10.

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Figure 17.6. Kidney: Glomerulosclerosis. H&E 40. Figure 17.7.—Kidney: Glomerulosclerosis. H&E 40. Figure 17.8.—Kidney: Infiltrate,interstitial. H&E 20. Figure 17.9.—Kidney: Interstitial nephritis. H&E 10. Figure 17.10.—Kidney: Mineralization (arrows). H&E 10.
Figure 17.11.—Kidney: Mineralization (arrows). H&E 20.

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Toxicologic Pathology 49(1)Figure 17.12. Renal pelvis: Infiltrate. H&E 5. Figure 17.13.—Renal pelvis: Infiltrate (eosinophilic granulocytes). H&E. 40. Figure 17.14.—
Renal pelvis: Vacuolation, urothelium of renal pelvis (arrows). H&E 4. Figure 17.15.—Renal pelvis: Vacuolation, urothelium of renal pelvis(arrows). H&E 10.

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Authors’ Note
All procedures used to prepare macroscopic and microscopic imagesof animal specimens for this article were performed in accordancewith regulations and established guidelines for humane treatment ofresearch animals and were reviewed and approved in advance by an
Institutional Animal Care and Use Committee.

Acknowledgments
The authors wish to express their thanks to the BSTP, ESTP, JSTP,
and STP membership for the comprehensive reviews, excellent comments, and helpful edits. The authors also thank Rupert Kellner for
manuscript review and Beth Mahler for image editing.

Declaration of Conflicting Interests
The author(s) declared no potential, real, or perceived conflicts ofinterest with respect to the research, authorship, and/or publicationof this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD
Mikala Skydsgaard
https://orcid.org/0000-0001-9352-8629
Keith Nelson
https://orcid.org/0000-0002-0417-7423

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Figure 17.16. Ureter: Vacuolation, urothelium of ureter (arrows).
H&E 20. Figure 17.17.—Urinary bladder: Hemorrhage. Infiltrate(arrows). H&E 20. Figure 17.18.—Urinary bladder: Hemorrhage.
H&E 10.

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