Nervous System Sampling Practices, p. 1
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STP Position Paper:

5

Recommended Practices for Sampling, Processing and Analysis of the6

Peripheral Nervous System (Nerves, Somatic and Autonomic Ganglia)

7

during Nonclinical Toxicity Studies1

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Brad Bolon (chair), Georg Krinke, Mark T. Butt, Deepa B. Rao, Ingrid D. Pardo,

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Bernard S. Jortner, Karl Jensen, Robert H. Garman, Lydia Andrews-Jones, James P.

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Morrison, Alok K. Sharma, Michael S. Thibodeau (co-chair)

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GEMpath, Inc., Longmont, CO, USA (BB); AnaPath GmbH, Oberbuchsiten, Switzerland

23(GK); Tox Path Specialists, LLC, Frederick, MD, USA (MB); U.S. Food and Drug

24

Administration, Center for Drug Evaluation and Research, Silver Spring, MD, USA

25(DBR); Pfizer, Groton, CT, USA (IDP); Virginia Polytechnic Institute and State

26

University, Blacksburg, VA (BSJ); U.S. Environmental Protection Agency, Research

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Triangle Park, NC, USA (KJ); Consultants in Veterinary Pathology, Inc., Murrysville, PA,

28

USA (RHG); Allergan, Inc., Irvine, CA (LAJ); Charles River Laboratories, Inc,

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Shrewsbury, MA, USA (JM); Covance Laboratories, Inc., Madison, WI, USA (AKS);

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Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA (MT)

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STP Working Group on Peripheral Nervous System Sampling:

Running title: PNS Sampling / Processing in Nonclinical Toxicity Studies

Nervous System Sampling Practices, p. 2
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Address correspondence to: Dr. Brad Bolon, GEMpath, Inc., 1100 East 17th Avenue, Unit

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M202, Longmont, CO 80504-3715, United States. Phone: (720) 684-6540; Email:42

bradgempath@aol.com

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1

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Protection Agency (U.S. EPA) and U.S. Food and Drug Administration (U.S. FDA) and48

approved for publication. The opinions expressed in this paper solely represent those of the49

authors and should not be construed as representing the official views or policies of the U.S.

50

EPA or U.S. FDA, nor should mention of trade names or commercial products constitute

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U.S. EPA or U.S. FDA endorsement or recommendation for use.

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This document has been reviewed in accordance with policies of the U.S. Environmental

Nervous System Sampling Practices, p. 3
55

Abstract(232 words)

56

These Society of Toxicologic Pathology “best” practice recommendations should ensure

57

consistent sampling, processing, and evaluation of the peripheral nervous system (PNS). For

58

toxicity studies where neurotoxicity is not anticipated (Situation 1), PNS evaluation may be

59

limited to one sensorimotor spinal nerve. If somatic PNS neurotoxicity is possible (Situation

602), analysis minimally should include three spinal nerves, cranial nerve V, and their sensory

61

ganglia. If autonomic PNS neuropathy is suspected (Situation 3), parasympathetic and62

sympathetic ganglia with associated autonomic nerves should be assessed. For dedicated63

neurotoxicity studies where neurotoxic activity is likely (Situation 4), PNS sampling follows

64

the strategy for Situations 2 and/or 3, as dictated by in-life data or other information for the65

compound/target. For all situations, bilateral sampling with unilateral processing is

66

recommended. For Situations 1, 2, and 3, PNS is processed conventionally (immersion in67

formalin, paraffin embedding, H&E staining). For Situation 4 (and if feasible Situations 2

68

and 3), perfusion fixation with methanol-free fixative (MFF) is recommended. Where PNS

69

neurotoxicity is possible, at least one (Situations 2 and 3) or two (Situation 4) nerve cross70

sections should be post-fixed with glutaraldehyde and osmium before hard plastic resin71

embedding; soft plastic embedding is not suitable. Special methods (axonal and myelin

72

stains, etc.) may be used to further characterize PNS findings. Initial PNS analysis should be73

informed, not masked (“blinded”). Institutions should explain the basis for their sampling,74

processing, and evaluation strategy.

75
76
77

Key Words: PNS, peripheral nervous system, neuropathology, neurotoxicity,

78

recommended practices, nerve, ganglia, autonomicNervous System Sampling Practices, p. 4
79Abbreviations

80

CNS

central nervous system

81

DRG

dorsal root ganglion

82

EPA

(U.S.) Environmental Protection Agency

83

FDA

(U.S.) Food and Drug Administration

84

GFAP

glial fibrillary acidic protein

85

GLP

Good Laboratory Practices

86

GMA

glycol methacrylate

87

H&E

hematoxylin and eosin

88

Iba1

ionized calcium-binding adaptor molecule 1

89

IENFD

intra-epidermal nerve fiber density

90

IHC

immunohistochemistry

91

MFF

methanol-free formaldehyde (or fixative)

92

MGG

medical-grade glutaraldehyde

93

MIE

molecular-initiating event

94

MMA

methyl methacrylate

95

MOA

mode of action

96

NBF

neutral buffered 10% formalin

97

NME

new molecular entity

98

NOAEL

no observed adverse effect level

99

OECD

Organisation of Economic Co-operation and Development

100

PNS

peripheral nervous system

101

PPD

paraphenylenediamine

102

QSAR

quantitative structure/activity relationship

103

RT

room temperature

104

SOP

standard operating procedure

105

STP

Society of Toxicologic Pathology

106

TEM

transmission electron microscopy

107

WOE

weight of evidence

108
109

Nervous System Sampling Practices, p. 5
110
111

I.

Background112

Neurological deficits due to toxicant-induced peripheral neuropathy are a recognized

113

consequence of accidental occupational or environmental exposures and some therapeutic114

treatments. Therefore, the neuropathology component of toxicity studies is a critical means

115

for identifying potential hazards and assessing risks posed to humans by contact with new116

biomolecular or chemical entities.

117

Different regulatory agencies offer independent guidance1 based on their distinct

118

mandates, variable scientific levels of concern, and diverse uses of the agents they oversee

119

regarding the specimens and procedures to be used in evaluating the integrity of the120

peripheral nervous system (PNS) when seeking to register new products (Bolon et al., 2011,

121

Salvo and Butt, 2011). The guidelines vary by the kind of industry (agrochemical vs.

122

chemical vs. pharmaceutical vs. biopharmaceutical), differences in potential exposure levels,123

and ages of the test subjects (e.g., developing animals (EPA, 1998b, OECD, 2007) vs. adults

124(EPA, 1998a, OECD, 1997)). Guidelines also differ based on the aim of the study (hazard

125

identification vs. safety assessment). For example, regulatory guidelines for performing the126

neuropathology analysis of Good Laboratory Practice (GLP)-type general toxicity studies

127(i.e., screening or “Tier I” surveys) are fairly general since such studies assess the PNS as

128

just one system among many organs and systems to be surveyed, while guidelines for GLP-

129

type dedicated neurotoxicity studies (i.e., advanced or “Tier II” studies) are fairly detailed

130

since assessment of the nervous system is the primary focus of the study (Bolon et al., 2011,

1

Guidance or guideline documents provided by regulatory agencies communicate current agency thinking on
topics governed by regulations. Guidances and guidelines represent legally unenforceable interpretations thatare designed to help institutions achieve compliance with legally enforceable regulations.

Nervous System Sampling Practices, p. 6
131

Salvo and Butt, 2011). However, substantial differences exist in the kinds of PNS toxicity

132

that might be encountered (Table 1), and current guidelines do not address variations in

133

approach that might be required to adequately investigate these divergent scenarios. Recent

134

compilations reviewing published regulatory guidance in this area (Bolon et al., 2011, Salvo

135

and Butt, 2011) and/or individual regulatory guidelines should be consulted because

136

guidance is reviewed and revised over time—as is presently occurring for the Toxic

137

Substances Control Act (administered by the U.S. Environmental Protection Agency [EPA])

138

and the “Redbook” guidance on food and color additives (overseen by the U.S. Food and139

Drug Administration [FDA]).

140

When sampling the PNS, considerable care must be given to selecting the appropriate

141

methodology (sampling scheme, fixatives, tissue orientation, embedding media, special

142

stains, etc.) to ensure that tissue morphology is optimally preserved. Basic PNS sampling143

and processing methods were promulgated recently by a Working Group of the Society of

144

Toxicologic Pathology (STP) tasked with establishing “best practice” recommendations for

145

sampling and processing the central nervous system (CNS) for nonclinical general toxicity

146

studies (Bolon et al., 2013b). Given the CNS focus, however, coverage of the PNS in this147

STP document was brief, and did not specifically include recommendations encompassing

148

different divisions of the PNS—somatic (sensorimotor) vs. autonomic (parasympathetic and149

sympathetic)—or effectors controlled by the PNS (e.g., glands, skeletal muscle, or viscera).

150

Accordingly, the STP established a new Working Group on PNS sampling, processing, and151

analysis to provide more specific recommendations appropriate to distinct varieties of152

neuropathies that might be encountered during the course of GLP-type toxicity studies.

Nervous System Sampling Practices, p. 7
153

The Working Group was given a charter with multiple specific aims. The first charge154

was to recommend what PNS structures should be regularly sampled during GLP-type

155

toxicity studies (“Tier I” and “Tier II”) performed in common vertebrate test species. The

156

second charge was to suggest tissue processing procedures and trimming schemes to157

facilitate analysis of these regions. The third charge was to define what routine stains and158

special neurohistology procedures, if any, should be used routinely in PNS evaluations. The

159

fourth charge was to consider when other special morphological techniques should be

160

undertaken to provide a more complete assessment of PNS lesions. The fifth charge was to161

define appropriate means for assessing whether or not PNS recovery has taken place. The

162

sixth charge was to propose what format should be used to most efficiently document163

histopathologic evaluation of PNS tissues in reports destined for review by regulatory bodies.

164

The recommendations given below with respect to particular neural structures to collect165(Table 2) and suggested sampling and processing procedures (Table 3), as well as the means

166

for documenting that they have been assessed, are based on the collective experiences and167

opinions of the Working Group members2 as well as selected input from the global

168

toxicologic pathology community3 received during a __-day-long public comment period in

169

the ____ quarter of 2017. Where consensus among Working Group members and/or STP

2

The Working Group consisted of 12 individuals with formal academic and/or industrial training in some
aspect of neuroscience and between 13 to 49 years of experience acquiring and analyzing neuropathology
data sets for nonclinical general (“Tier I”) toxicity studies and/or dedicated neurotoxicity (“Tier II”) tests
while working in contract research organizations; government agencies (research laboratories or regulatory
bodies); industrial firms (biotechnological, chemical, or pharmaceutical companies); universities; and/or
private consulting practices.

3

The draft recommendations devised by the Working Group received several levels of internal review by
STP committees before being circulated for comment to the entire STP membership. The final draft also
was sent to multiple other societies of toxicologic pathology representing nations in Asia, Europe, andLatin America to obtain international feedback on the proposal. At the time of publication, these practices
have been endorsed by the STP, _____.

Nervous System Sampling Practices, p. 8
170

members was lacking on certain points, several options have been included and discussed171with respect to their potential advantages and disadvantages.

172
173

II. Situation-specific Recommendations for Sampling, Processing, and Analysis of174

the PNS during Toxicity Studies

175
176

Basic Philosophy

177

The Working Group concluded that a rigid “one-size-fits-all” approach to sampling,178

processing, and evaluating PNS tissues is inappropriate due to the variety of situations,179

modes of action (MOAs), molecular-initiating events (MIE), and potential target sites that180

might be encountered. Instead, the Working Group is of the unanimous opinion that the181

appropriate and achievable objective is to delineate a strategy for evaluating key PNS

182

structures to differentiate common classes of neurotoxic lesions, but let the experiences and183

needs of individual institutions drive selection of the specific battery of sampling, processing,184

and analytical methods undertaken to provide a suitable survey of the PNS. The rationale for185

such decisions should be articulated clearly in the study report. Such institutional decisions

186

should be made using a “weight of evidence” (WOE) approach, where expanded sampling187

and evaluation of the PNS is considered only when evidence of PNS neurotoxicity is

188

substantial enough to be an important factor in the final risk assessment. In general, such

189

WOE decisions incorporate such factors as the degree of PNS neurotoxicity vs. toxicity to

190other target systems (i.e., how sensitive is the PNS to the test item4 relative to other systems)

4

Or test article

Nervous System Sampling Practices, p. 9
191

for non-target species, including humans, as well as the extent of PNS neurotoxicity that192

develops at relevant levels of exposure.

193
194

Scenarios for PNS Neurotoxicity

195

Four general situations during which PNS tissues may be sampled in the course of196

toxicity studies were considered (Table 1). Each utilizes a slightly different sampling197

strategy, based on the different locations in which the PNS is affected. The first three198

situations involve general (“Tier I” or “screening”) toxicity studies, while the last scenario

199

relates to dedicated neurotoxicity (“Tier II” or “advanced”) evaluations.

200

Situation 1 is a general toxicity study in which (1) no potential for PNS neurotoxicity was

201

detected in data obtained during prior studies (in vivo, in vitro, and/or in silico) and (2) no in-

202

life behavioral or neurological deficits are seen in the current study. This strategy represents

203

a rational default approach when analyzing new molecular entities (NME) for which no or204

few prior in vivo toxicity studies have been done. Situation 2 is a general toxicity study in

205

which in-life signs of peripheral neuropathy or other data reflect damage mainly to the206somatic (motor and/or sensory) nerves and/or their associated ganglia. Situation 3 represents

207

a general toxicity study in which in-life signs of peripheral neuropathy or other data suggest

208

injury to autonomic nerves and/or ganglia, which collectively regulate involuntary, visceral

209

homeostatic functions. For both Situations 2 and 3, other data that might trigger an expanded

210

PNS analysis include known or presumed MOA and quantitative structure–activity

211

relationships (QSAR) models for the test item, its metabolites, and/or related compounds or212

molecules. Situation 4 is the dedicated neurotoxicity study, which usually is required for test

213

items in which human epidemiological data, experimental findings from animal studies (inNervous System Sampling Practices, p. 10
214vivo and/or in vitro), and/or MOA or QSAR similarities to known neurotoxic agents indicates215

a high probability that PNS neurotoxicity may occur under likely exposure scenarios. Some

216

agents may simultaneously impact the somatic and autonomic PNS, and thus may require

217

increased sampling (combining Situations 2 and 3) and evaluation to fully assess both arms

218of the PNS.

219

A side-by-side comparison of PNS specimens to collect as well as baseline tissue

220

sampling and processing recommendations for the four situations are given in Table 2 and221

Table 3, respectively. The Working Group recommends that this information be used to222

define one or more institutional standard operating procedures (SOPs) that describe the223

collection and processing practices for PNS tissues. These documents should be detailed but

224

sufficiently flexible so that the study director and study team may adjust the PNS practices as

225

needed to meet the recommendations for all four situations.

226
227

Best Practice Recommendations for All Four Situations

228

The PNS sampling strategy should be guided by observed in-life neurological signs or229

other information for the compound/target. The choice of which PNS samples to collect and230

whether or not special histology processing and/or investigative techniques should be used

231

for a given toxicity study should be decided by the institution using a WOE approach. For all

232

situations, PNS structures (nerves, ganglia, and effector organs) typically should be collected

233

bilaterally but may be processed and evaluated unilaterally. Nerves and skeletal muscle (an234

effector organ) should be evaluated in both cross and longitudinal orientations. All PNS

235

specimens from the treatment groups selected for initial evaluation (e.g., high-dose andNervous System Sampling Practices, p. 11236

control animals) should be processed in the same time frame to avoid systematic variation in237

processing conditions.

238

Where plastic embedding is required by regulatory guidelines (EPA, 1998a), hard plastic

239

resin is the recommended medium. Soft plastic (e.g., glycol methacrylate [GMA] or methyl

240

methacrylate [MMA]) is not an acceptable substitute for hard plastic resin.

241

The recommended best practice for light microscopic evaluation is to undertake a tiered,242

semi-quantitative analysis with foreknowledge of the study design. A subsequent masked

243(“blinded” or “coded”) analysis of PNS tissues with findings of concern may be conducted at244

the discretion of the study pathologist (or peer review pathologist), but usually is done only245

to aid in defining the dose-response and/or establishing a no observed adverse effect level

246(NOAEL).

247
248

Best Practice Recommendations for Situation 1

249

For general toxicity studies with no specific concern for PNS neurotoxicity (Situation 1),

250

the majority of the Working Group concurs that one large, mixed (i.e., sensorimotor) somatic251

nerve, such as the sciatic nerve (or tibial nerve if the sciatic trunk has been traumatized), is a252

suitable baseline PNS survey. Additional peripheral nerves and dorsal root ganglia (DRG),

253

either in situ in vertebral column segments (rodents only) or isolated, should be collected at

254

necropsy but need not be assessed unless nerve or spinal cord lesions require additional

255

characterization. Standard processing—immersion fixation in conventional (i.e., methanol-Nervous System Sampling Practices, p. 12
256containing) neutral buffered 10% formalin (NBF), paraffin embedding, and hematoxylin and257

eosin (H&E) staining—usually is acceptable.5

258
259

Best Practice Recommendations for Situation 2

260

For general toxicity studies where somatic PNS neurotoxicity is a concern under likely

261

exposure scenarios (Situation 2), three spinal nerves—typically the sciatic nerve and two or

262

more of the following nerves (most of which are distal branches of the sciatic nerve): tibial,

263

fibular (i.e., common peroneal), plantar, saphenous, sural, or (in rodents) caudal nerves—as264

well as cranial nerve V (trigeminal nerve) should be evaluated. The sciatic, tibial and fibular

265

nerves in all species, and the sural and caudal nerves in rodents are mixed sensorimotor

266

structures; the saphenous, plantar, and (in nonhuman primate) sural nerves are sensory-only267

branches. Nerve selection generally should be based on in-life findings. At least four DRG

268(two each associated with the species-specific locations of the cervical and lumbar269

intumescences [Table 4], collected in situ or isolated); the associated dorsal and ventral

270

spinal nerve roots; and the trigeminal (Gasserian [cranial nerve V]) ganglion should be271

evaluated. Conventional processing conditions (immersion fixation in formalin, paraffin272

embedding, H&E staining) are suitable for PNS tissues, with three exceptions. First,

273

methanol-free formaldehyde (MFF6) or medical-grade glutaraldehyde (MGG, typically 2.5%)274

rather than NBF ideally should be employed to minimize processing artifacts. The Working

5This recommendation represents the majority view of Working Group members, with the understandingthat special post hoc processing (i.e., glutaraldehyde and osmium post-fixation, hard plastic embedding) ofat least one nerve cross section, as described for Situations 2, 3, and 4 where PNS neurotoxicity is possible,
may be helpful in further characterizing the PNS findings for Situation 1, especially the nature of changes
observed in myelin.
6
Methanol-free 4% formaldehyde is made from paraformaldehyde pellets or powder and thus often isreferred to in the scientific literature as “4% paraformaldehyde” (PFA) (Kiernan, 2000). MFF may bepurchased commercially or prepared in the laboratory shortly before use.

Nervous System Sampling Practices, p. 13
275

Group recognizes that this first adjustment may not be feasible on short notice, especially if276

the in-life PNS-related signs develop late in the course of a large study. Second, if nerve277

lesions are seen in H&E-stained sections, acquisition of serial sections for at least one mixed

278

nerve should be considered for special neurohistological staining to highlight axonal279

morphology (silver stain) and explore myelin integrity (myelin stain). Third, at least one

280

nerve cross section (usually a mixed-function distal trunk like the tibial or fibular nerve, or a281

mainly sensory branch like the sural or caudal nerve) should be post-fixed by immersion in

282

MGG followed by osmium (to stabilize myelin during the processing steps with lipid-283

solubilizing organic solvents), processed into hard plastic resin, and then stained with284

toluidine blue for light microscopic evaluation. The last two adjustments should be feasible

285

regardless of whether MMF or NBF is utilized.

286
287

Best Practice Recommendations for Situation 3

288

For general toxicity studies where autonomic PNS neurotoxicity is a concern at relevant289

levels of exposure (Situation 3), elements of the parasympathetic, sympathetic, and enteric7

290

PNS should be evaluated, including nerves (vagus and sympathetic chain) and multiple

291

autonomic ganglia. Common ganglia to assess include one post-ganglionic parasympathetic

292

site (i.e., those in the walls of protocol-specified hollow organs [commonly the heart and293

urinary bladder], but ideally at sites related to in-life findings); at least two sympathetic sites

294(e.g., cranial cervical, cervicothoracic, cranial mesenteric, and/or sympathetic chain ganglia);

295

and several enteric sites (i.e., submucosal [Meissner’s] and myenteric [Auerbach’s] ganglia).

7Enteric ganglia, which serve parasympathetic-like functions, form a neural net with independent reflex
activity and thus are considered by some investigators to be distinct from the autonomic nervous system(Furness, 2006).

Nervous System Sampling Practices, p. 14
296

In addition to autonomic PNS nerves and ganglia, somatic PNS nerves and ganglia should be297

collected as described in Situation 2. Conventional processing (immersion fixation in NBF

298

or ideally MFF, paraffin embedding, H&E staining) is suitable for most autonomic PNS

299

samples. Post-fixation with MGG and osmium followed by hard plastic embedding may be

300

useful despite the lower myelination of most autonomic nerves.

301
302

Best Practice Recommendations for Situation 4

303

For dedicated neurotoxicity studies where PNS neurotoxicity is likely or certain

304(Situation 4), expanded sampling includes at least three spinal nerves (sciatic, tibial, and305

fibular, saphenous, sural, plantar, or caudal); trigeminal (cranial n. V) nerve; DRG and their306

associated spinal nerve roots; and a trigeminal ganglion. At least six DRG should be307

examined (two or more DRG for each of the cervical, thoracic, and lumbar spinal cord308

divisions). In general, DRG should be removed from the vertebral column rather than309

processed and evaluated in situ to avoid soft tissue degradation associated with skeletal

310

decalcification, but in rodents in situ analysis following vertebral column decalcification is

311

acceptable. Fixation is undertaken by whole-body perfusion fixation with a methanol-free

312

fixative (typically MFF or mixtures of MFF and MGG). Paraffin embedding is suitable for313

most nerves and ganglia, although at least two distal nerve cross sections (typically the tibial

314

nerve and a more distal branch) should be post-fixed in MGG and osmium and then315

embedded in hard plastic resin. Paraffin-embedded nerves should be stained with H&E and,316

if warranted, axonal and myelin stains, while plastic-embedded nerves are stained with

317

toluidine blue. Ganglia usually are stained only with H&E, although silver and myelin stains

Nervous System Sampling Practices, p. 15
318

may be beneficial. Other special methods (see below) may be considered at the discretion of319

the institution to better characterize any neurotoxic lesions.

320

The Working Group recommendations for PNS sampling in Situations 1, 2, 3, and 4 are321

designed to be applicable to cases where test items have been delivered systemically (i.e.,322

where all PNS tissues are liable to some degree of test item exposure), and thus may need to be323

modified for selected scenarios and/or unusual test items. Decreased PNS evaluation may be

324

warranted if the pattern and severity of PNS lesions for the doses and/or the dosing regimen

325

used in a study have been well defined in one or more previous studies, although the Working

326Group recommends that all PNS tissues described in Situation 4 be collected and archived as327

wet tissue. Additional PNS samples (e.g., forelimb nerves) may have to be evaluated if clinical328

signs suggest that PNS damage has occurred at these sites. Local delivery of a minimally329

diffusible test item8 generally warrants increased collection and prioritized analysis of nerves

330

near the administration site, while more distal PNS elements may be collected but retained as

331

wet tissue. Such modifications in sampling and evaluation may be made at the discretion of the332

institution. The rationale for such adjustments should be given in the study report.

333
334

III. Rationale for Recommended PNS Sampling, Processing, and Analysis Practices

335

Regulatory guidelines are fairly generic with respect to prescribing the PNS sampling

336

strategy (Bolon et al., 2011, Salvo and Butt, 2011), so common sense is an essential attribute337

when selecting the PNS tissues to collect and evaluate. Selection of PNS sites to sample

338

depends on the situation (Table 2). Reasonable flexibility is possible in the choice of PNS

8

An example of this situation is onabotulinumtoxinA (BOTOX®), which disrupts the function of motor
nerve endings at the nerve/skeletal muscle interface at the site of injection, but not the structure of PNS
axons and ganglia elsewhere in the body.

Nervous System Sampling Practices, p. 16
339

tissues, depending on institutional preference. A “weight of evidence” (WOE) approach

340

should be employed in deciding whether or not expanded PNS evaluation will provide data341

relevant to the risk assessment. Situations in which PNS toxicity is judged to represent a

342

modest hazard relative to more substantial test item-related findings that are observed in

343

more sensitive systems and/or in which PNS toxicity at high dose will not be used to define344

the dose response and NOAEL may preclude the need for a substantial expansion, or permit

345

only a modest expansion, in PNS sampling and examination.

346
347

A. Situation-specific PNS Sampling Strategies

348

Basic Considerations

349

For screening in the absence of PNS neurotoxicity (Situation 1), evaluation of one large

350

mixed (sensorimotor) nerve is a suitable survey for PNS involvement. If PNS neurotoxicity is351

a concern (Situations 2, 3, and 4), PNS evaluation is expanded to include additional nerves

352

and ganglia, with the choice depending on the nature of the in-life signs. Therefore, study

353

protocols and institutional SOPs should facilitate collection of any PNS tissues that might be354

needed to explain the constellation of PNS-related clinical signs seen during the in-life

355

portion of the study.

356

Collection of PNS samples (nerves, ganglia, and effector organs) for all four situations

357

usually should be done bilaterally unless such an approach would impact another endpoint358(e.g., collection of unfixed tissue for biochemical or molecular analysis). The rationale for359

this recommendation is that bilateral sampling can be done quickly by skilled technicians,

360

and the retention of such specimens may permit additional characterization of unexpected361

findings without having to repeat the entire study; again, the choice of bilateral vs. unilateralNervous System Sampling Practices, p. 17362

PNS collection should remain with the institution. Sample acquisition should be undertaken

363

in a fashion that minimizes structural artifacts produced by manipulation, compression, and364

traction of incompletely fixed PNS tissue. The keys to curtailing artifacts are to limit

365

handling (pressure and stretching applied to neural tissues during sampling), to promptly

366

place tissues into properly prepared fixative and buffer solutions, and to maintain tissues at367

an appropriate temperature (generally room temperature [RT] for GLP-type toxicity studies)

368

until additional processing may be undertaken.

369

In general, PNS samples should be individually identified. Sample identity may be

370

assured by either placing each specimen in its own tissue cassette, applying it to a labeled371

index card (to which it will adhere due to the inherent stickiness of epineurial connective

372

tissue), or stapling it (through one end, not the middle) to an acetate strip prior to fixation to373

maintain it in an extended (but not “stretched”) orientation (Jortner, 2000). Stapling is the374least desirable method due to the likelihood for “crushing” the tissue. The orientation of the375

proximal and distal ends of nerves can be identified by labeling one end.

376
377

Situation 1

378

In general toxicity studies where no neurotoxic potential is expected (Situation 1), the379

minimal list of PNS tissues to be evaluated in all species is a readily accessible, large, spinal-

380

origin somatic nerve and the autonomic ganglia within the walls of major viscera. This PNS

381

sampling strategy is identical to that proposed in the STP best practices document for CNS

382

sampling in nonclinical general toxicity studies (Bolon et al., 2013b) and reflects the current

383

practice for general toxicity studies.

Nervous System Sampling Practices, p. 18384

Nerves. The usual PNS sample for Situation 1 is sciatic nerve. The rationale for selecting

385

this nerve is that it contains both sensory and motor nerve fibers, which permits analysis of386

major peripheral sensorimotor structures in a single sample. The sciatic nerve is exposed by387

reflecting and/or removing the overlying skeletal muscle (Figure 1). Sciatic nerve samples

388

commonly are acquired at a distal location (i.e., just proximal to where the tibial and fibular389

nerves branch, which occurs near the femorotibial joint). Sciatic nerve collection more390

proximally, typically mid-way between the vertebral column and knee, is a frequent391

alternative. Proximally collected sciatic nerve is populated by bigger Schwann cells covering

392

longer axonal lengths, and these large cells appear to be more sensitive to neurotoxic agents

393

than are distal Schwann cells (Friede and Bischhausen, 1982, Krinke, 2011). Therefore,394

damage to proximal Schwann cells may make myelin disruption easier to detect since395

damage to the larger cells tends to leave longer expanses of denuded axons. The choice of396

sciatic nerve site to be sampled (proximal vs. distal) is left to the discretion of the institution.

397

A sciatic nerve branch, typically the tibial nerve (another trunk carrying both sensory and398

motor nerve fibers), may be evaluated instead of the sciatic nerve if likely artifactual changes

399

might confound sciatic nerve analysis. A common scenario in which this substitution may be

400

warranted is in nonhuman primates that have received intramuscular injections of ketamine

401in the region where sciatic nerve is routinely collected. Chemical and mechanical trauma

402

associated with such injections has been shown to damage the nearby sciatic nerve trunk

403(Carrier and Donnelly, 2014).

404

While sciatic nerve (or tibial nerve) commonly is the only PNS structure evaluated for

405

Situation 1, additional spinal-origin somatic nerves may be collected at necropsy. Retaining406

other nerves in the archived wet tissues may prevent the need to repeat studies in the eventNervous System Sampling Practices, p. 19
407that changes observed in the sciatic nerve necessitate evaluation of other portions of the PNS.

408

A simple means for accomplishing this task in rodents is to retain an entire hind limb (after409

removing the skin) and the proximal tail. In non-rodent species, the distal nerve trunks

410

should be removed at necropsy. Other nerves to consider for collection are listed below

411(under Situations 2-4, and in Table 2). The choice of which additional nerves to harvest, or

412

whether more PNS tissue should be sampled at all, should remain the decision of the413

institution.

414

Ganglia. A majority of Working Group members, with some dissent, recommend that415

DRG need not be evaluated routinely for Situation 1. The Working Group does endorse

416

collection and archiving of at least one DRG location associated with the origin of the sciatic

417

nerve against the possibility that an explanation might need to be sought for lesions observed418

in the nerve. The rationale for this recommendation is that DRG, as well as the nerves they419

serve, lack effective neurovascular barriers (Olsson, 1990, Abram et al., 2006, Sapunar et al.,4202012) and thus may be exposed to test items that are excluded from the CNS by the blood-421

brain barrier. Usually, the chosen DRG is associated with the spinal cord segments from422

which the sampled spinal nerve arises (i.e., the lumbar intumescence for the sciatic nerve and423

its branches) (Table 4). A fast and simple means for retaining the DRG (and their associated424

spinal nerve roots) in the wet tissues is to harvest an extended portion (rodents) or region-425

specific segments (all species) of the vertebral column (after removing the musculature and426skin). The DRG may be processed and evaluated as isolated ganglia (all species) or in situ in

427

decalcified vertebral column sections (rodents only). Autonomic PNS ganglia to be assessed428

in Situation 1 are limited to the enteric and parasympathetic ganglia already present withinNervous System Sampling Practices, p. 20
429

protocol-specified hollow viscera (e.g., heart, intestines, urinary bladder). Specific sampling430

of additional autonomic ganglia is not needed.

431

Effector Organs. In Situation 1, skeletal muscle typically is examined as a protocol-

432

specified tissue. Reductions in myofiber diameter may serve as indirect evidence of PNS

433

damage due to nerve fiber (i.e., motor axon) degeneration if direct evidence of myopathic

434

injury is not seen. Although tongue is a common choice for histologic evaluation of skeletal

435

muscle (as a means of assessing many myofibers in several orientations in a single section),436

other skeletal muscle groups can be collected along with their innervating nerves. Muscles

437

commonly selected for sampling are composed mainly of type I (“slow twitch,” fatigue-

438

resistant) fibers (e.g., diaphragm and soleus) and/or type II (“fast twitch,” glycolytic) fibers

439(e.g., biceps femoris, quadriceps femoris, and gastrocnemius) (Schiaffino and Reggiani,

4402011). Some investigators substitute biceps brachii (if the forelimb appears to be affected).

441

The Working Group recommends the gastrocnemius as the default sample since it has a442

mixed (but mainly type II fiber) composition (Armstrong and Phelps, 1984); is a common

443

site of neurogenic atrophy in both humans (Spencer and Schaumburg, 1977) and animals

444with peripheral neuropathy; and the size of the muscle can be assessed qualitatively during

445

life by palpation. The biceps femoris is a suitable alternative sample as it also is a common

446

location for detecting neurogenic atrophy. The exact choice of muscles should be left to the447

discretion of the institution.

448
449

Situation 2

450

In general toxicity studies where in-life clinical signs or other data (e.g., MOA and451

QSAR similar to known PNS toxicants) suggest the potential for somatic (sensorimotor) PNS

Nervous System Sampling Practices, p. 21452

effects (Situation 2), the number of PNS specimens subjected to light microscopic analysis

453

should be expanded. Specific neurological evidence warranting additional sampling of the454

somatic PNS includes local or generalized signs of paresis, paralysis, proprioceptive defects,

455

or muscle atrophy (Table 1). Non-specific clinical observations related to possible somatic

456

nervous system dysfunction (e.g., abnormal movement, circling, difficulty walking, lameness457

of unknown origin, and generalized skeletal muscle weakness) also may trigger collection of458

additional PNS samples, at the discretion of the institution.

459

Nerves. Multiple mixed (sensory and motor) spinal nerves are sampled bilaterally during460

the initial tissue analysis (Figure 1) (Spencer and Schaumburg, 1977). In addition to the461

sciatic nerve, the choice of other nerves to collect may be dictated by the spectrum of

462

neurological signs observed in-life or may conform to a pre-defined battery specified in an

463

institutional SOP. Typically, distal nerve branches are preferred for evaluation since they464

usually contain a high proportion of sensory axons, and clinical cases of peripheral

465

neuropathy often present as altered sensation (Martyn and Hughes, 1997, Azhary et al.,4662010). Furthermore, hind limb nerves rather than forelimb nerves usually are sampled in467

toxicity testing because the longer nerve fibers that serve the hind limb usually are affected468

first during neuropathies (Krinke, 2011). That said, forelimb nerve branches also should be469

harvested if the in-life neurological signs suggest that forelimb function has been affected.

470

Evaluation of nerves near the administration site may be prioritized in instances where a471

locally delivered test item has limited systemic bioavailability.

472

At least three spinal-origin nerves (usually sciatic nerve and two of its branches) are473

evaluated, but the decision regarding which nerves to assess should be left to the discretion of474

the institution. The tibial (all species), fibular (all species), and/or sural (rodents (Peyronnard

Nervous System Sampling Practices, p. 22475

et al., 1986)) nerves are common choices as they are mixed sensorimotor tributaries of the476

sciatic nerve. In rodents, the caudal nerve (a mixed nerve that extends the entire length of the477tail) also may be considered for evaluation as electrophysiological testing (e.g., nerve

478

conduction velocity) combined with light microscopic examination of this nerve affords an

479

opportunity to correlate structural and functional findings related to PNS neurotoxicity

480(Schaumburg et al., 2010). Some Working Group members have found that aldehyde

481

fixation of the proximal to middle tail (via intravascular perfusion or immersion) allows for

482

later harvest and analysis of caudal nerve. In general, nerves are evaluated unilaterally (in483which case nerves that are to be examined for a given animal typically are harvested from the484

same side), but bilateral evaluation may be considered at the discretion of the institution or if

485

necessitated when iatrogenic nerve damage is likely due to in-life trauma (e.g., intramuscular

486

injection sites).

487

Collection of dedicated sensory-only or motor-only nerves is not necessary for safety488

assessment since the approach to microscopic evaluation is similar for both mixed and489

single-modality nerves. If observed clinical signs are indicative of a sensory neuropathy

490(which is the most common presentation of peripheral polyneuropathy in humans and491

animals), the Working Group recommends that at least one PNS specimen be a sensory-492

predominant (often termed “sensory-only”) nerve. Readily accessible sites include the plantar

493(usually the lateral branch in dogs (Ghoshal, 1975a) but the medial branch in rodents

494(Sant'Anna et al., 2016) and pig (Ghoshal, 1975b)); saphenous (dogs (Braund et al., 1980)

495

and rodents (LaMotte et al., 1991)); sural (rodents and primates [including humans] (Butt et496al., 2014)); or caudal (rodent (Schaumburg et al., 2010)) nerves. The only motor-specific497

nerves in all species are the ventral spinal nerve roots, which may be assessed individually orNervous System Sampling Practices, p. 23
498

in sections that also include the sensory-only dorsal spinal nerve root and its associated DRG.

499

For this purpose, serial DRG sections may be necessary to ensure that the desired nerve root500

is examined as their morphologic features are identical. The choice regarding whether or not

501

to sample sensory-only and/or motor-only nerves should be left to the institution.

502

Cranial nerve V (trigeminal nerve) often is considered for evaluation since this mixed

503

somatic nerve may be readily collected once the brain has been removed. In addition, several

504

trigeminal nerve branches also may be evaluated in situ if present within standard nasal

505

sections taken for inhalation toxicity studies (usually done only for rodents). Other cranial506

nerves typically are analyzed only if in-life neurological signs suggest that their function has

507

been compromised (reviewed in (Bolon and O'Brien, 2011). The optic nerve (or cranial508

nerve II), while routinely included in the list of protocol-specified tissues for GLP-type509

general toxicity studies, develops as an evagination arising from the forebrain and is510

myelinated by oligodendrocytes and not Schwann cells (Butt et al., 2004, Garman, 2011b),

511

and so is not a part of the PNS.9

512

Ganglia. If evidence of a somatic peripheral neuropathy is observed, at least two DRG

513

should be evaluated for both the cervical and lumbar divisions of the spinal cord (i.e., at least

514

four total DRG). The best practice is to remove DRG from the vertebral column (Figure 2)

515

to preclude the induction of handling artifacts associated with vertebral decalcification

516

needed for in situ examination. However, an acceptable practice in rodents is to assess DRG

517

in situ to avoid trauma produced during their removal. Because soft tissue gathered when

9Best practices for sampling optic nerve have been published previously Bolon, B., Garman, R.H., Pardo,
I.D., Jensen, K., Sills, R.C., Roulois, A., Radovsky, A., Bradley, A., Andrews-Jones, L., Butt, M. andGumprecht, L. (2013b). STP position paper: Recommended practices for sampling and processing thenervous system (brain, spinal cord, nerve, and eye) during nonclinical general toxicity studies. Toxicol
Pathol, 41, 1028-1048.

Nervous System Sampling Practices, p. 24
518

seeking DRGs sometimes represents connective tissue or fat, more than two DRG should be519

harvested to ensure that at least two DRG from each specified spinal cord level actually are

520

available for histologic evaluation. Even more ganglia may need to be collected and521

examined when the test item is delivered directly nearby (e.g., epidural or intrathecal

522

injection) or when clinical signs suggest that nerves arising from a particular spinal cord523

segment or segments have been affected. The DRG typically are chosen from those524

associated with the origins of the brachial plexus (i.e., origin of the brachial nerve) and525

lumbosacral plexus (i.e., origin of the sciatic nerve) because axons emanating from these526

ganglia are some of the longest (and thus among the most susceptible) in the body. The

527

locations of DRG serving the brachial and sciatic nerves vary by species and sometimes

528

strain (Table 4).

529

In addition to DRG, the trigeminal ganglion (i.e., the sensory ganglion of cranial nerve V)

530

should be collected for evaluation. Ganglia of the autonomic PNS are assessed when seen in

531

situ within routinely sampled organs (e.g., intramural parasympathetic and enteric ganglia in532

the heart, intestines, and urinary bladder). Similar to Situation 1, additional autonomic

533

ganglia need not be sampled for this scenario.

534

Effector organs. Skeletal muscle from sites other than the tongue should be examined

535

from two or more distinct muscles. The specific sampling location(s) may be left to536

institutional preference and the parameters of the study design (e.g., muscle near sites of537

locally delivered test items also should be sampled). The Working Group recommends that538

gastrocnemius serve as the default choice for one of the two specimens.

539

Muscle weights acquired at necropsy may provide an indirect but quantitative means of540

discriminating peripheral neuropathic effects. Weights typically are acquired from isolatedNervous System Sampling Practices, p. 25
541

biceps brachii, biceps femoris, gastrocnemius, and/or quadriceps femoris, which can be

542

easily identified and collected in a consistent fashion (Greene, 1935, Vleggeert-Lankamp,543

2007, Magette, 2012). The Working Group recommends the gastrocnemius for weighing

544

since peripheral neuropathies usually occur first in longer axons (which in the hind limb are545

most distant from their supporting neurons (Krinke, 2011)). Care is required in interpreting546

the relevance of muscle weights if they have been gathered from samples taken near the site547

of local test item administration. Where present within muscle sections, muscle spindles (i.e.,548

sensory end-organs) and intra-muscular nerves should be assessed, leaving the choice to the549

institution regarding how to record test item-related findings observed in these structures.

550
551

Situation 3552

In general toxicity studies where autonomic PNS neurotoxicity is a concern (Situation 3),553

expanded sampling of autonomic PNS structures is necessary. Evidence warranting more

554

extensive autonomic PNS sampling includes signs of visceral dysfunction including

555

abnormalities in gastrointestinal motility, heart rhythms, micturition (urinary retention or556

incontinence), ocular responsiveness (mydriasis and miosis), salivation, or vascular tone

557

(Mathias, 2003) (Table 1). A WOE approach is especially important in deciding whether or558

not to engage in expanded sampling and analysis of the autonomic PNS. In general, isolated559

signs of visceral distress (e.g., affecting one or two autonomic functions) usually reflect signs

560

of toxicity to extra-neural organs rather than to the autonomic PNS, and thus would not serve561

as an automatic trigger for increased autonomic PNS sampling. Instead, expanded autonomic562

PNS collection would be undertaken if a generalized autonomic dysregulation was suggested563

by multiple anomalous signs originating in the autonomic CNS or PNS.

Nervous System Sampling Practices, p. 26
564

When autonomic PNS neurotoxicity is suspected, care should be taken to properly define565

the extent to which the histopathologic evaluation of the nervous system should be increased.

566

Sometimes multiple autonomic divisions (i.e., enteric, parasympathetic, sympathetic) may be

567

affected at once, which would warrant more sampling of all these divisions. In addition,

568

autonomic neuropathies also may be accompanied by somatic neuropathies, in which case569

expanded sampling of the somatic PNS (as defined for Situation 2 above) also is required. As

570

noted above, the final PNS sampling strategy should be driven by the constellation of PNS-

571

related in-life neurological signs.

572

Nerve. Though the number of autonomic nerves conducive for sampling may be

573

limited, multiple autonomic (Figure 3) nerves should be assessed during the initial tissue574

analysis for Situation 3. Autonomic PNS sampling may include parasympathetic (e.g.,575

cranial nerve X [vagus]) and/or sympathetic (e.g., sympathetic chain branches) structures.

576

Somatic nerve sampling often mirrors that described above for Situation 2 (Figure 1).

577

Ganglia. Intramural autonomic (parasympathetic) ganglia in protocol-specified hollow

578

organs (e.g., gastrointestinal tract, heart, urinary bladder) should be evaluated. Ganglionic579

sampling should be based on in-life findings (i.e., visceral dysfunction), but enteric ganglia

580

should be included for evaluation whenever autonomic neuropathy is suspected as they are

581

readily identified in intestinal sections. If enteric ganglia are missing from routine sections,582

then preparation of additional tissue sections of protocol-specified viscera may be

583

considered.

584

In addition, several sympathetic ganglia should be obtained. Frequently sampled sites

585

include the cranial (superior) cervical ganglion, cervicothoracic ganglion, cranial (superior)586

mesenteric ganglion, and the celiac/cranial mesenteric ganglion. The caudal vagal (nodose)Nervous System Sampling Practices, p. 27
587

ganglion—which is a sensory [visceral afferent] portion of cranial nerve X—is easily

588

confused with the cranial cervical ganglion since both are located in proximity to the589

bifurcation of the carotid artery (Figure 3). Somatic sensory PNS ganglia, such as multiple

590

DRG (cervical and lumbar) and trigeminal (cranial nerve V) ganglia, also should be591

considered for sampling.

592

Effector organs. In most toxicity studies, the list of protocol-specified tissues will593

include multiple effector organs that are innervated by the autonomic PNS (e.g., glands,

594

heart, hollow organs with abundant smooth muscle like the digestive tract and urinary

595

bladder).

596

Lesions of the autonomic PNS have been linked on occasion to structural changes in597

some effector organs. For example, systemic administration of ganglioplegic drugs (i.e.,

598“ganglionic blockers,” which inhibit transmission between pre-ganglionic and post-599

ganglionic autonomic neurons in both the parasympathetic and sympathetic systems) can

600

induce sperm granulomas in the epididymis of rats (Bhathal et al., 1974). However, sperm

601

granulomas are a common incidental background finding in this species, so their presence

602

should not be interpreted as confirmation that a test item produces autonomic dysfunction in603

the absence of additional evidence to support this conclusion.

604

Central (CNS) autonomic centers. Preganglionic neurons for autonomic nerves reside in605

various brain nuclei (parasympathetic role) and the lateral (intermediate) column of the606

thoracic ± rostral lumbar spinal cord (sympathetic role). The hypothalamus serves many

607

significant autonomic tasks. The most important autonomic structure in this region is the608paraventricular nucleus (PVN) of the hypothalamus, which contains neuroendocrine cells609

that innervate the median eminence and pituitary gland (Ulrich-Lai and Herman, 2009). InNervous System Sampling Practices, p. 28
610

rodent brains trimmed according to current STP “best practices” for CNS sampling (Bolon et611

al., 2013b), the PVN should be present in Level 3. Cranial nerves III, VII, IX, and X carry612

both somatic motor and parasympathetic nerve fibers; the parasympathetic components

613

innervate involuntary functions of multiple muscles and glands. Locations of these614

brainstem parasympathetic nuclei reside outside the seven levels recommended for615

assessment under current STP “best practices” for CNS sampling (Bolon et al., 2013b), and616

instead will need to be localized using a species-specific neuroanatomy atlas (Paxinos et al.,617

2000, Paxinos and Franklin, 2001, Paxinos and Watson, 2007, Palazzi, 2011) if in-life signs

618

warrant their assessment. The lateral column of the sacral spinal cord also contains

619

preganglionic autonomic neurons. Dogma for the past century has classed these sacral620

neurons as parasympathetic, but recent functional and molecular data indicates that these621

neurons may actually regulate sympathetic functions in pelvic viscera (Espinosa-Medina et622al., 2016). These CNS sites may be considered for sampling and evaluation if the potential

623

for an autonomic neuropathy is present, at the discretion of the institution.

624
625

Situation 4626

In dedicated neurotoxicity studies where a CNS or PNS liability is likely (Situation 4),627

expanded sampling is required to more fully characterize neurotoxic hazards. Because the628

nervous system is the main focus of the study, more extensive sampling of the PNS (and629

CNS) is expected by regulatory agencies. This approach is applicable to both adult (Rao et630

al., 2011, Pardo et al., 2012, Bolon et al., 2013b) and developmental (Bolon et al., 2006,

631

Garman et al., 2016) neurotoxicity studies in mammals, and to organophosphate-induced

632

delayed neurotoxicity in hens (Krinke et al., 1979, Krinke et al., 1997).

Nervous System Sampling Practices, p. 29
633

Nerve. Multiple (three or more) spinal-origin nerves and cranial nerve V are sampled, as

634

defined in Situation 2 above. The precise choice of spinal-origin nerves is left to the635

discretion of the institution, although more distal locations and predominantly sensory nerves

636

should be emphasized due to their early involvement in toxicant-induced peripheral

637

neuropathies. Where nerve conduction velocity is tested (e.g., in dogs, the fibular nerve for638

motor fibers and the sural nerve for sensory fibers; in rats, the caudal nerve), the same nerves

639

for the ipsilateral and/or contralateral limb should be considered for microscopic examination640

to permit structure-to-function correlations. Autonomic nerves typically are not collected641

unless in-life neurological signs suggest that lesions may exist in the autonomic PNS, in

642

which case additional autonomic nerves as defined in Situation 3 should be collected as well.

643

Ganglia. Multiple DRG (more than the four collected in Situation 2) should be644

examined. At least two should be harvested and assessed bilaterally for each spinal cord

645

division (cervical, thoracic, and lumbar); some institutions collect a dozen or more,

646

especially in studies that involve direct epidural or intrathecal delivery or in which in-life

647

neurological signs show that the sensory PNS represents a sensitive target organ. In studies

648

where the PNS findings seen at relevant exposure levels are likely to contribute to the risk

649

assessment, the Working Group members concur that it is impossible to assess too many650

DRG since neurotoxic changes in these structures do not develop in a uniform manner in

651

these organs. The Working Group recommends removal of the DRG from the vertebral

652

column as the best practice (to avoid decalcification-related tissue artifacts). In rodents,

653

DRG may be evaluated in situ following vertebral decalcification.

654
655

Sites for collecting cervical and lumbar DRG are the same ones recommended above for
Situation 2 (Table 4). The thoracic DRG typically are collected from the middle of that

Nervous System Sampling Practices, p. 30
656

division. For DRG investigations, it is important to remember that while all DRG are located657

immediately adjacent to the vertebra of the same designation (i.e., DRG L5 is immediately

658

caudal to vertebra L5), the spinal cord segment associated with a DRG frequently is present

659

cranial to the vertebra bearing the same designation (i.e., spinal cord segment L5 is located in660

vertebra L1-2 in rodents (Bolon et al., 2013b)).

661

As with Situation 2, the trigeminal ganglion (for cranial nerve V) and autonomic

662(parasympathetic) and enteric ganglia as available in other protocol-specified organs should663

be examined. If neurological signs suggest that autonomic dysfunction may be present,

664

sampling of autonomic ganglia may be expanded to include the specimens listed for Situation

665

3.

666

Effector organs. If the known potential for neurotoxicity suggests that neural lesions are667

localized to somatic nerves and/or ganglia, skeletal muscle should be examined for at least668

two distinct sites, as defined above for Situation 2. Organ weights may be obtained after

669

whole-body perfusion fixation for one or more isolated muscle bellies, at the discretion of the670

institution, and the isolated muscles may be employed thereafter for histopathologic analysis.

671
672

B. Situation-specific Fixation Options for PNS

673

Situation 1. For general toxicity studies in which PNS neurotoxicity is not known,

674

suspected, or observed during life, the PNS is fixed using the same regimen applied to the675

non-neural tissues: immersion in NBF, commercial formulations of which contain 3.7 to 4%676

formaldehyde and approximately 1% (v/v) methanol (included as a stabilizer to extend the677

shelf-life by slowing polymerization of formaldehyde monomers into paraformaldehyde

678

polymers (Kiernan, 2000, Kiernan, 2008)). Methanol is a solvent and therefore may induceNervous System Sampling Practices, p. 31
679

morphologic artifacts in PNS, especially vacuoles and splitting of myelin sheaths (Garman,

680

2011a). Nonetheless, due to cost and ready availability, NBF is still the preferred PNS

681

fixative for general toxicity studies without a pre-defined need for a special assessment of the682

nervous system.

683

Immersion fixation in NBF is conducted at RT for at least 24 hours. The ratio of fixative684

solution to tissue should be at least 10 volumes of fluid to one volume of tissue. The quality

685

of PNS preservation using methanol-containing NBF is acceptable provided that tissues are

686

harvested quickly and not handled excessively (to avoid crush and stretch artifacts). If

687

desired, MFF may be utilized for selected specimens at the discretion of the institution to688

preserve methanol-sensitive antigens for later immunohistochemical (IHC) detection, but this689

practice is not undertaken for entire studies for Situation 1.

690

Situations 2, 3. For general toxicity studies in which a concern for somatic (Situation 2)

691

or autonomic (Situation 3) PNS neurotoxicity is projected by in-life neurological signs, PNS

692

fixation typically is identical to that employed in Situation 1: immersion in NBF (3.7%693

formaldehyde with 1% methanol). Where feasible (e.g., where in-life neurological signs694

develop early enough in the course of a study to allow bulk acquisition of specialty reagents),695

a preferred choice for immersion fixation is MFF (e.g., methanol-free 4% formaldehyde) as

696

the absence of methanol improves myelin integrity.

697

Some institutions may prefer to employ whole-body perfusion fixation if PNS

698

neurotoxicity is suggested by in-life neurological signs (Table 3) and providing that699

additional study endpoints do not preclude this manner of fixation. Perfusion fixation may

700

alter certain parameters commonly included in the data sets of GLP-type toxicity studies,

701

particularly organ weights and the microscopic integrity of highly vascular organs (e.g., lung,Nervous System Sampling Practices, p. 32
702

spleen). Except for the lungs and possibly the spleen and heart, comparison of organ weights

703

among groups should be possible for perfusion-fixed tissues from animals in the same study,704

if the laboratory has an established track record of successfully performing the perfusion

705

procedure. Comparison of organ weights from perfusion-fixed animals with historical

706

control data from immersion-fixed animals is not recommended. Technical details for whole-707

body perfusion fixation are given below under Situation 4.

708

Situation 4. For dedicated neurotoxicity studies in which an impact on the nervous system

709(PNS or CNS or both) is likely or certain (Situation 4), whole-body perfusion using MFF or

710

another methanol-free fixative (e.g., 2.5% MGG) is recommended. Because perfusion

711

fixation can impact the ability to assess other protocol-specified organs, collection of PNS

712(and CNS) samples commonly is done on a satellite group specifically slated for713

neuropathology evaluation.

714

For intravascular perfusion, fixative is introduced into either the left cardiac ventricle or715

aorta of a deeply anesthetized animal through a blunt metal needle or plastic cannula at a716

pressure of 120 to 150 mm Hg (approximately equal to vertebrate systolic blood pressure) by

717

perfusion pump or a gravity drip system (Fix and Garman, 2000). Species-appropriate needle

718

sizes are 21-25 gauge in mice and young rats, 19 to 21 gauge in adult rats, and 14 to 18 gauge

719(or even greater) in non-rodents (Hancock et al., 2005, Bolon and Butt, 2014). A pre-flush of720

physiological saline may be given to prevent thrombi from forming in small blood vessels as

721

the fixative contacts blood cells and plasma proteins. Inclusion of a vasodilator (e.g., sodium

722

nitrite, 1 mg/ml) and/or anti-coagulant (e.g., sodium heparin, 1000 IU/L of solution) in the723

pre-flush maximizes vessel patency. The choice of using a pre-flush (with or without anti-

724

coagulants and vasodilators) should be left to the institution’s discretion. The volumes ofNervous System Sampling Practices, p. 33
725

pre-flush and fixative to infuse usually are determined by the need to adequately preserve the726brain and spinal cord, and vary by the species. Each laboratory should develop their own

727

protocols for intravascular perfusion especially concerning the duration, volume, and rate of728

perfusion. Fifty to 100 mL in adult mice, 500 to 1000 mL in adult rats, and 3 to 5 L (or more)729

in non-rodents are suggested as starting points for the amount of fixative solution to instill;730

the amount of pre-flush typically is between 30% to 50% of these volumes. Both pre-flush

731

and fixative solutions may be perfused at either RT or 4°, but RT solutions may produce732

fewer artifacts (Hancock et al., 2005, Bolon and Butt, 2014).

733

The consensus recommendation of the Working Group is that MFF is a perfusion fixative734

of choice for preserving PNS (and CNS) tissues for routine light microscopic analysis. If

735transmission electron microscopy (TEM) also is to be undertaken, inclusion of MGG is736

recommended as another component of the perfusate to better preserve cytoarchitectural

737

details and reduce artifactual changes in myelin. These two aldehydes may be applied738

sequentially (usually using MFF to begin) or in combination. Two common mixtures are739

modified Karnovsky’s solution (2% MFF and 2.5% MGG) and McDowell/Trump solution

740(4% MFF and medical-grade 1.0% MGG); in the Working Group’s experience, the most741

common choice is modified Karnovsky’s solution. Fixatives for TEM often are made in 0.1742

M cacodylate or phosphate buffer (pH 7.4). Cacodylate solutions have a longer shelf-life but

743

contain arsenic and thus require extra care during use and disposal. For combination

744

fixatives, intact ganglia or nerves are post-fixed by immersion in fresh fixative at 4°C for 2 to745

24 hours, after which tissue is transferred to fresh, ice-cold buffer. The reason for reduced

746

fixation length with glutaraldehyde is that this agent renders tissues hard and brittle through747

its ability to more effectively cross-link molecules (Kiernan, 2000). Extended storage inNervous System Sampling Practices, p. 34
748

glutaraldehyde-containing fixatives results in excessive tissue hardening that may lead to749

fragmentation of the samples during sectioning.

750

Post-fixation. For settings in which PNS neurotoxicity is suspected (Situations 2 and 3) or

751

likely (Situation 4), or where regulatory guidelines require plastic embedding of nerve (EPA,

752

1998a), selected nerve samples require additional fixation to stabilize myelin lipids. For this753

purpose, one (Situations 2 and 3) or at least two (Situation 4) nerves—usually spinal-origin

754

somatic trunks rather than autonomic branches—are post-fixed in glutaraldehyde and then755

osmium tetroxide10 (Bolon et al., 2008, Raimondo et al., 2009). Osmium must be used with756

glutaraldehyde to best maintain cellular structures (Penttila et al., 1974).

757

Isolated PNS samples (typically nerve cross sections) first are immersed in MGG for at758

least two hours (Dyck, 2005, Bilbao and Schmidt, 2015). A common composition is 2.5%759

MGG in 0.025 M cacodylate buffer, pH 7.4, at an osmolarity of 300-330 mOsm. Fixation

760

may be done at RT or 4°C, after (usually overnight to 24 hours) which fixed tissue may be

761

stored in buffer. Post-fixation in MGG is utilized for tissues fixed in NBF or MFF but is not762

needed for samples in which MGG was part of the perfusate. Subsequently, samples are

763

immersed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at RT for 1 to 4 hours,

764

after which tissues are shifted to buffer. Osmium penetrates poorly (approximately 1 mm

765

total (Dykstra, 1992)), so prior to osmication PNS samples must be cleaned of surrounding

766

adipose and connective tissue—without injuring the neural elements. Large samples (e.g.,

767

sciatic nerves of non-rodents) may need to be trimmed into thin slices to facilitate osmium

768

permeation into the nerve center.

769
10

Fixation in osmium is typically termed “osmication” (though sometimes is rendered as “osmification”).

Nervous System Sampling Practices, p. 35
770771

C. Strategies for Trimming PNS Samples
Great care should be exercised when handling nerves and ganglia (even when fixed) as

772

even subtle manipulation may cause artifactual changes. Tissue trimming of the PNS

773

includes one or more nerve trunks and skeletal muscle (an effector organ) in all four

774

Situations as well as DRG (including spinal nerve roots) and/or autonomic ganglia for

775

Situations 2, 3, and 4. Nerves and skeletal muscle should be trimmed to permit analysis of776

fibers in both cross (transverse) and longitudinal orientations. Particular attention should be

777

given to evaluating skeletal muscle in cross sections because the morphological features of778

myofibers affected by PNS lesions (e.g., “fiber group atrophy” from denervation) are779

assessed most readily in this orientation. Myofibers in the diaphragm and tongue are arranged

780

in crisscrossing patterns that preclude most fibers from being viewed in truly longitudinal781

and cross orientations, thereby adding to the challenge of detecting “fiber group atrophy.”782

A properly prepared nerve cross section (Figure 4) allows for an assessment of the783

density and numbers of myelinated axons, and to a lesser extent those of unmyelinated axons

784(Raimondo et al., 2009). The cross-section also allows for an evaluation of myelin integrity785(including discrimination between demyelination and remyelination), and may reveal

786

Schwann cell changes not readily seen on a longitudinal section. The longitudinal section

787

provides a means for demonstrating axonal or myelin damage spanning several internodes

788(Figure 4) and may, due to the length of nerve examined, allow for a better assessment of789

associated changes such as inflammatory reactions. Longitudinal nerve samples should be790

approximately 1 cm long if feasible (Bolon et al., 2013b) to ensure that sufficient numbers of791

nerve fibers will be visible over extended lengths. Spinal nerve roots may be isolated if

Nervous System Sampling Practices, p. 36
792

necessary for evaluation (after embedding in plastic resin, but generally are embedded along

793

with their associated DRG, typically in longitudinal orientation (Figure 5).

794
795

For Situation 3, isolated sympathetic ganglia should be processed in a fashion similar toother ganglia.

796
797

D. Situation-specific PNS Embedding Strategies

798

Embedding of PNS tissues is a critical factor in determining the data quality derived from799

evaluation of PNS tissues. Paraffin allows detection of primary degenerative and infiltrative

800

processes and therefore is a suitable embedding medium for PNS samples in general toxicity

801

studies where PNS neurotoxicity is not a concern (Situation 1). Paraffin also is used for most802

specimens in general toxicity studies where PNS neurotoxicity is a concern (Situations 2 and803

3) as well as in dedicated neurotoxicity studies where neurotoxicity (CNS and/or PNS) is

804

likely or certain (Situation 4) due to its low cost and ready availability. One neurotoxicity

805

testing guideline states that “[p]lastic embedding is required for tissue samples from the806

peripheral nervous system” (EPA, 1998a). The intent of this recommendation is to improve807

discrimination of fine cellular detail in myelinated and unmyelinated fibers. Use of plastic

808

embedding media permits acquisition of thinner sections, thus providing improved resolution809

of cellular features.

810

Plastic embedding is expensive and labor-intensive. In studies where it is deemed that811

plastic embedding will be to costly for use with all PNS samples, the Working Group advises

812

the following adaptation of regulatory guidance requiring plastic embedding for the PNS.

813

The Working Group recommends plastic embedding for at least one (Situations 2 and 3)

814

or two (Situations 4) nerve cross sections (Figure 4), which are scenarios in which a concern

Nervous System Sampling Practices, p. 37
815

exists that a test item may elicit PNS neurotoxicity. Indeed, for Situation 3, nerve fibers (and816

especially the myelin sheaths) of autonomic nerves often are so small that plastic sections of817

osmicated nerves may be essential for light microscopic assessment. In such cases, PNS

818

specimens slated for plastic embedding have been post-fixed in glutaraldehyde and osmium.

819

Cross sections of these nerve samples permit ready evaluation of the features and diameters

820

for both axons and complete nerve fibers (i.e., axons plus myelin). Plastic embedding of

821

longitudinal nerve sections is used less often as osmium deposition in myelin may obscure822

features in superimposed PNS nerve fibers due to overlap of the metal-impregnated myelin

823

sheaths; however, plastic-embedded longitudinal nerve sections may be useful for evaluating824

nodes of Ranvier. Several Working Group members suggest that laboratories and sponsoring

825

institutions be encouraged to consider adjusting their PNS processing procedures for

826

Situation 1 to incorporate routine preparation of osmicated, plastic-embedded nerve cross827

sections as a means of attaining ideal morphologic preservation for PNS samples. However,828

the majority of the Working Group accepts that this proposed modification, while technically829

correct, may not be practical for the many general toxicity studies where no concern exists

830

that the test item has induced PNS neurotoxicity.

831

Plastic embedding for nerve samples usually employs one of two variants: “hard plastic”

832(hydrophobic) resins such as araldite, epon, or Spurr’s, or combinations thereof (e.g., epon-

833

araldite); or “soft plastic” (hydrophilic) materials like glycol methacrylate (GMA) and834

methyl methacrylate (MMA). Section thicknesses that are reproducibly attainable for PNS

835

using hard plastic (<1 μm, Figure 4) and soft plastic (2 μm, Figure 6) are considerably

836

reduced relative to that which is readily achievable for paraffin (4-6 μm, Figure 4 and837

Figure 6). Soft plastics are more expensive than paraffin but are less costly and easier toNervous System Sampling Practices, p. 38
838

process and section than are hard plastics. However, hard plastics can be used with839

osmicated PNS samples while soft plastics are not compatible with osmium; thus, myelin

840

lamellae are only imperfectly conserved in soft plastic sections, which negates the original841

reason why plastic embedding of PNS tissues was required (EPA, 1998a). The Working

842Group is of the unanimous opinion that soft plastic embedding media offer little

843

improvement in cytological resolution over paraffin embedding for non-osmicated nerve844

samples (Figure 6), and that soft plastic offers substantially inferior tissue preservation

845

relative to hard plastic combined with osmication (Figure 4). Accordingly, the Working

846Group recommends hard plastic resin (of osmicated samples) as the best practice for plastic

847

embedding of PNS, and further advises that the use of soft plastic is not a suitable alternative

848

for PNS embedding. Methodological details for hard plastic embedding are found in the849

manufacturer’s instructions available with commercially available kits.

850

Osmium-impregnated nerves may be embedded in paraffin (Bolon et al., 2013a). The

851

preservation and visualization of myelin is enhanced in osmicated, paraffin-embedded nerve852

sections in comparison to non-osmicated, paraffin-embedded nerve sections but remains

853

inferior to osmicated, hard plastic resin-embedded sections. Therefore, the Working Group854

recommends that paraffin embedding of osmicated tissues be avoided as a substitute for hard

855

plastic resin embedding.

856

For dedicated neurotoxicity studies (Situation 4), the Working Group recommends that857

nerves and DRG should be embedded in individual blocks (with or without other tissues) so

858

that lesions may be tracked to identifiable PNS sites. Alternatively, some institutions place859

many DRG from all spinal cord divisions in one cassette (Figure 5), or group DRG from

Nervous System Sampling Practices, p. 39
860

specific spinal cord segments into separate cassettes. The Working Group recommends that861

sampling more DRG is preferable, even if exact locations of individual DRG are not tracked.

862

For both paraffin- and plastic-embedded specimens, the Working Group recommends

863

that all PNS tissues from the treatment groups selected for initial evaluation (e.g., high-dose864

and control animals) should be processed in the same time frame to avoid any systematic

865

variation in such technical factors as the lengths of time spent in fixative or dehydrating866

solutions. If a quantitative endpoint has been built into the study design, tissues from all

867

study groups for which the endpoint might need to be collected should be processed into

868

blocks at the beginning of the study, even if sectioning of the blocks for intermediate doses

869

groups will be delayed; this strategy will greatly reduce the likelihood that variations in

870

processing will impact the quantitative data. A key means of standardizing the effects of871

fixation and processing across treatment groups is to include cassettes from animals in the872

different cohorts within each processing “run” so that handling-related artifacts are balanced873

by mixing samples from all dose groups.

874
875
876

E. Situation-specific PNS Staining Strategies
Staining of PNS tissues employs hematoxylin and eosin (H&E) for paraffin sections and877

toluidine blue for hard plastic resin sections (Table 3). The Working Group recommends

878

these two stains as a suitable initial screen for PNS specimens in toxicity studies. When879

preparing PNS tissues, an important consideration is that delayed processing of some880

treatment groups (as opposed to immediate processing of all groups into blocks) may result881

in altered tinctorial intensity in H&E-stained nerve sections, which might confound any

882

quantitative or post hoc coded histopathologic evaluations. For studies in which PNS

Nervous System Sampling Practices, p. 40
883

neurotoxicity represents a possible concern (Situations 2, 3, and 4), other neurohistological884

methods may be undertaken in non-osmicated, paraffin-embedded nerves at the discretion of885

the institution to further characterize any PNS findings discerned during the initial analysis.

886

Stain quality for such special procedures varies depending on many factors, including section

887

thickness, technician experience, and regularity with which the procedure is performed.

888

When needed, the Working Group recommends stains for axons and myelin as the most889

useful special methods for further characterizing PNS findings related to test item exposure.

890

Silver stains (e.g., Bielschowsky’s [Figure 7], Bodian’s, or Holmes) are helpful to highlight891

neurofilament-rich structures, including axons and cytoplasmic organelles in neurons, and for

892

demonstrating damaged axons in axonopathy (e.g., fragmentation) or neuroaxonal dystrophy893(e.g., axonal spheroids). Myelin-staining methods used in PNS tissues include Luxol fast894

blue (LFB; Figure 7) and Sudan black, which are especially beneficial for intact myelin, and895

the Marchi stain, which often is used to reveal demyelination (Strich, 1968). The reasons for896

recommending these procedures are that axons and myelin are the two key components of897

PNS structures, and thus many laboratories routinely perform these stains.

898

Special neurohistological methods used to showcase neurotoxic damage in the CNS

899

typically are not utilized when evaluating PNS neurotoxicity. Routine IHC methods to900

demonstrate glial fibrillary acidic protein (GFAP, upregulated in reactive astrocytes and in901

some satellite glial cells) and ionized calcium-binding adapter molecule 1 (Iba1, expressed902

by microglia and macrophages) may be used in DRG to detect satellite glial cells and903

activated macrophages, respectively (Patro et al., 2010, Ton et al., 2013), but such techniques904

typically are deployed in the research setting rather than in toxicity testing. Glutamine905

synthetase (a preferred marker for satellite glial cells (Miller et al., 2002, Schaeffer et al.,

Nervous System Sampling Practices, p. 419062010)) and CD68 (a macrophage marker (Jimenez-Andrade et al., 2006)) may be used with907

or in place of anti-GFAP and anti-Iba1 procedures; leukocyte biomarkers (e.g., anti-CD3 for908

T-lymphocytes) may be helpful in differentiating inflammation from increased satellite cell

909

numbers. Fluoro-Jade, a fluorescent stain used to detect necrotic neurons in the CNS

910(Schmued and Hopkins, 2000, Schmued et al., 2005), may be attempted in the PNS to detect

911

degenerating neurons in ganglia (Marmiroli et al., 2009). In the experience of several

912

Working Group members, Fluoro-Jade does not specifically highlight necrotic ganglionic

913

neurons, presumably because dead neurons in DRG do not express the as yet unidentified

914

marker labeled by Fluoro-Jade stains in dead CNS neurons. Accordingly, the Working Group

915

does not recommend the routine use of these CNS-oriented special methods for evaluating

916

PNS lesions.

917

While the use of soft plastic is not recommended for PNS tissues, archival samples

918

embedded in this medium may be stained routinely with H&E. Other procedures that may be

919

undertaken in soft plastic-embedded PNS tissues include silver stains for axons and920

histochemical stains (e.g., Sudan black; (Cerri and Sasso-Cerri, 2003)) or IHC methods (e.g.,

921

myelin basic protein (Mueller et al., 2000)) to reveal myelin lipids. The experience of922

Working Group members is that special techniques are applied to soft plastic-embedded PNS

923

specimens mainly in the research setting.

924

Hard plastic-embedded nerve cross sections usually are osmicated during processing and925

then stained with toluidine blue (Figure 1). The concentration of toluidine blue used for this926

purpose varies among laboratories but typically is set at 1% (1:100) (Hancock et al., 2005).

927

Paraphenylenediamine (PPD) also may be employed to highlight lipid-rich cell membranes

Nervous System Sampling Practices, p. 42928

in osmicated, hard plastic resin-embedded sections (Shirai et al., 2016), including myelin

929

sheaths (Sadun et al., 1983).

930

Special histochemical procedures may be applied to differentiate various myofiber types

931

in skeletal muscles (Armstrong and Phelps, 1984, Kremzier, 1984, Staron et al., 1999).

932

These methods generally are not used to evaluate muscle samples in situations where PNS

933

neurotoxicity is a concern as muscle lesions due to PNS damage (i.e., fiber group atrophy)934

may be seen easily by H&E.

935
936
937

F. Special Procedures for Evaluating PNS Neurotoxicity
If warranted, additional techniques may be undertaken to better characterize PNS lesions.

938

Examples include TEM (Peters et al., 1991), morphometry (Diemer, 1982, Kristiansen and939

Nyengaard, 2012, Butt et al., 2014), stereology (Hyman et al., 1998, Butt et al., 2014), teased

940

fiber preparations (Krinke et al., 2000), and quantification of intra-epidermal nerve fiber

941

ending density (IENFD; (Lauria et al., 2005a, Lauria et al., 2005b, Myers and Peltier, 2013,

942

Mangus et al., 2016)), motor end plates (Francis et al., 2011), and muscle spindles (Krinke et943al., 1978, Muller et al., 2008). A detailed consideration of such special procedures is beyond

944

the scope of this paper. Decisions regarding whether or not to deploy these methods should945

be guided by data showing the neurotoxic potential of a test item to the PNS, usually the946

presence of in-life neurological signs or prior knowledge that the test item or a related

947

molecule produces morphological effects in the PNS. Another important reason for

948

quantifying IENFD, motor end plates, and/or muscle spindles may be to show an absence of949

neuropathy, to provide evidence that nerve signaling is intact. The choice regarding whether

950

or not to use these special procedures should be left to the discretion of the institution.

Nervous System Sampling Practices, p. 43
951
952

IV. Strategy for PNS Neuropathology Analysis

953

The approach used for initially evaluating the PNS during nonclinical toxicity studies in954

all four situations is a tiered, semi-quantitative light microscopic examination (i.e., assigning955

lesion grades) equivalent in concept to that for any other organ or tissue. Criteria used to set

956

histopathologic grades for unusual findings should be defined using text descriptions and/or

957

visual illustrations of concrete features, or should be established by citing published, well-958

established grading schemes. The analysis should identify the existence of morphological959

changes in PNS tissues and characterize the lesion pattern so that the cell populations (e.g.,960

neurons and/or Schwann cells) and structures (e.g., cell body vs. axon vs. myelin) targeted by961

the test item can be determined.

962

The initial microscopic evaluation of PNS tissues from nonclinical toxicity studies for all

963

four situations generally should be conducted in an informed (“unblinded” or “unmasked”)

964

fashion. In other words, the study pathologist should receive in advance full knowledge of965

the dose level and group assignment for each animal as well as other data (macroscopic

966

findings, organ weights, clinical observations and outcomes of behavioral testing), all of

967

which might help in interpreting the microscopic pathology data. This recommendation

968

represents a consensus opinion among experienced toxicologic pathologists (Gosselin et al.,9692011), including members of the Working Group, and conforms to STP recommended “best970

practices” for histopathologic evaluation of tissues from toxicity studies (Crissman et al.,971

2004). The initial uncoded assessment may be limited to the control and high-dose groups or

972

may include all dose groups at the discretion of the institution. The rationale for this973

recommendation is that informed examination greatly enhances the quality of the pathology

Nervous System Sampling Practices, p. 44974

data set by (1) permitting development of more objective criteria for grading changes, (2)975increasing the likelihood of detecting subtle PNS findings, and (3) enhancing the speed with976

which the analysis may be undertaken (which reduces diagnostic drift). This logic is no977

different from that used when designing the assessment for any other organ or system.

978

Once a PNS finding has been identified, a masked (“blinded” or “coded”) post hoc

979

assessment of specific changes may be performed at the discretion of the study pathologist

980(or peer review pathologist (Morton et al., 2010)). Such blinded evaluations should be981

limited, performed only as needed to clarify the incidence of subtle findings, tighten severity

982

grade assignments, discern treatment-associated exacerbation of background lesions, and/or

983

establish a dose-effect relationship (including definition of a no observed adverse effect level

984[NOAEL]). The choice of which dose groups and findings to include in a masked evaluation985

is not defined by existing regulatory guidelines but rather is chosen by the pathologist; for986

example, the “blinded” assessment may be limited to the control and low-dose animals and987

ignore any other dose groups if clear neuropathologic changes are evident in the mid-dose988

and high-dose animals. This same strategy—informed initial analysis followed if necessary989

by a supplemental masked evaluation—also should be the usual practice for neuropathology

990

peer reviews oriented toward PNS lesions.

991
992
993

IV. Neuropathology Documentation
With respect to communication of PNS neuropathology data, the final report for a

994

toxicity study should contain all the parts of a conventional pathology report (e.g., a narrative

995

together with individual animal and summary data tables) while providing detailed

996

descriptions of the particular neuropathology techniques (e.g., fixative solutions andNervous System Sampling Practices, p. 45
997

methods, embedding and staining procedures) used for the study, and ideally the reasons why

998

they were chosen by the institution. Regulatory scientists have repeatedly expressed a999

preference that PNS lesions in the individual animal data tables be referenced to specific1000

anatomical sites (e.g., “sciatic nerve” or “dorsal root ganglion”) rather than more generic1001

terms (e.g., “nerve” or “peripheral nerve” or “ganglion”), and that the key PNS structures that

1002

were sampled are explicitly stated in the report. When assessing autonomic ganglia,

1003

institutions retain the discretion regarding whether or not findings in non-protocol-specified

1004

neural structures (e.g., intramural autonomic ganglia in protocol-specified organs, muscle

1005

spindles) are to be recorded as a separate tissue (i.e., “enteric ganglia”) or as a notation under1006

the tissue in which they reside (i.e., “heart”, “jejunum”, or “urinary bladder”). This choice1007

may differ for Situation 1 (general toxicity study with no in-life PNS signs) vs. Situations 2,

1008

3, or 4 (studies for which PNS neurotoxicity is a concern). The Working Group concurs that1009

the use of specific terms for protocol-specified PNS sites represents the optimal practice for1010

reporting PNS lesions. If full methodological details are not included specifically in the final

1011

report, they should be made available in an institutional reference document (e.g., SOP)

1012

detailing the PNS sampling and trimming scheme.

1013
1014

V. Discussion

1015

The Working Group unanimously holds that these “best practice” recommendations for1016

PNS sampling, processing, and evaluation are detailed enough to provide for a systematic

1017

analysis of the PNS in GLP-type toxicity studies for four distinct neurotoxicity scenarios and1018

yet still sufficiently flexible to allow their implementation via relatively modest revisions of1019

existing institutional practices. The experiences of Working Group members suggest that

Nervous System Sampling Practices, p. 46
1020

PNS sampling at many institutions already approaches or conforms to the recommendations

1021

set forth here, especially for Situations 1, 2, and 4—with the likely exception of the preferred

1022

plastic embedding medium (as discussed below). Therefore, adoption of these best practice1023

recommendations should not represent a major departure from current practice for these three1024

scenarios. The sampling recommendations where autonomic PNS neurotoxicity is a concern

1025(Situation 3) likely will require adjustments to existing institutional practices; given the1026

extensive autonomic control of many physiologic processes, a discussion of what spectrum

1027

of clinical signs might suggest a general effect on the autonomic nervous system warranting

1028

increased autonomic PNS sampling also will be in order. Common sense will need to be1029

utilized during implementation of these recommendations as certain common clinical1030

observations (e.g., emesis in dogs and nonhuman primates) occurring in isolation seldom will1031

indicate the existence of autonomic PNS neurotoxicity, and should not automatically be1032

investigated as such. In short, the decision regarding which PNS tissues to sample and1033

evaluate should be made using a “weight of evidence” approach where expanded PNS

1034

sampling and evaluation is done only in Situations where the PNS represents an important

1035

target system that is likely to be an important factor in the risk assessment.

1036

The principal adjustment that may be needed at many institutions to conform to the1037

Working Group’s “best practice” recommendations is to modify the plastic embedding

1038

protocol for PNS tissues. Current practice where PNS neurotoxicity is a concern (i.e.,1039

Situations 2, 3, and 4) often employs soft plastic media (e.g., GMA or MMA) for routinely

1040

fixed (i.e., NBF only) tissue. However, the Working Group unanimously agrees that optimal

1041

PNS preservation (especially of myelin) requires initial fixation in MFF, post-fixation in

1042

glutaraldehyde followed by osmium, and embedding in hard plastic resin. The Working

Nervous System Sampling Practices, p. 47
1043

Group recognizes that many test facilities and contract histology laboratories may not be1044

equipped at present with the specialized microtomy and hazardous waste reclamation

1045

equipment and procedures required to prepare hard plastic blocks and sections. Nonetheless,1046

the Working Group unanimously judges that the data quality obtained using hard plastic-1047

embedded cross sections of osmicated nerves offers the most effective means for meeting the1048

intent of regulatory guidelines that require plastic embedding (EPA, 1998a). Indeed, the1049

Working Group consensus is that time spent evaluating one optimally processed nerve1050

sample—a cross section post-fixed in both glutaraldehyde and osmium tetroxide, embedded1051

in hard plastic resin, and then cut at 1 m—will be of greater value in understanding the1052

nature and mechanism of toxicant-induced lesions in the PNS than will be the examination of1053

multiple sections made using routine methods (i.e., NBF-fixed, non-osmicated, paraffin-1054

embedded, 5 m thick) or currently accepted specialty techniques (i.e., NBF-fixed, non-1055

osmicated, soft plastic-embedded, 2 m thick). A majority of the Working Group agrees that1056

hard plastic embedding of nerve for general toxicity studies where PNS neurotoxicity is not

1057

expected (Situation 1) is not feasible as a routine practice.

1058
1059

VI. Concluding Remarks

1060

Current approaches to investigating PNS neurotoxicity during GLP-type toxicity studies

1061

vary to some degree across institutions, and appear to be distinguished more by application

1062

of a few time-tried methods rather than a reasoned exploration of the PNS as a potential

1063

target site for toxicity. The procedures for PNS collection, processing, analytical, and1064

reporting practices should depend on the aims of the study, and thus to a fair degree on1065

institutional preference. However, a substantial improvement in the risk assessment for PNS

Nervous System Sampling Practices, p. 48
1066

neurotoxicity may be gained by improving the consistency of PNS sampling, processing, and1067

evaluation. The STP believes that adoption of these “best practice” recommendations will1068

provide a systematic yet malleable strategy for increasing the consistency, and thus the1069

quality, of PNS sampling, processing, and analysis among institutions and across geographic

1070

regions over time.

1071

Continuing advances in diverse fields like computational biology and non-invasive1072

imaging (structural and functional) are transforming the modern practice of toxicologic

1073

neuropathology and human risk assessment. The STP believes that these “best practice”

1074

recommendations for PNS collection, processing, and evaluation may serve as a logical1075

morphological “gold standard” against which emerging technologies and experimental

1076

neurotoxicity models may be measured.

1077
1078
1079
1080
1081
1082

VII. Acknowledgments
The authors thank Dr. William Valentine for the generous provision of images used inthis paper.

Nervous System Sampling Practices, p. 49
1083
1084

VIII. References

1085

Abram, S.E., Yi, J., Fuchs, A. and Hogan, Q.H. (2006). Permeability of injured and intact

1086
1087
1088

peripheral nerves and dorsal root ganglia. Anesthesiology, 105, 146-153.
Armstrong, R.B. and Phelps, R.O. (1984). Muscle fiber type composition of the rat
hindlimb. Am J Anat, 171, 259-272.

1089

Azhary, H., Farooq, M.U., Bhanushali, M., Majid, A. and Kassab, M.Y. (2010).

1090

Peripheral neuropathy: differential diagnosis and management. Am Fam Physician,1091

81, 887-892.

1092

Bailey, C.S., Kitchell, R.L., Haghighi, S.S. and Johnson, R.D. (1988). Spinal nerve root

1093

origins of the cutaneous nerves of the canine pelvic limb. Am J Vet Res, 49, 115-119.

1094

Bhathal, P.S., Gerkens, J.F. and Mashford, M.L. (1974). Spermatic granuloma of the

1095
1096
1097

epididymis in rats treated with guanethidine. J Pathol, 112, 19-26.
Bilbao, J.M. and Schmidt, R.E.(2015). Biopsy Diagnosis of Peripheral Neuropathy,
Springer, New York.

1098

Bolon, B., Anthony, D.C., Butt, M., Dorman, D., Green, M.V., Little, P., Valentine,

1099

W.M., Weinstock, D., Yan, J. and Sills, R. (2008). Current pathology techniques

1100

symposium review: Advances and issues in neuropathology. Toxicol Pathol 36,

1101

871-889.

1102

Bolon, B., Bradley, A., Butt, M., Jensen, K. and Krinke, G. (2011). Compilation of1103

international regulatory guidance documents for neuropathology assessment during

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Nervous System Sampling Practices, p. 59
VII. Tables
Table 1: Different GLP-type Nonclinical Toxicity Studies in Which PNS Neurotoxicity is Evaluated“Tier I” Studies

Situation 1

Situation 2

Situation 3“Tier II” Studies
Dedicated
Neurotoxicity
StudiesSituation 4

None

Somatic(suspected)

Autonomic(suspected)

Somatic and/or
Autonomic (expected)

X

X

X



X

X

X

±



N/A

N/A

N/A

N/A



±



N/A

±



±

General Toxicity Studies
Parameter
PNS Neurotoxic Presentation
Historical Evidence of Neurotoxicity
 Known potential for neurotoxicity (CNS or PNS) Suspected potential for neurotoxicity based on the putativemode of action (MOA) and/or quantitative structure-activity
relationships (QSAR) modeling
In-life Evidence of PNS Neurotoxicity
 None Signs suggest somatic (sensorimotor) PNS neurotoxicity—
abnormal movement, circling, difficulty walking, lamenessof unknown origin, generalized muscle weakness) Signs suggest autonomic PNS neurotoxicity—anomalies ingastrointestinal motility, heart rhythms, micturition (urinary
retention or incontinence), ocular responsiveness (mydriasis
and miosis), salivation, or vascular tone, and formation of
sperm granulomas

Abbreviations: CNS = central nervous system, N/A = not applicable, PNS = peripheral nervous system
Symbols:  = present, X = not present, ± = may be present

Nervous System Sampling Practices, p. 60
Table 2: Recommended Sampling for Peripheral Nervous System (PNS) Tissues During Nonclinical Toxicity Studies

Situation 3
Autonomic(suspected)

Dedicated
Neurotoxicity Studies
Situation 4
Somatic and/or
Autonomic (expected)



















X




d
d
X
X
d
d
 d
X

2 or more (for both
C and L)







X
X


X
X

2 or more (forboth C and L)




d
d
d
d
Assess if present
d
d
d
d

2 or more (for all
regions: C, T, L)




X
X
X
As available insitu in protocol‐
specified organs(e.g., intestines)

X
XX
As available in situ
in protocol‐
specified organs(e.g., intestines)X
As available in situ inprotocol‐specified
organs (e.g.,
intestines)

As available insitu of protocol‐
specified organs(e.g., urinary
bladder)

As available in situ
in protocol‐
specified organs(e.g., urinary
bladder)

Examine at least 2
specific ganglia (e.g.,
in walls of heart or
urinary bladder)

Assess if present
Assess if present
As available in situ inprotocol‐specified
organs (e.g.,
intestines)
As available in situ inprotocol‐specified
organs – use scheme
for Situation 3 if in‐life autonomic signs
are observed

General Toxicity Studies

Parameter

Parameter Options

PNS Neurotoxic Presentation
Sampling (nerves and ganglia)
Somatic PNS
Mixed nerves

Motor-only nerve
Sensory-only nerve

Cranial nerve
Ganglia
Other
Autonomic PNS
Autonomic Nerve

Autonomic Ganglia

Bilateral (all species)
Sciatic
TibialFibular (common peroneal)
Sural (rodent)
Caudal (rodent)
Ventral spinal nerve root
Muscle spindles (in large skeletal muscles)
Saphenous (canine, rodent)
Sural (nonhuman primate)
Plantar (canine, rodent)
Dorsal spinal nerve root
V (trigeminal) a
Dorsal root ganglia (DRG) – cervical (C) andlumbar (L) ± thoracic (T) regions a
Trigeminal (cranial nerve V) a
Vertebral column b
Hind limb (intact) c
Cranial nerve X (vagus = parasympathetic)
Sympathetic chain (sympathetic)
Nerve trunks attached to autonomic ganglia
Enteric ganglia

Parasympathetic ganglia

Situation 1

Situation 2

None

Somatic (suspected)


Alternative(instead of sciatic)
X
X
X
X
X
X
X
X
X
X
X

d

Nervous System Sampling Practices, p. 61

Autonomic CNS centers

Effector Organs
Skeletal Muscle

As available in situ inprotocol‐specified
organs – use scheme
for Situation 3 if in‐life autonomic signs
are observed

X

X

Sample at least 2
distinct sites (e.g.,cervicothoracic,
cranial cervical,
cranial mesenteric, or
sympathetic chain)









X

X

Sample (using
neuroanatomic atlas)as needed based onneurological signs

Use scheme for
Situation 3 if in‐life
autonomic signs areobserved

Spinal cord, lateral/intermediate column inthoracic division (sympathetic)

X

X





One or more (biceps femoris, a specific headof the quadriceps femoris, gastrocnemius,
soleus, and/or diaphragm)
Muscle weights

One site(gastrocnemius
recommended)X

Two or more sites(gastrocnemius
recommended)

One site(gastrocnemius
recommended)X

Two or more sites(gastrocnemius
recommended)

Sympathetic ganglia

Hypothalamus: paraventricular nucleus(PVN) – present in routine brain sections
Brain nuclei (parasympathetic) for cranial
nerves III, VII, IX, and X

As needed c

As needed c

Abbreviations: C = cervical, DRGs = dorsal root ganglia, L = lumbar, T = thoracic

Symbols:  = collected, X = not collecteda

May be prepared in situ in rodents followed by decalcification of vertebral cross sections or skull.

b

Vertebral column (intact in rodents, cervical and lumbar segments in non-rodents) should be kept in case DRGs and spinal nerve roots areneeded.

c

A hind limb (intact in rodents, intact or distal in non-rodents) should be kept in case additional somatic nerves are needed; overlying muscle
should be reflected to expose nervesdDenotes examples of distal nerve branches that may be evaluated (along with sciatic and tibial nerves) as the minimal set needed for systematic
evaluation of neurotoxicity affecting the somatic PNS; in general, a sensory-only nerve (i.e., more distal branch) will be the preferred choice.
Situations 2, 3, and 4 suggest sampling at least three spinal nerve locations (inclusive of sciatic and/or tibial nerves), at least one of which should
be sensory-only if a sensory neuropathy is suspected.

Nervous System Sampling Practices, p. 62
Table 3: Recommended Baseline Processing Strategies for Peripheral Nervous System (PNS) During Nonclinical Toxicity Studies

Situation 1

Situation 2

Situation 3

Dedicated
Neurotoxicity
StudiesSituation 4

NoneSomatic (suspected)

Autonomic(suspected)

Somatic and/or
Autonomic (expected)

Immersion
ImmersionNerves andganglia
As needed d(for nerves)
Used as needed
for TEM and/or
prior to osmium
post‐fixation
At least 1 somatic
N (C section for
hard plastic
embedding)
Ganglia
Somatic N (C/L)
Cranial N
ImmersionPerfusion
X

Nerves and ganglia

As needed d(for nerves)
Used as needed for
TEM and/or prior
to osmium post‐
fixation
At least 1 somatic N
and 1 autonomic N(C section for hardplastic embedding)Ganglia
Somatic N (C/L)
Cranial N
1 Somatic N (C)and if possible
1 autonomic N (C)– after osmium
post‐fixation

As needed d(for nerves)
Used as needed for
TEM and/or prior
to osmium post‐
fixation

General Toxicity Studies
Parameter
PNS Neurotoxic Presentation

Parameter Options

Sampling (nerves and ganglia)
Processing (nerves and ganglia)
Trimming Orientation – Nerve
Fixation Method
Fixative

Bilateral (all species)
Unilateral (all species)
Longitudinal and cross (transverse)

Post-fixative (by immersion)

Neutral buffered 10% formalin (standard)
4% formaldehyde (methanol-free) a
TEM fixative (with glutaraldehyde at 1%
or greater concentration) b
Glutaraldehyde (at concentration of 1% or
greater) b

X
X
X

Osmium tetroxide (at 1%) b
X
Embedding Medium

ParaffinGanglia
Somatic N (C/L)

Hard plastic resin c

Staining (paraffin sections)

Hematoxylin and eosin (H&E)
Silver stains (for axons – Bielschowsky’s,
Bodian’s. or Holmes)
Myelin stains (e.g., Luxol fast blue,
Marchi)Cell type-specific biomarkers (e.g.,X

1 Somatic N (C) –after osmium
post‐fixationdAs needed

At least 2 somatic N(C sections for hardplastic embedding)Ganglia
Somatic N (C/L)
Cranial N
At least
2 Somatic N (C) –after osmium post‐
fixationd

As needed dXAs neededXAs needed d

As needed d

As needed dXAs needed d

As needed d

As needed dNervous System Sampling Practices, p. 63

Staining (hard plastic sections)intermediate filaments, neurotransmitters)
Toluidine blue

X







Abbreviations: C = cross (transverse) orientation, L = longitudinal orientation, N = nerve, TEM = transmission electron microscopy

Symbols:  = utilized, X = not utilizedaMethanol-free 4% formaldehyde is prepared from paraformaldehyde powder to avoid the presence of methanol (a stabilizing agent that can
induce myelin vacuolation as an artifact)b

Post-fixation in glutaraldehyde (e.g., modified Karnovsky’s solution: methanol-free 2% formaldehyde [from paraformaldehyde powder]
combined with medical-grade 2.5% glutaraldehyde) followed by osmium are required for optimal myelin preservationcSoft plastic (e.g., glycol methacrylate [GMA]) is not an acceptable substitute for hard plastic resin (e.g., araldite, epon, or Spurr’s)d“As needed” decisions remain at the discretion of the institution

Nervous System Sampling Practices, p. 64
Table 4: Spinal Cord Origins of the Principal Forelimb (Brachial) and Hind Limb (Sciatic) Nerves for Common Vertebrate Species
Species

Nerve

Main Source Segments of Spinal Cord

ChickenBrachial

C12 – C15

(Jungherr, 1969)

Sciatic

Syn3 – Syn8

(Jungherr, 1969)

Brachial

C4 – T2(Kaufman and Bard, 1999)

Sciatic

L3 – L4 **(Rigaud et al., 2008)

Brachial

C4 – T1 (± T2)

Sciatic

L4 – L5 **Brachial

C6 – T2 (± C5)(Ghoshal, 1975a, Sharp et al., 1990)

Sciatic

L4 – S2(Ghoshal, 1975a, Bailey et al., 1988)

Brachial

C5 – T1(Ghoshal, 1975b)

Sciatic

L5 – S2 (± L4)(Ghoshal, 1975b)

Brachial

C5 – T1(Turnquist and Minugh-Purvis, 2012)

Sciatic

L1 – S2(Turnquist and Minugh-Purvis, 2012)

Mouse
Rat
Dog
Pig
PrimateReference(Greene, 1935)(Rigaud et al., 2008)

Abbreviations: C = cervical, L = lumbar, S = sacral, Syn = synsacral (representing the fused lumbar and sacral vertebral segments)** Denotes that the origin varies with the strain

Nervous System Sampli
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1363
1364
1365
1366
1367
1368
1369
1370
1371
1372
1373
1374
1375
1376
1377
1378
1379
1380
1381
1382
1383
1384
1385
1386
1387
1388
1389
1390
1391
1392
1393
1394
1395
1396
1397
1398
1399
1400
1401
1402
1403
1404
1405

VII. Figures
Fig 1.

Locations for harvesting somatic nerves from the rodent hind limb. Nerves: a =sciatic; b = tibial; c = common peroneal (fibular); d = lateral sural; e = plantar.
Muscles: 1 = gluteus medius; 2 = biceps femoris; 3 = semitendinosus; 4 = quadriceps
femoris; 5 = gastrocnemius lateralis; 6 = rectus femoris; 7 = gastrocnemius medialis; 8= tibialis cranialis. Bones: P = patella; T = tibia. (Schematic diaphragm adapted from(Popesko et al., 2003) by permission of the Publisher).

Fig 2.

Diagrams show how to approach and localize dorsal root ganglia (DRG) associatedwith the origin of the sciatic nerve. Left panel: Note that caudal segments of lumbar
spinal cord (L4 to L7) are displaced cranially relative to the DRG and vertebrae of thesame number. Regional anatomy is based on the dog vertebral column. Right panel:
DRG are best approached via removing the vertebral arches (at the location of thedotted lines at 2 o’clock and 10 o’clock) using bone-cutting rongeurs. Images craftedby Mr. Tim Vojt.

Fig 3.

Two cervical ganglia, the cranial (superior) cervical ganglion (C, a part of thesympathetic division) and the caudal vagal (nodose) ganglion (X, a visceral afferent[i.e., sensory] element), may be isolated adjacent to the trachea in the vicinity of thebifurcation of the carotid artery. Samples: left column = adult rat (provided courtesyof Dr. Magalie Boucher, Pfizer, Inc.); right = adult Beagle dog showing collection ofboth the cranial cervical ganglion (C) and caudal vagal ganglion (X) in the same
histologic section. Processing (right image): immersion fixation in neutral buffered
10% formalin, paraffin embedding, sectioning at 4 μm, H&E staining.

Fig 4.

Nerves should be available for histopathologic analysis in both cross (top row) andlongitudinal (bottom row) orientations. The cross (transverse) view allows comparisonof the numbers and densities of myelinated nerve fibers (large-caliber, pale blue axons
bounded by thick, dark myelin sheaths) and possibly unmyelinated fibers (smallcaliber axons with minimal myelin [often found in small clusters]), although such finediscrimination is only possible in specially prepared nerves exhibiting high contrast
between axons (pale) and myelin sheaths (dark) (upper left panel) and not in routinely
processed sections (upper right panel) where contrast is modest and extensive clear
space exists between as a very common processing artifact. The longitudinal plane
permits axonal and myelin integrity to be assessed over extended distances. Samples:
sciatic nerve from normal (i.e., control) adult rat. Processing: left column = wholebody perfusion fixation with 4% glutaraldehyde, post-fixation in 1% osmium tetroxide,
hard plastic resin (epon) embedding, sectioning at 1 μm, toluidine blue staining; rightcolumn = immersion fixation in neutral buffered 10% formalin, no glutaraldehyde or
osmium post-fixation, paraffin embedding, sectioning at 4 μm, H&E staining. [Imagesof hard plastic-embedded nerves (left column) were provided courtesy of Dr. William
Valentine, by permission.]Nervous System Sampli
1406
1407
1408
1409
1410
1411
1412
1413
1414
1415
1416
1417
1418
1419
1420
1421
1422
1423
1424
1425
1426
1427
1428
1429
1430
1431
1432

Fig 5.

Placement of multiple ganglia in a single cassette ensures that sufficient tissue isavailable for histopathologic evaluation. The grouping may be ganglia from all spinal
cord divisions (as shown here) or alternatively grouping as a single spinal cord
division (e.g., cervical, thoracic, or lumbar). Sample: dorsal root ganglia and spinal
nerve roots (arrows) from adult control rat. Processing: immersion fixation in neutral
buffered 10% formalin, paraffin embedding, sectioning at 4 μm, H&E staining.

Fig 6.

Soft plastic (left panel) is not a suitable medium to comply with regulatory guidelinesthat mandate plastic embedding of nerves, as soft plastic provides no significant
improvement in resolution relative to conventional paraffin embedding (right panel).
Samples: sciatic nerve from normal (i.e., control) adult rat. Processing: left column =whole-body perfusion fixation in neutral buffered 10% formalin, no osmication, soft
plastic (glycol methacrylate) embedding, sectioning at 2 μm, H&E staining; rightcolumn = immersion fixation in neutral buffered 10% formalin, no osmication,
paraffin embedding, sectioning at 4 μm, H&E staining.

Fig 7.

Special methods used to highlight nerve fibers include silver and myelin stains. Upper
panel: Bielschowsky’s silver stain demonstrates axons and neuronal cytoplasm as dark
profiles against a pale background. Arrows indicate swollen axons. Lower panel:
Luxol fast blue stain reveals myelin as intact dark blue sheaths. A single degenerating
axon is revealed at the bottom of the image as a series of vacuoles containing
fragmented debris. The myriad tiny, clear vacuoles in the myelin sheaths represent a
processing artifact. Processing: immersion fixation in neutral buffered 10% formalin,paraffin embedding, sectioning at 4 μm.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7