bluuebunny/crossref_metadata_2025 huggingface dataset:
Esophageal Anastomosis Pressure Testing to Failure: Full‐thickness or Submucosa‐Mucosal?
Authors: Aho Johnathon Michael , Manzar Shahid A , Wise Kevin B , Wigle Dennis A , Tschumperlin Daniel J , Urban Matthew W
Published: November 2023

Background

Esophageal pathology such as perforation or malignancy often necessitates esophageal reconstruction. However, the optimal method of esophageal primary repair and anastomosis are unclear. We aim to determine stress to failure of esophageal anastomosis for full thickness and mucosal‐submucosal anastomotic techniques.

Methods

Six esophagi from freshly sacrificed 60 kilogram swine were transected at the mid thoracic portion of the esophagus. Esophageal anastomoses were created and subsequently primarily anastomosed using 2‐0 running continuous polyglactin (Vicryl, Ethicon, Somerville, NJ) suture either as full thickness or partial thickness (mucosa‐submucosa alone). The sutured specimens were then mounted on cannulae. Using a syringe pump system, the esophagi were infused with colored saline while monitoring pressure continuously with optical tracking for detection of fluid leak. Data acquisition software was used to collect stress‐to‐failure data. Synchronized video and pressure data were analyzed post hoc for visualization of the leak and concurrent pressure to determine failure pressure.

Results

The full‐thickness anastomosis failed at a mean ± (SEM) pressure of 15.33 ± (1.45) mmHg while the mucosal‐submucosal anastomosis failed at a mean ± (SEM) pressure of 36.33 ± (3.76) mmHg, (P = 0.02, unpaired t‐test with Welch's correction). MATLAB (MathWorks, Natick, MA) was used calculate the statistical results.

Conclusion

While the optimal in vivo method for esophageal anastomosis is unclear, we present evidence that an anastomosis incorporating only the mucosa and submucosa may be superior to full thickness. Additional research is needed to further clarify optimal technique and post‐operative outcomes.

Support or Funding Information

Dr. Aho reports funding from a Grant from the National Heart, Lung, and Blood Institute T32 HL105355, a component of the National Institutes of Health (NIH).
Nonrotation of the Intestine: Embryological and Clinical Correlation
Authors: Velavan Sumathilatha Sakthi , Castellanos Bedia , Gulfam Nida , Rich Sushama
Published: November 2023

The Embryology of the gastrointestinal tract involves a well‐orchestrated rotation of midgut to assemble the parts of the gut in the normal anatomical location. The nonrotation is a stage II anomaly and is a very rare variation with an incidence of 1:500. This case is reported for its extreme rarity and its clinical implications that result from the completely altered architecture of the organs in the peritoneal cavity. During routine dissection of the abdomen in an adult female cadaver, abnormal location of the small intestine was noted. A detailed analysis of the intestines was done by tracing from pyloric end of the stomach to the rectum. The vasculature of the abdominal organs, the liver, pancreas, spleen, and the genitourinary tract were dissected to note any variation. The entire small intestine was found to lie on the right side of the abdominal cavity. The duodenum was coiled, located to the right of the superior mesenteric artery and was not crossed by the artery. The ileocecal junction was traced to the hypogastrium where cecum and appendix were situated. The ascending and transverse colon formed a loop on the left half of the abdominal cavity, while the descending colon and sigmoid colon were typically located. A fibrous Ladd's band was found to extend from posterior abdominal wall to the colon, crossing anterior to the duodenum. It held the intestines coiled and had to be released to trace the viscera. The left kidney was normally situated while the right kidney was located close to the midline. The clinical presentation associated with nonrotation of gut ranges from vague intermittent pain to symptoms of acute bowel obstruction, although it may be asymptomatic too. The abnormally located appendix may lead to misdiagnosis of appendicitis. The peritoneal bands may lead to intestinal compression or volvulus. Adequate knowledge of Embryology and resultant variations aid in understanding the abnormal findings during diagnostic interventions prevents surgical complications and also assists in anatomical correction surgery of nonrotation of the intestine.
Classification of Type II Diabetes Mellitus Progression Based on Quantification of Pancreatic Histology
Authors: Papazian Lucine A , Pan Zenggang , LaRosa Francisco , Lee Lisa
Published: November 2023

Type II Diabetes is a metabolic disorder characterized by insulin insensitivity resulting in a decreased ability to transport glucose into cells systemically. While type II diabetes is well understood both clinically and in the renal system, there is a gap in documentation of the histological changes that occur in pancreatic tissue throughout disease progression. Thus, the objective of this study was to investigate the morphological changes in pancreatic tissues and to establish a set of histological criteria associated with the progression of type II diabetes. The systemic nature of type II diabetes provides opportunity to use the already established renal progression schematic to stage the disease into Diabetic Kidney Disease (DKD) I–IV. In this project, 100 matched pancreatic and kidney tissue slides from autopsy cases, with a pre‐mortem diagnosis of type II diabetes, were obtained from the existing autopsy repository. Kidney tissues were stained with periotic acid schiff to assess alternations in the glomerular basement membrane thus allowing each case to be staged into DKD I–IV. Matched pancreatic tissue slides were digitally scanned using Aperio slide scanner. Each digitized image file was then analyzed using the imagescope software; size and density of pancreatic islets and parenchymal adipose infiltrates were quantified. The quantified metrics and characteristics of the pancreatic tissues were compared across the 4 DKD groups. The results illustrate a general pattern of increased adipose infiltration and decreased islet density that correspond with type II diabetes disease progression. Our findings are consistent with other studies documenting incidental observations of increased adipose infiltration, and beta‐cell deficits in diabetic patients. For the first time, a classification schematic based on pancreatic histology for type II diabetes disease progression, is proposed.

Support or Funding Information

Capstone Funding MSMHA
Phylogenetic and dietary patterning in the morphology of avian colic ceca
Authors: Hunt Andrew , Lee Andrew , Al‐Nakkash Layla , Riede Tobias , Smith Heather F.
Published: November 2023

In birds, the colic ceca appear as a pair of blind sacs that arise from the junction of the ileum and colon. The length of the ceca is variable among species, ranging from absent to as long as 50% the length of the small and large intestine. We tested the hypotheses that cecal length is related to diet composition, and that phylogenetic relationships are not reflected in cecal morphology. We collated published data on relative cecal length, dietary category, and body mass from 150 avian taxa. To test whether these data patterned phylogenetically, we mapped the character states for each of the 150 avian taxa onto a recently published supertree and recorded the number of evolutionary steps. We then generated 1000 and 10,000 random simulated trees, and mapped the data onto them. None of the simulations required as few evolutionary steps as the real supertree (p<0.001), indicating a significant phylogenetic signal in avian colic cecal morphology. The correlation between diet composition and relative cecal length was not significant (pairwise correlation and Pagel's analyses). However, this could be due to the absence of clades with a large variability in both cecal length and diet. We also found that birds within a given clade have a similar relative cecal size, which further suggests that phylogenetic relationship dominates dietary function. Future work within Galloanserae will examine the relationship between dietary function, cecal size, and phylogenetic relationships. We will specifically test whether plasticity in cecal length is a response to dietary alterations.
AMPK: A Novel Link Between E‐cadherin Mechanotransduction And Cell Metabolism
Authors: DeMali Kris , Bays Jennifer , Miller Hannah , Heidema Christy
Published: November 2023

The response of cells to mechanical force is a major determinant of cell behavior and is an energetically costly event. How the cell derives energy to resist mechanical force is unknown. Here, we show that application of force to E‐cadherin stimulates Liver Kinase B1 (LKB1) to activate AMP‐activated protein kinase (AMPK), a master regulator of energy homeostasis. LKB1 recruits AMPK to the E‐cadherin mechanotransduction complex, thereby stimulating actomyosin contractility, glucose uptake, and ATP production. The increased ATP provides energy for reinforcing the adhesion complex and actin cytoskeleton so that the cell can resist physiological forces. Together, these findings reveal a paradigm for how mechanotransduction and metabolism are linked and provide a framework for understanding how diseases with contractility and metabolic disturbances arise.

Support or Funding Information

Research reported in this publication was supported by The National Institutes of General Medicine (Award Number R01GM112805 to K.A.D) and the National Cancer Institute of the National Institutes of Health (Award Number P30CA086862). Predoctoral fellowships from the American Heart Association (Award Number AHA 16PRE26701111) and National Institutes of Health (Award T32 GM067795) support J.B. and C.H., respectively.
Loading and Form in the Mammalian Jaw Joint
Authors: Rafferty Kathy , Herring Sue
Published: November 2023

The synovial articulation between the dentary and squamosal bones, usually referred to as the temporomandibular joint (TMJ), is a defining feature of the Class Mammalia. The mandibular component of this joint is a secondary cartilage that develops in a membranous matrix and contributes to growth of the mandible. While resembling the growth cartilage of long bones, the mandibular condylar cartilage differs in that it also must function as the articular surface of the mammalian jaw joint, a function mediated by a fibrous thickening of the perichondrium. Another difference from typical growth cartilage is that cells are closely packed and not aligned in columns. This feature permits the direction of growth to be altered. Perhaps because of its origin from membranous tissue, the mandibular condyle is notably sensitive to mechanical loading, particularly during the period of growth. Loading of the TMJ arises from contraction of the jaw muscles, but the magnitude and orientation of loading vary according to the muscle combination used. In this contribution we summarize and synthesize studies on condylar loading assessed using strain gages affixed to the condylar neck of rabbits (Oryctolagus cuniculus) and pigs (Sus scrofa) under normal and altered conditions, correlating the findings with condylar morphology. During normal mastication young adult rabbits evince nearly vertical compressive strain on the condylar neck. Reduction of muscle force using botulinum toxin diminished and reoriented the strain, and these changes were accompanied by a striking loss of bone in the condylar head and neck. The paralyzed side condyle tended to be smaller than that of the healthy side, suggesting that condylar surface area did not increase normally, but the thickness of the cartilage layer was unaffected, implying no change in length of the cartilage process. Like rabbits, pigs show compressive strain on the condylar neck during mastication. During the growth process, the orientation of masticatory compressive strain alters from posterior‐superior in younger animals to anterior‐superior in older pigs. Studies correlating condylar strain with cartilage proliferation showed a tendency for proliferation to decrease as condylar compressive strain increased. A procedure (distraction osteogenesis) used clinically to increase mandibular length was found to lower the magnitude and reorient condylar strain. Unlike the adult rabbits, these juvenile pigs responded to lowered loads with increased growth, but the direction was altered in correspondence with the reorientation of masticatory strain. Collectively, these studies illustrate the dependence of mandibular morphology on the specifics of species, age, and especially masticatory mechanics.

Support or Funding Information

Supported by DE08513, DE14336, DE018142 from NIDCR.
Histological Evaluation of Olive Oil Effects on Skin of Chronically Stressed Mice
Authors: Romana‐Souza Bruna , Monte‐Alto‐Costa Andréa
Published: November 2023

Psychological stress‐induced oxidative damage may be an important factor that influences skin aging. However, neither study demonstrated if olive oil administration might inhibit the histological alterations induced by chronic stress in skin aging. The aim of this study was to investigate histologically the effects of olive oil administration on skin of chronical stressed mice. Mice were daily submitted to rotational stress, for 28 days, until euthanasia. Another group of animals did not submit to stress model. In the same period, animals were orally treated with olive oil or water. After 28 days, mice (5 animals per group) were killed and skin was analyzed. All experiments was approved by the Ethical Committee for Animal Use of State University of Rio de Janeiro (CEUA/044/2014). In mice skin, chronic stress reduced dermis and epidermis thickness, but it did not alter collagen fiber organization when compared to nonstressed mice after 28 days. The chronic stress also increased the number of proliferating and apoptotic cells in dermis when compared to nonstressed group after 28 days. The olive oil administration inhibited the effect of chronic stress on epidermis and dermis thickness and cell proliferation and apoptosis in dermis after 28 days. In addition, collagen fibers were thicker in the skin of the stressed mice treated with olive oil than that of stressed mice. In conclusion, olive administration may inhibit the histological alterations induced by chronic psychological stress on mice skin.

Support or Funding Information

The Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ) and the National Council of Research (CNPq)
Immunohistochemical and Cytoarchitectonic Studies of Testes in Triticum aestivum Linn (Poaceae) Lectin‐Administered Sprague‐Dawley Rats
Authors: Okoko Ini‐Ibehe Essien , Osiagwu Daniel , Mbagwu Smart , Duru Francis I. , Osinubi Abraham
Published: November 2023

This study investigated the reproductive sequelae of administration of common wheat lectin on the cytoarchitecture and immunohistochemically localized androgen receptors (AR) sites in prepubertal rat testes. Forty‐five male prepubertal rats weighing 50–60 g and approximately 30 days old were randomized into three groups represented by different time intervals of two, five and ten weeks, respectively. Each study time frame consisted of three sub‐groups of five rats each. Each sub‐group consisted of control which received 10 mL/kg BW/day of distilled water, reference standard group which received doses of 250 mL/kg BW of pure common wheat lectin while the third group received equal dose of common wheat lectin fraction. All administrations were given daily by gavage via oral cannula. At the end of each study time frame, the rats were sacrificed. The testes were excised and processed for immunohistochemical and cytoarchitectonic studies. Cytoarchitectural assessment of the testes of common wheat lectin exposed rats was characterized by varying degrees of tubular degenerations including germ cells depletion, segmental partial germ cell loss, exfoliation of germ cells into the lumen, appearance of residual bodies in the lumen as well as degeneration and distortion of interstitial (Leydig) cells. These changes were more significant in prepubertal rats administered with a dose of 250 mg/kg BW/day of pure common wheat lectin for 10 weeks. Common Wheat Lectin gradually decreased the androgen receptor expression in the Leydig, Sertoli and peritubular myoid cells, which could induce male factor infertility in rats. The results of this study indicated that lectins from Triticum aestivum Linn. disrupted reproductive parameters in prepubertal male rats time dependently through alteration of the testicular cytoarchitecture, markedly and gradually decreased the androgen receptor expression in the Leydig, Sertoli and peritubular myoid cells, which could induce male factor infertility in rats.
THE ANATOMY OF PEDIATRIC LAPAROSCOPIC AND THORACOSCOPIC PROCEDURES ‐ NEWER CONCEPTS AND PROCEDURES
Authors: GHOSH TAMOJIT
Published: November 2023

Laparoscopic pediatric procedures have revolutionized the treatment of tracheo‐oesophageal fistulas with oesophageal atresia, duodenal atresia, anorectal malformations, appendicitis, hirschsrpung disease and inguinal hernias. They can be grossly divided into abdominal and thoracic procedures and a basic knowledge of body wall and surface anatomy is required.

Abdominal laparoscopy requires gas insufflation, the use of surface ports in various sites and and a keen understanding of upper and lower abdominal anatomy. Iatrogenic abdominal injuries include vascular trauma, retroperitoneal heamorrhage, bladder injuries and stomach injuries especially when it is over inflated. Most procedures use a camera port with 2 or more ports for instrumentation. Lower abdominal laparoscopies have a risk of hernias, damage to the inferior epigastric blood vessels and bowel damage. A detailed knowledge of the abdominal body wall musculature and underlying vascular structures is required. The anatomy reinforcing single port laparoscopy procedures is presented in this work too. Transuterine and cul‐de‐sac approaches are revisited and explained.

Thoracospic pediatric surgical procedures have a risk of injury to the lung, pleura and thoracic wall vasculature. Newer anatomical approaches through the posterior thoracic wall ports are also proposed and explained.

To correct spinal and vertebral defects newer sites of laparoscopic port entry are proposed with an eye to access and maximum utilization of small working space in such procedures.
The gross‐anatomical plates of the DU LAURENS book, 1627 “Historia anatomica humani corporis”
Authors: LE FLOCH‐PRIGENT Patrice Pierre , FROMANGER Nadine , GILLOT Jean‐Bernard
Published: November 2023

The 478 pages of the in folio, gross‐anatomy book of Du Laurens contained 27 copper‐engraved plates with 1 to 13 figures in each one. We studied the sample (Francfort, Bekker, 1627) of the National Academy of Medicine in Paris (D684) and compared it to two other samples (one = 1600) of the Alain Brieux Scientific Library, Paris 6th. The drawings were very similar between the three editions, and were inspired by Vesalius (1543) and also by Valverde. However they had a good originality by their own and the knowledge had advanced at the time of du Laurens (1558–1609), an ordinary physician and counselor of Henri IV of France who was initially Professor of Medicine in Montpellier.
HIND LIMB PERIMETRY AND MUSCLE WEIGHT IN HIGH INTENSITY INTERVAL TRAINED RATS
Authors: Fazan Frederico Sassoli , Almeida Lucas Ferreira , Francescato Heloisa Della Colleta , Giorgetto Carolina , Fazan Valéria Paula Sassoli
Published: November 2023

Interval‐training can be divided into high‐intensity interval training (HIIT – near maximal efforts) and sprint interval training (supramaximal efforts) [1]. Physiological adaptations to interval‐training are well known, such as increased aerobic capacity (VO2max). Nevertheless, skeletal muscle adaptations, that is clearly dependent on excercise intensity, still need investigation, particularly on HIIT protocols. Male Wistar rats (N=17), weighing 150–200g, were submitted to a HIIT that consisted in a nine week time and intensity incremental protocol starting at 50% Vmax during 20 minutes and ending at 70% Vmax during 60 minutes (Vmax being the maximum velocity, in meters per minute that the animals could individually reach). At the end of the nine‐week protocol, animals were weighed and anesthetized with Sodium Pentobarbital (50mg/kg) and hind limb perimetry was performed below the knee, at mid‐leg level, using a stainless steel Vernier Caliper (300mm – 0.02mm accuracy) and a flexible and inextensible thin cord. Rats were them perfused with buffered saline (PBS 150mM) and had their right and left triceps sural carefully dissected, removed in one piece and weighed using a digital scale Mettler Toledo™ (320g capacity – 0.1 mg readability). Average final body weight was 565 ± 12g. Average leg perimetry was 6.91 ± 0.14cm on the right and 6.81 ± 0.12cm on the left sides with no differences between sides. Average triceps sural weight was 3.61 ± 0.12g on the right and 3.72 ± 0.12 on the left sides, also with no differences between sides. There was no correlation between final body weight and right or left legs perimetry. Also, no correlation was found between final body weight and right or left triceps sural weight. Finally, no correlation was present between leg perimetry and triceps sural weight for both sides. Thus, triceps sural weight was normalized by body weight and the correlation with leg perimetry was again investigated. Still no correlation was found either for right or left sides between the normalized muscle weight and leg perimetry. Despite the absence of positive correlations in the parameters investigated in this study, further histological evaluation of these muscles will be performed since there is still little evidence of the role of exercise intensity in mediating skeletal muscle alterations such as capillary density and mitochondrial content.

Support or Funding Information

FAPESP, CNPq, CAPES and FAEPA
Exploration and Description of Accessory Foot Evertor Muscles
Authors: Greiner Thomas M
Published: November 2023

Anatomical texts commonly describe three muscles as foot evertors: fibularis longus, fibularis brevis and fibularis tertius. During dissection accessory muscles are frequently found that would also serve to evert or invert the foot. A survey of the legs and feet from 110 cadavers shows that accessory foot invertors are rare – only one example was found. However, accessory foot evertors appear with some regularity – in approximately 25% of subjects.

Six different versions of an accessory evertor were identified in the leg, two from the anterior compartment and four from the lateral compartment. About half of the identified muscles occurred bilaterally. Most of these muscles attached to the lateral calcaneus, although some attached to the base of the fifth metatarsal. Many of these muscles are small and would contribute little to the forceful eversion of the foot. Therefore they are probably best regarded as proprioceptive organs. However, some variants are comparable in size to the other deep leg muscles and probably provide a significant evertor contribution. Fibularis longus and brevis were found in all subjects, while 7% of the subjects lacked a fibularis tertius. Only one example was found where the “accessory” evertor was present when the fibularis tertius was absent. This suggests that these “accessory” muscles are indeed accessory, and not functional replacements for the more standard fibularis muscles.

The biological significance of these muscles is unclear. The frequency of their appearance is higher than several other anatomical variants that are consistently mentioned in anatomy textbooks. By this standard alone, it would seem that these muscles should be part of a standard presentation of human anatomy. A literature review was unsuccessful in finding reports of these accessory evertors among non‐humans. Therefore, it is unclear if these muscles represent muscular spandrels, atavistic traits, or attempts at muscular innovations that are perhaps associated with an improved adaptation to bipedal locomotion.
Morphometric Analysis of Commonly Grafted Costal Cartilages: Considerations for Reconstructive Surgery
Authors: Ganoe Maria R , Lynch Hannah L , Bender‐Heine Adam N , Holmes J Scott , Rickards Allen A , Armeni Mark A , Lambert H Wayne , Zdilla Matthew J
Published: November 2023

Over the past seventy‐five years, autologous costal cartilage has been used for diverse surgical reconstructions including reconstruction of the ear, nose, and midface. Despite the wide utilization of costal cartilage grafts, there is little agreement regarding which cartilage level and which particular portion of each costal cartilage is the most optimal for grafts of varying sizes. Furthermore, there have been few studies dedicated to understanding the size of costal cartilage. Therefore, the aim of this cadaveric study was to provide salient measurements that will aid in the selection of optimal costal cartilage harvests. The study analyzed the left and right sides of the most commonly utilized costal cartilages (the 5th, 6th, and 7th costal cartilages) from sixteen cadaveric rib cages. The data presented in this study provides a starting‐point for the development of an algorithm that will enable selection of optimal cartilage for facial reconstructive surgery based upon the desired graft size.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.14.13]

WVU Initiation to Research Opportunities (INTRO) Summer Research Program
Distribution of CGRP and bone remodeling markers in the mandible of senescence accelerated mouse
Authors: Miwa Yoko , Sunohara Masataka , Sato Iwao
Published: November 2023

Objectives

Calcitonin gene related peptide (CGRP) has been proposed to contribute the transmission of pain in peripheral and central neurons. Mandibular condyle of SAMP8 mice is early‐onset age‐related degenerative changes in TMJ's articular surface (Chen et al., 1989). Osteopontin effect the anchoring osteoclasts in bone remodeling regions. Osteocalcin is also a marker for the bone remodeling produced by osteoblasts. Our study is to identify the expression change of mRNA in CGRP, Osteopontin and Osteocalcin for senescence accelerated mouse model.

Methods

The senescence‐accelerated SAMP8 mouse (male, 12 weeks of age n=4, 24 weeks of age n=4) and SAMR1 (male, 12 weeks of age n=4, 24 weeks of age n=4) were purchased for this study. All sampled tissues were flash frozen in liquid nitrogen and put in‐80 degree freezer for RNA extraction.

Results

Quantitative Real time PCR was performed using an Applied Biosystems 7300 Fast Real Time PCR Systems for the detection of CGRP, Osteocalcin and Calcitonin. Our data gave an information for mandible bone and cartilage degeneration accompanied by CGRP expression levels between 12 weeks and 24 weeks. The statistical significance of differences in the percentages of positive cells was analyzed by two‐tailed, unpaired Student's t test (p<0.01).

Conclusion

These different expression of each marker also may indicate pain mechanism related to CGRP in mandibular bone.

Support or Funding Information

none
Femoral Fracture Pattern and Its Role in Determining Direction of Impact in a Pedestrian Case
Authors: Porta David J. , Kress Tyler A.
Published: November 2023

There is a long history of anatomical and biomechanical research on human cadaver long bones for the worthy goal of determining tolerance to impact. Such data can assist design engineers with the enhancement of safety features in motor vehicles, sporting equipment, etc. In addition to collecting valuable force data, details learned about anatomical fracture patterns can benefit investigators who reconstruct mechanisms of injury in forensic cases. For example, fracture pattern can be used to distinguish torsional versus bending type injuries. After hundreds of long bone impact experiments, and detailed observations on the resulting fracture patterns, it is clear that when an impact occurs to the diaphysis of a long bone, the fracture initiates on the opposite side. While the impact compresses the tissue on the contact side, the shaft of the bone is placed in tension on the opposite side. The result is fracture lines that radiate from the side in tension back toward the contact side which is in compression. When one or more of these tension lines travels the entire diameter of the bone, a fragment or fragments are formed and one can note a pattern‐ e.g. oblique, transverse, wedge, segment, etc. In addition to the basic fracture pattern, observation of the incomplete fracture/tension lines (which may not be visible on standard X‐ray) can sometimes be valuable. We present a case in which a pediatric pedestrian was struck and killed by a car and there were no witnesses to the event. Femoral fragments were retained from the autopsy. The question of direction of impact became important as an attempt was made to determine if the boy had run into traffic or if he was struck while walking parallel to the lane of travel. Tension lines on the femur indicated the boy was struck from the lateral aspect. In conclusion, the study of nearly microscopic fracture anatomy can be a useful endeavor when evidence of direction of impact might enhance injury reconstruction.
Histological identification of cells directly participating calcified tissue resorption and destruction
Authors: Nakamura Masanori , Otsuka Hirotada
Published: November 2023

Introduction

It is well established that multinucleated osteoclasts are the main cells to resorb calcified tissues. However, it still remains the possibility that the mononuclear cells other than osteoclasts directly resorb or destruct calcified tissues. In this study, we examined the ontogenic development of long bones in vivo and in vitro and the process of the bone destruction during the experimentally induced mouse arthritis model to detect the calcified tissue destruction by mononuclear cells.

Materials and Methods

Humeral bones from E16 Balb/c mice were dissected and processed for transmission electron microscopy (TEM). To inhibit resorption by osteoclasts, pregnant mice were treated with a daily administration of nitrogen‐containing bisphosphonate (alendronate) from E15–E17 before the formation of fetal humeral bone marrow cavities. Metatarsals at E16 were organ cultured for 7 days and processed for TEM. Ten‐weeks old male DBA/1 mice were treated with type II collagen to induced collagen‐induced arthritis. Alendronate was administered once a week before the onset of arthritis to inhibit osteoclastic bone resorption. At the indicated time, knee joints were dissected and processed for the histological and immunohistochemical analyses.

Results

During the development of the humeral primary bone marrow cavity formation, the density of type II collagen fibers in the cartilage decreased with the progress of the calcification. Finally, the calcified cartilage directly became fragmented and macrophages invading from the perichondrium phagocytosed the fragmented particles. The number of primary bone trabecula of new born mice increased with alendronate treatment. However, bone marrow was formed in humeral bones of alendronate –treated newborn mice where active hematopoiesis was observed. In the organ culture of mouse E15 metatarsals, the primary bone marrow was also formed where fibroblasts phagocytosed calcified cartilage particles. In the mouse collagen–induced arthritis model, alendronate induced more severe inflammation than the control, which leaded to the severe articular cartilage and bone destruction. Many osteoclasts actively resorbed bone in the control group whereas many neutrophils were accumulated on the bone surface in the alendronate‐treated group. Ultrastructural observation clearly showed the accumulation and rupture of neutrophils on the bone surface. No collagen fibers were detected in the bone matrix.

Discussion

Bone homeostasis is maintained by the functional coupling between osteoblasts and osteoclasts. It has been accepted that the aberration of these two cells lead to bone diseases. In this study, we strongly indicated the direct resorption and destruction of calcified tissues by mononuclear cells other than osteoclasts. The results may change the concept for bone diseases and help the way to the understanding and the development of new therapies for the disease mainly involving bone destruction.
Unravelling the Role of the Vasculature and Extracellular Matrix in Intramembranous Bone Development
Authors: Franz‐Odendaal Tamara Anne , Hammer Christine
Published: November 2023

The relationship between bone development and the developing vasculature is well understood for endochondral bones but not for intramembranous bones. Intramembranous bones develop without a cartilage precursor from an aggregation of preosteogenic cells. Here we study, a series of intramembranous bones, known as the scleral ossicles, in order to unravel these relationships. The scleral ossicles are induced by specialised placodes of the conjunctival epithelium. We injected hydrocortisone into the developing embryo and observed the effect on the scleral bones and on the epithelial placodes. We show that there is a greater effect on the scleral bones than on the placodes, the structures that induce them. To further explore this effect, we next analysed the effect of the treatment on the developing scleral vasculature and scleral extracellular matrix. We show that both are affected indicating that vasculature and extracellular matrix within the sclera make a critical contribution to intramembranous bone development. This research provides important insights into understanding the role of the scleral mesenchyme in supporting scleral ossicle development and contributes to our understanding of sclerotic ring loss over vertebrate evolution.

Support or Funding Information

Natural Sciences and Engineering Research Council, Canada
Factors Associated with Developmental Instability in the Dental Arches of Children
Authors: Miller Steven F. , Moreno Uribe Lina M. , Bay Camden , Broffitt Barbara , Levy Steven M.
Published: November 2023

Dental arch asymmetry is often associated with malocclusion and is implicated in numerous functional deficiencies including speaking and mastication. While the existence of asymmetric dental arch shape has been well documented in malocclusion, less is known about the potential factors tied to its expression. Developmental instability refers to the suite of epigenetic factors that can disrupt the normal growth pattern of an individual and cause an increase in asymmetry. In this study, we examine the relationship of numerous health and dietary factors with developmental instability in the dental arches of children.

A sample of 406 dental casts of children at age 5 from the Iowa Fluoride Study were digitized with 22 landmarks along the gingival papillae and then submitted to a Procrustes ANOVA to quantify dental arch Fluctuating Asymmetry (FA). Using FA as a metric of developmental instability, we separated individuals into low and high FA groups and then ran a series of two sample T‐Tests to see if individuals with high FA had poorer dietary or health related factors when compared to individuals with low FA.

Results indicate that antibiotic usage was higher on average in children in the high FA group when compared to children with low dental arch FA (p=0.037), indicating that higher instances of childhood infection and disease may result in more asymmetric dental arches. Likewise, children with lower Vitamin D consumption had increased FA compared to children with higher levels of Vitamin D in their diet (p=0.039).

These results confirm that infectious disease and diet can play a role in the normal development of the dental arches. Deficits in important nutrients, like Vitamin D, as well as elevated exposure to infectious disease, can be tied to increased dental arch asymmetry. These factors should be considered not only for the general health of a growing child, but also with regard to their proper masticatory development.

Support or Funding Information

Supported by a Carver Charitable Trust Grant #12‐4058 and the NIH through Grants 2 UL1 TR000442, 1T90DE023520, R01‐DE09551 and U01‐DE018903.
Contrast Enhanced Imaging as an Alternative to Dissection in Tree Shrew (Tupaiidae: Scandentia) Chewing Musculature
Authors: Kristjanson Heather L , Perry Jonathan MG
Published: November 2023

A burgeoning technique in the field of comparative biology involves staining specimens with iodine, followed by CT scanning. Along with providing excellent 3D visualization of soft tissue by giving it a higher attenuation, iodine staining enables digital isolation of muscles and the potential for measuring individual muscle fascicles. It is well suited for visualizing muscles of mastication, especially the medial and lateral pterygoid muscles; located internally, they are often difficult to dissect in small mammals. Here, we test the reliability of this method as an alternative to traditional dissection for obtaining accurate volume and muscle fascicle length measurements. This was accomplished by digitally isolating the masticatory muscles in three tree shrew cadaver specimens that had been subjected to iodine staining. These specimens represent the largest (Tupaia tana), smallest (T. minor), and average (T. belangeri) sizes of tree shrew. All specimens were frozen from death. After fixing with 10% buffered formalin, specimens were stained in a 3% solution of I2KI (T. tana; T. minor) or 5% solution of I2KI (T. belangeri) for 14 days. The heads were microCT scanned (at resolutions of 44.5 μm, 26.5 μm, and 32.2 μm respectively), enabling their muscles to be digitally isolated. Fascicles were visible only in T. minor due to a higher resolution, and were not measurable in any of the specimens. Larger bundles of fascicles contained within connective tissue compartments were identified and used to delineate separate muscles layers (eg. superficial vs deep masseter). These results indicate that while muscle fascicles are not measureable using these scans, they may be measurable if resolution could be increased. Isolating the muscles digitally provided more accurate measures of volume as compared to traditional dissection, while also preserving their orientation. Future work will focus on using these data to digitally map tree shrew muscles of mastication onto fossil primate mandibles as an analogue for studying early primate chewing systems.

Support or Funding Information

This work was supported through an American Association of Anatomists Short‐term Visiting Scholarship to H. Kristjanson
Tracing Cell Movement in the Bladder During Regeneration After Subtotal Cystectomy
Authors: Dettman Robert W , Devine Megan , Delos Santos Grace B , Wetterlin Jessica J , Kukulka Natalie A , Firmiss Paula R , Bowen Diana K , Gong Edward M
Published: November 2023

Introduction and Objectives

It has been shown that the bladder has significant regenerative capabilities. In accordance with previous studies, there are differences between these regenerated organs and their original, healthy counterparts. The objective of this study was to determine the bladder regeneration pattern in mice with bladder resections.

Methods

50% of the bladder was removed in a subtotal cystectomy (STC), performed on 6–10 week female mice. DiI, a red dye, was applied to the suture site to mark the incision location. EdU was injected into STC and sham surgery mice 3 days post‐op. 4 days after injecting the EdU, Thymidine was injected to chase the remaining EdU from the mice. Bladders were fixed in blocks, cryosectioned and stained for analysis at 1, 4, and 8‐week time points. Images were taken on the Zeiss 880 Confocal microscope at 10× objective and tiled together. Using the tiled images, EdU pixel density was analyzed in Adobe Photoshop CS5. The bladders were divided into four cross‐sections: the suture site, both areas adjacent to the suture site, and bladder neck. Within each cross‐section, EdU labeled cells (yellow pixels) were counted and divided by each section's total number of cells (DAPI pixels) to determine the density of EdU per region.

Results

It was observed that the sham bladder had EdU evenly distributed throughout the bladder. STC bladders originally had most of the EdU in the suture site, however by 4 weeks post‐op, there was a decrease in EdU at the suture site and an increase in the middle and neck areas. Overall EdU decreases over time as it gets metabolized.

Conclusions

We conclude that cell division is a response to STC. Within the 8wk study, labeled cells are migrating with a net movement away from the incision towards the bladder neck.

Support or Funding Information

Research Support Source: The Hartwell Foundation to EM Gong
Can androgens have significant effects on mesenchymal stem cells?
Authors: Firmiss Paula R , Bowen Diana K , Kukulka Natalie A , Gong Edward M , Dettman Robert
Published: November 2023

Introduction and Objectives

We have identified a mesenchymal stem cell (MSC) population in the urinary bladder. In a model of fibrosis secondary to partial bladder outlet obstruction (PO) we observed that fibrosis was not as severe in female mice as compared with male mice. We hypothesized that male androgen may function in the bladder in response to obstruction.

Methods

8–10wk male CD‐1 mice were surgically obstructed at the bladder neck to create a partial obstruction model (PO), with and without surgical castration (CPO). Fluorescence‐activated cell sorting (FACS) was utilized to isolate MSCs (Sca‐1+/CD34+/lin− (PECAM−, CD45−, Ter119−)) in the sham, PO, and CPO bladders after 7 days of obstruction. BSCs were cultured for 24 hours and expression of androgen receptor (AR) and NKX3.1 on the BSCs was determined with immunofluorescence. Quantitative PCR was performed to determine levels of gene expression. Testosterone was replaced in castrated mice with the administration of testosterone or dihydrotestosterone.

Results

Bladder function was decreased and fibrosis increased after PO. Function was normal and fibrosis reduced in CPO. Administration of testosterone or dihydrotestosterone to PO mice resulted in reduced function and increased fibrosis. The bladder MSC population was significantly decreased by PO. Castration restored this population as determined by FACS (Figure 1C). MSCs were found to express AR and twice as many sorted MSCs expressed AR (80%) by immunofluorescence after obstruction when compared to sham (40%) (Figure 1A). Microarray analysis identified the transcription factor NKX3.1 as an androgen response gene increased after PO. This was validated by qPCR and immunofluorescence staining of MSCs (Figure 1B).

Conclusions

Our data suggest that testosterone directly effects bladder resident MSCs through AR and the AR target gene NKX3.1. NKX3.1 is a transcription factor that was previously thought to be expressed exclusively in the prostate gland. In fact, NKX3.1 is proposed to be expressed in and perform important functions in epithelial progenitor cells in the prostate. Activation of NKX3.1 in MSCs after obstruction may identify a maladaptive mechanism occurring in these cells that leads to fibrosis. We are currently studying a knockout model of NKX3.1 to test these ideas.

Support or Funding Information

Source of Funding: The Hartwell Foundation award to EM Gong
Mesodermal differentiation of human pluripotent stem cells increases Mitofusin‐2 mediated Mitophagy
Authors: Krantz Sarah , Wang Li , Toth Peter , Marsboom Glenn , Rehman Jalees
Published: November 2023

Pluripotent stem cells rely on glycolysis for their energy needs and shift their metabolism towards mitochondrial oxidative phosphorylation during differentiation in order to adapt to the metabolic needs of mature differentiated cells. Oxidative phosphorylation generates reactive oxygen species and can thus lead to collateral protein oxidation and damage. Excessive mitochondrial damage leads to mitochondrial autophagy or mitophagy during which mitochondria are marked for degradation. A key regulator of mitophagy is the outer mitochondrial membrane protein Mitofusin‐2 (Mfn2) which either directs mitochondria towards fusion with other mitochondria to optimize mitochondrial function or towards mitophagy. We hypothesized that differentiation of pluripotent stem cells would necessitate increased mitophagy to contain the oxidative damage due to enhanced oxidative phosphorylation.

Human induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) were treated with Bone Morphogenetic Protein 4 (BMP4) to induce differentiation to the mesodermal lineage. These cells exhibited an expected increase in the mesodermal transcription factor Brachyury. Cells were stained with MitoTracker to visualize mitochondria and assess mitochondrial network formation. We observed an increase in mitochondrial fusion during mesodermal differentiation. There was also a two‐fold increase in Mitofusin 2 protein levels with BMP4 treatment. The ratiometric fluorescent sensor mitoKeima which detects the pH of mitochondria and assesses mitophagy indicated significantly higher basal mitophagy after three days of BMP4 induced differentiation with an increase in the relative mitophagy index from 2.7 to 9.7 (p<0.01) within three days.

These findings suggest that mitophagy and mitochondrial networking are critical Mitofusin‐2 mediated adaptive processes required for the metabolic reprogramming of differentiating stem cells.

Support or Funding Information

NIH T32 Training Grant