Myelin Methods: A Mini-Review

Abstract

Hematoxylin and eosin (H&E) staining is a suitable approach for detecting substantial structural changes in neural tissues but is less sensitive for identifying subtle alterations to subcellular structures and various chemical constituents, including myelin. Neurohistological methods to better evaluate myelin integrity by light microscopy include acidophilic dyes (eg, eriochrome cyanine R, toluidine blue [used with hard plastic sections]); lipoprotein-binding dyes (eg, Luxol fast blue [LFB], Weil’s iron hematoxylin); lipid impregnation with metals (eg, Marchi’s, which uses osmium tetroxide for en bloc staining before embedding); and immunohistochemical (IHC) methods to highlight various antigens (eg, myelin basic protein [MBP] and peripheral myelin protein 22 [PMP22]). Some IHC methods reveal enhanced marker expression in damaged myelin (eg, matrix metalloproteinase-9 [MMP9], S100). In neuropathology investigations, H&E is the first-tier screening method, whereas myelin stains (often LFB alone or in combination with dyes that highlight other structural elements) are second-tier procedures performed in combination with other neurohistological procedures to examine neuroaxonal injury and/or glial responses. The choice of myelin method depends on such considerations as cost, institutional preference, the procedure (fixation and embedding medium), and the study objective.

Keywords

myelin nervous system neurohistology neuropathology nonclinical toxicity safety testing

Neurological diseases affect millions of people globally, and myelinating cells or myelin are common targets in both the central and peripheral nervous systems (CNS and PNS, respectively). Principal myelin-damaging conditions in humans and animal models include leukodystrophies and multiple sclerosis (MS) in the CNS as well as Charcot-Marie-Tooth disease (CMT), chronic inflammatory demyelinating polyneuropathy (CIDP), and Guillain-Barré syndrome (GBS) in the PNS. Demonstrated pathogeneses of myelin diseases include autoimmune attack (CIDP, GBS, MS); inherited genetic defects (CMT, hypomyelinating and metachromatic leukodystrophies); or pathogen reactivation due to immunosuppression (progressive multifocal leukodystrophy [PML] due to JC virus). Neuropathological evaluation is an important tool when diagnosing and staging these diseases in basic investigations of neural disease models and in nonclinical safety testing. From a neuropathology standpoint, myelin diseases usually result from either primary damage to myelinating cells or myelin or secondary disintegration of myelin following primary neuroaxonal injury.

In the nonclinical setting, multiple neurohistological methods are available to assess myelin integrity. This mini-review highlights some common options, comparing and contrasting their qualities and costs.

Myelin Biology

Myelin is a lipid-rich (approximately 80%) insulating sheath that forms around nerve fibers throughout the nervous system.²⁴ Myelin is produced by plasma membrane extension from oligodendrocytes in the CNS, myelinating Schwann cells in the PNS, and olfactory ensheathing cells (OECs, a CNS macroglial cell) in cranial nerve I (CN I [olfactory nerve]).

Myelin represents the laminar arrangement of concentric membrane layers around a central nerve fiber, compressed to remove the cytoplasm between the adjacent laminae (Figure 1). Myelin sheaths are arranged as alternating layers of hydrophobic constituents (hydrocarbon tails of lipid molecules and hydrophobic segments of transmembrane proteins) and hydrophilic components (anionic heads of polar lipids and basic proteins). Myelin thickness varies from a single layer of glial membrane for “unmyelinated” autonomic nerve axons to many layers of glial membrane (several hundred) in thick myelin sheaths surrounding high-speed proprioceptive nerve fibers in the CNS and PNS.²² Depending on their location, myelinating cells encompass different numbers of nerve fibers. In general, oligodendrocytes myelinate multiple nerve fibers (up to 50 per glial cell) while myelinating Schwann cells envelop a single fiber.¹⁸ However, non-myelinating Schwann cells can encircle small clusters of multiple axons (a neurite-glial complex termed a Remak bundle or Remak fiber). Myelination (formation of myelin) is regulated by trophic signaling mediated by interactions between axon proteins with receptors on myelinating cells.²⁹

Figure 1.

Appearance of myelin surrounding myelinated and unmyelinated nerve fibers in the peripheral nervous system. Panel A: A large-caliber axon (Ax) with six dark, oval to round mitochondria and pale, granular axoplasm is enveloped by a thick myelin sheath (M). This plane of section is located at the mid-internode (ie, halfway between two nodes of Ranvier) as shown by the nucleus (N) and abundant cytoplasm (C) of the myelinating Schwann cell that is generating the myelin sheath. [Image courtesy of Dr. William Valentine.] Panel B: Multiple nerve fibers are evident, including highly (H) and lightly (L) myelinated axons and a cluster of unmyelinated (U) axons (the group of which is termed a Remak fiber). Note that a single Schwann cell (S [lower middle]) is producing the myelin sheath of the lightly myelinated axon, while another Schwann cell (S [right]) is enveloping all of the unmyelinated fibers. Arrows denote the basement membrane surrounding the Schwann cell and its axon(s). Transmission electron micrographs of a sciatic nerve cross-section. Species: adult rat (strain unspecified). Processing (both panels): glutaraldehyde (4%) perfusion, osmium tetroxide (1%) immersion, hard plastic resin embedding, 600-nm-thick “thin” section, uranyl acetate and lead citrate. [This two-panel figure is reproduced with a modified legend from ID Pardo et al,²² “Atlas of normal microanatomy, procedural and processing artifacts, common background findings, and neurotoxic lesions in the peripheral nervous system of laboratory animals,” Toxicol Pathol 48(1): 105-131, 2020, by courtesy of Sage.].

The structural organization and electrophysiological properties of myelin are comparable in the CNS and PNS although the molecular composition (ie, differing complements of proteins detectable as immunohistochemical [IHC] markers) differs between the two sites. The g-ratio of myelinated fibers, calculated as the axon diameter divided by the nerve fiber (axon plus myelin) diameter, generally falls between 0.6 and 0.7. This ratio is highly conserved regardless of the axon diameter. The length of axon covered by myelin sheath derived from a single glial cell is about 1 mm (in the PNS). Adjacent myelin segments are separated by small gaps (“nodes of Ranvier”) that are approximately 1 μm in length; the myelin segment between two nodes of Ranvier is termed an “internode.” Electrical impulses traveling along nerve fibers jump between nodes by saltatory conduction due to the insulating properties of myelin sheaths while at nodes the impulse slows substantially since it must cross the node by conventional membrane depolarization.²⁹ Any loss of myelin integrity deprives the axon of insulation, thereby thwarting saltatory conduction and greatly slowing neurotransmission until myelin sheaths can be rebuilt. Remyelination of intact axons after primary myelin injury is slow at best, less effective in the CNS, and produces thinner sheaths with shorter internodes (ie, an increased number of nodes of Ranvier); a greater number of nodes reduces the efficiency of saltatory conduction. Reduced myelin quality related to myelin injury and/or insufficient repair may yield no clinical evidence of disease, but more severe myelin injury may lead to altered electrophysiological conduction or neurological signs because denuded axons transmit impulses slowly or disintegrate due to the loss of neurotrophic support to the damaged myelinating cells.²⁹

Myelin Methods

In safety testing, neuropathological evaluation is one means for surveying whether a novel product has neurotoxic potential. With respect to myelin damage, morphological responses to primary myelin damage observed in test species may include demyelination (loss of previously formed myelin), dysmyelination (reduced myelin production during development), vacuolation (fluid accumulation between myelin laminae [“splitting”] or among myelinated nerve fibers), and variable proliferation of myelinating glial cells (seen mainly in the PNS). Several morphologic methods are available to examine myelin integrity in animal (and human) tissues. The choice of myelin method depends on such factors as the study objectives, histological processing, and the procedure cost (Table 1).

Table 1.

Examples of common neurohistological methods for light microscopic evaluation of myelin integrity.

						Comparison of method attributes
Tier	Method		Method principle	Fixation	Embedding	Cost	Simplicity	Speed
1	Hematoxylin and eosin	H&E	Tinctorial dye	Immersion or perfusion	Paraffin^a	Low	Easy	Fast
2	Luxol fast blue	LFB	Metal-enhanced dye	Immersion or perfusion	Paraffin^a	Low intermediate	Intermediate	Intermediate
	Myelin basic protein	MBP	Immunohistochemistry^b	Immersion or perfusion^c	Paraffin^a	High intermediate	Intermediate	Intermediate
	Toluidine blue	TB	Tinctorial dye	Immersion or perfusion^c	Hard plastic^d	Low	Easy	Fast
3	Lead citrate		Metal impregnation (for TEM)	Perfusion^c	Hard plastic	High	Complex	Slow
	Uranyl acetate		Metal impregnation (for TEM)	Perfusion^c	Hard plastic	High	Complex	Slow

Abbreviation: TEM = transmission electron microscopy.

The stain is conventionally used for paraffin-embedded tissues but can be employed on samples embedded in “soft plastic” (glycol methacrylate or methyl methacrylate).

Other antigens for myelin-focused immunohistochemical (IHC) assays include myelin-associated glycoprotein (MAG), peripheral myelin protein 22 (PMP22), proteolipid protein 1 (PLP1), and S100, among others.

Perfusion-fixed tissues to be embedded in “hard plastic” (epoxy resin) are routinely post-fixed by immersion in osmium tetroxide.

Toluidine blue may be used on paraffin-embedded tissues but is usually reserved for hard plastic-embedded sections in conventional animal toxicity studies (where tissue processing for hard plastic embedding offers better preservation of fine myelin structural detail compared with tissue processing for paraffin embedding).

Routinely Processed Tissue

Animal toxicity studies rely on batch processing of multiple tissues to assess the safety of novel products. In this regard, CNS and PNS tissues represent only some of the components to be evaluated, so histology processing typically proceeds using an “average” protocol designed to accommodate all tissues rather than the specific needs of neural tissues. If the nervous system is the main target organ of interest, such common protocols may be modified to optimize histology processing of the lipid-rich CNS and PNS tissues.^4,8,13

By design, the pathology evaluation for routine animal toxicity studies uses conventional histological processing and bright-field microscopic evaluation. Conventional processing entails fixation by immersion in neutral buffered 10% formalin (NBF, which includes ~1% methanol as a stabilizer in commercial formulations¹⁶) followed by embedding in paraffin. Importantly, such “formalin-fixed, paraffin-embedded” (FFPE) tissues preserve myelin structure but may lead to various processing artifacts^10,11,22 (eg, myelin vacuolation [Figures 2A, 2B and 2C] and neurokeratin formation [Figure 2D]) that represent confounders when interpreting subtle structural changes indicative of potential myelin damage. Moreover, routine tissue processing includes dehydration in graded solvents such as ethanol and xylene, which can extract myelin lipids.^22,32 Multiple stains may be used to examine myelin in FFPE tissue sections.

Figure 2.

Processing artifacts in myelin typically present as spaces (vacuoles) within neural tissues. In the CNS, focally extensive collections of large vacuoles with irregular margins often occur in deep white matter tracts of the cerebellum (Panel A), and less frequently thalamus (Panel B) and spinal cord, of immersion-fixed, paraffin-embedded sections. Some artifactual vacuoles may contain pale, variably homogeneous material (dotted circle of Panel B); alternate names for such entities are “Buscaino bodies” or “mucocytes.” These vacuoles are reported to be more extensive if tissues were exposed to alcohol for an extended period—such as retention in the alcohol bath on an automated tissue processor over the weekend—possibly due to lipid extraction. In the PNS, myelin in immersion-fixed, paraffin-embedded nerve sections may exhibit extensive vacuolation (“bubbling,” Panel C) or neurokeratin formation (Panel D). Staining: H&E. Species: Beagle dog (Panels B and D); mouse, Panels A and C). [Panels A and C are reproduced with modified legends from B Bolon et al,⁵ “Nervous system,” in Haschek and Rousseaux’s Handbook of Toxicologic Pathology, 3^rd ed., Vol 3 (Haschek WM, Rousseaux CG, Wallig MA, eds), San Diego, Academic Press, 2013:2005-2093, by courtesy of Elsevier. Panel D is repeated with a modified legend from ID Pardo et al,²² “Atlas of normal microanatomy, procedural and processing artifacts, common background findings, and neurotoxic lesions in the peripheral nervous system of laboratory animals,” Toxicol Pathol 48(1): 105-131, 2020, by courtesy of Sage.]

Hematoxylin and eosin

Routine hematoxylin and eosin (H&E) is the preferred method for examining myelin in animal toxicity studies. The reasons for this preference are that the two dyes are inexpensive, can be bought readily in large quantity and high quality from many vendors, and in many cases can be applied to FFPE sections of neural tissues produced using the same histology processing protocol that applies for non-neural tissues. In addition, H&E offers good contrast between basophilic (hematoxylin-binding) cell nuclei and eosinophilic cytoplasm and membranes. Thus, myelin in CNS white matter and PNS ganglia and nerves is eosinophilic, while myelinating cell nuclei and sometimes myelin-wrapped neurites (axons) are basophilic (Figure 3A). The main features of primary myelin damage noted above can be detected in H&E-stained sections. However, for end-stage lesions (where both axons and myelin are disintegrating concurrently), H&E cannot reliably distinguish between primary neuroaxonal and primary myelin insults.

Figure 3.

Tinctorial (dye) methods for demonstrating myelin. Panel A: Hematoxylin and eosin (H&E) of the deep cerebellar nucleus of a cynomolgus macaque. Panel B: Luxol fast blue (LFB) in the human striatum. Panel C: Weil’s iron hematoxylin in the human brainstem. Panel D: Toluidine blue (TB) in the distal sciatic nerve of a rat. Processing conditions: formalin fixation by immersion, paraffin embedding (Panels A, B, C) or plastic resin embedding (Panel D).

Luxol fast blue

Additional tinctorial (colored) dyes may be used if warranted on routine FFPE sections to emphasize myelin.¹³ The rich color of these dyes is enhanced by metals within the dye solutions, where the metal ions act as mordants to better fix the dyes to lipid-rich myelin membranes.

The Luxol fast blue (LFB, also called Klüver-Barrera [KB]) method is the most common myelin method for nonclinical studies. The LFB method is a copper-based phthalocyanine dye that stains myelin a brilliant blue (Figure 3B). Protocols for this method are easy, readily available online and in publications,^13,14,17 and reliable. In practice, LFB may be used without a counterstain or together with a counterstain. Typical LFB counterstains include hematoxylin, to permit an assessment of myelin-rich domains in the context of general tissue architecture; cresyl violet (CV^13,14), to show Nissl substance (rough endoplasmic reticulum) in neurons, thus providing a clearer distinction of gray matter and white matter domains in the CNS and ganglia; periodic acid–Schiff (PAS⁹), to demonstrate myelin degradation products; or picrosirius red (PR), to detect collagen production. In our experience with animal toxicity studies, LFB is used most often in combination with CV or hematoxylin.

Other metal-based tinctorial methods

Other metal-based staining methods to highlight myelin sheaths are available for use in FFPE tissues. Eriochrome cyanine R (alternate designations: chromoxane cyanine R, solochrome cyanine R, and Mordant blue 3) is an iron-based anionic dye that imparts a blue color to myelin comparable to that of LFB;²⁷ this dye may be used with eosin (to produce an H&E-like staining pattern) or neutral red as a counterstain. Gallyas impregnation (silver-based^15,23), Marchi en bloc staining (osmium-based²⁸), and Weil’s iron hematoxylin (Figure 3C) all impart a black hue to lipid-rich tissues. These additional metal impregnation methods are rarely employed in nonclinical studies because of their high cost (Gallyas); prolonged incubation time (~2 weeks for Marchi); problematic protocols (Gallyas [as with all silver stains] is technically difficult, and Weil’s cannot be used with a counterstain); the toxicity of the reagents (Marchi); and the potential for dark black metal deposits to obscure subtle cell and tissue features in the densely stained myelinated regions.

Toluidine blue

This thiazine cationic dye is a versatile, water-soluble reagent employed in many biological applications.²⁶ The dye binds to anionic elements (cartilage, mast cell granules, mucins, nucleic acids, polar lipids, etc). Importantly, toluidine blue (TB) is metachromatic, a property whereby the final hue of a stained tissue component shifts with the local ionic chemistry. Adjusting the pH of the staining solution alters the ionic state of biomolecules, thus determining the quantity of TB bound by a structure and the final resulting color.

In principle, TB may be used with paraffin-embedded material, in addition to or in place of H&E. In general, however, TB is typically employed for tissues embedded in hard plastic resin (Figure 3D) for neuropathological evaluations performed in the course of routine animal toxicity testing.¹²

Immunohistochemical methods

In some situations, detection of specific myelin components may be warranted to address particular scientific questions. In our experience, such markers are usually examined during hypothesis-based experiments rather than animal toxicity studies due to the availability of LFB as an easy, inexpensive, and reliable procedure to investigate myelin integrity.¹³ In principle, however, the immunolabeling pattern for myelin proteins mimics the distribution of myelin-bound dyes.

Different antigens are appropriate for the detection of CNS and/or PNS myelin. Myelin-associated glycoprotein (MAG), myelin basic protein (MBP (Figure 4)), and proteolipid protein 1 (PLP1) are found in myelin produced by both oligodendrocytes and Schwann cells. Some myelin markers occur in particular nervous system domains. The CNS-specific myelin proteins include 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) and myelin oligodendrocyte protein (MOG), whereas PNS-specific myelin proteins include peripheral myelin protein 22 (PMP22) and S100. Certain markers are upregulated after myelin injury, including matrix metalloproteinase-9 (MMP9)^19,25 when assessed in tissue sections and MBP in cerebrospinal fluid or serum.^30,31 In our experience, MBP is the most common marker assessed by IHC for animal toxicity studies.

Figure 4.

Myelin staining varies with the neural domain. In gray matter, individual myelinated nerve fibers occur as strongly labeled (brown), variably branching linear arrays dispersed among pale neuron bodies. In white matter, the tissue consists chiefly of closely apposed, strongly labeled myelinated fibers with occasional pale basophilic oligodendrocyte nuclei scattered among them. Site: cerebral cortex. Species: cynomolgus macaque. Processing conditions: formalin fixation by immersion, paraffin embedding, indirect immunohistochemistry for myelin basic protein (MBP) with hematoxylin counterstain.

Specially Processed Tissue

Animal toxicity studies to investigate substances intended to produce pharmacologic effects in the nervous system or that are anticipated to induce neurotoxicity may require special fixation and processing procedures. With respect to myelin, optimal preservation is frequently provided by intravascular perfusion (to introduce the fixative solution deep into the nervous system) and plastic embedding (to minimize lipid extraction during processing).

The perfusion fixation procedure may be adjusted depending on the study objectives. Whole-body perfusion is used if both the CNS and PNS are to be evaluated, whereas head-only perfusion may be elected if brain is the primary organ of interest. The most commonly perfused fixatives are methanol-free 4% formaldehyde (MFF, colloquially referred to as “paraformaldehyde”) and mixtures of MFF and medical-grade glutaraldehyde such as Karnovsky’s solution or McDowell and Trump’s solution. Conventional NBF may be employed for perfusion if tissues are destined for paraffin embedding as the solvents in NBF (methanol) or the dehydration steps of histology processing (eg, graded ethanols followed by xylene) are presumed to produce no artifacts that will substantially impede analysis of neural components (especially lipid-rich myelin). Post-fixation of perfused neural tissue by immersion in osmium tetroxide (OsO₄, 1% in phosphate-buffered saline or sodium cacodylate buffer) for 1 to 3 hr at room temperature provides the best preservation of myelin sheaths but is typically not used in routine safety testing due to the limited penetration of osmium into routinely trimmed CNS and PNS tissues and the requirement that this fixative must be handled in a fume hood due to the marked toxicity of OsO₄ vapors.^4,6 Post-fixation with osmium may be used for both paraffin (for nerve) and plastic (for all neural tissues) embedding media.

Peak stabilization of myelin structure is afforded by embedding in plastic. This fact is recognized by inclusion of plastic embedding for nerves (and for CNS tissues when warranted) as a recommendation in guidelines for animal toxicity studies published by the Organisation for Economic Co-operation and Development (OECD)²¹ and U.S. Environmental Protection Agency (EPA).⁷ Importantly, these guidance documents do not specify the type of plastic to use. Soft plastic (eg, glycol methacrylate, methyl methacrylate) has been demonstrated to provide no improvement in myelin stabilization compared with paraffin.⁶ Accordingly, while use of soft plastic technically meets the letter of the guideline, this embedding medium does not permit truly high-resolution evaluation of myelin structure. Both LFB and MBP immunohistochemistry may be performed on neural tissues embedded in soft plastic though these approaches are seldom used since the same methods work in routine FFPE sections (and more consistently so in the case of MBP). In contrast, hard plastic (eg, epoxy resin) provides optimal stabilization of myelin lamination, especially if combined with perfusion fixation or/and OsO₄ post-fixation. Hard plastic may be employed for routinely trimmed neural tissues, most often whole nerves oriented in cross-section and stained with TB;¹² this metachromatic thiazine dye imparts a deep blue color to myelin compared with more pale blue hues for axons and interstitial tissues (Figure 3D). Azure methylene blue basic fuchsin (AMBF) may be substituted for TB when staining nerve.² However, maximal preservation of myelin structure (eg, for transmission electron microscopy [TEM]) involves perfusion fixation with a glutaraldehyde-based fixative initially, subsequent mincing of the tissue into minute (1-mm³) cubes, OsO₄ post-fixation, and epoxy resin embedding. For TEM, optimal myelin stabilization is further enhanced by metal stains such as lead citrate and/or uranyl citrate. The best TEM preparations show intact myelin lamination (Figure 5) without artifactual distortion or vacuolation (where such artifacts are visible in Figure 1 as a few crescentic intra-laminar spaces [Panel A] or curvilinear black deposits [Panel B] in thick myelin sheaths).

Figure 5.

Laminar arrangement of compact myelin in a thick myelin sheath demonstrating the major dense (dark) and intraperiod (light) lines. Transmission electron micrograph of a sciatic nerve cross-section of an adult rat (unspecified strain). Processing: Glutaraldehyde (4%) perfusion, osmium tetroxide (1%) immersion, hard plastic resin embedding, 600-nm-thick “thin” section, uranyl acetate and lead citrate. [This figure is reproduced from B Bolon et al,³ “Current pathology techniques symposium review: Advances and issues in neuropathology,” Toxicol Pathol. 36:871-889, 2008, by courtesy of Sage.]

Myelin Evaluation in Animal Toxicity Studies

The choice of myelin method for animal toxicity studies depends on the situation. For routine general toxicity studies, the default choice for screening myelin integrity is H&E since this stain is cheap, reliable, technically easy, and permits neural tissues to be processed in parallel to other protocol-specified tissues without disrupting the work flow of a histology laboratory. This stain can be used for both paraffin-embedded and soft plastic-embedded sections. In most cases, H&E alone provides an acceptable survey of myelin integrity.

More specific myelin methods are added when evidence of myelin injury is detected in H&E-stained sections or when the test article is anticipated to produce neurotoxicity. The typical stains chosen for animal toxicity studies are LFB or, infrequently, MBP. These two methods are both reliable and also demonstrate good concordance in identifying myelin when applied to serial FFPE sections, but LFB is preferred as it is less expensive and simpler. Toluidine blue is the preferred stain for hard plastic sections.

When assessing sections microscopically, several staining patterns reflect potential injury to myelin. At the sub-gross level, demyelinating diseases such as MS or GBS are visible as regions of myelin pallor, evident as reduced or absent eosin or LFB intensity in affected regions (Figure 6).¹ Lesions resulting from inflammatory processes may be focal or multifocal and are typically asymmetric around the body axis (Figure 6B). In contrast, myelin lesions resulting from neurotoxicity tend to be bilateral albeit with variable symmetry. During light microscopic evaluation, myelinotoxicity may present as degeneration or necrosis of myelinating cells or as increased space within myelin sheaths; in our experience, altered myelin lamination is more common than effects localized to myelinating cells. Common presentations of myelin-associated injury include myelin splitting (Figure 7, left column) or myelin vacuolation (Figure 7, right column). Vacuoles related to myelin injury usually have regular (oval or round) contours and smooth margins, typically as a consequence of fluid accumulation (Figure 7, right column), whereas vacuoles resulting from lipid extraction (ie, processing artifacts) commonly have irregular contours (Figures 2A and 2B).

Figure 6.

Appearance of altered myelin integrity in microscopic sections. Panel A: Brain from a multiple sclerosis (MS) patient showing a typical MS “plaque” characterized by pallor (essentially complete loss of myelin). Processing conditions: formalin fixation by immersion, paraffin embedding, Luxol fast blue. [This figure is reproduced from D Agamanolis, “Neuropathology: An Illustrated Interactive Course for Medical Students and Residents,” 2011,¹ https://neuropathology-web.org/chapter6/chapter6aMs.html, by courtesy of Dr. Dimitri Agamanolis.] Panel B: Spinal cord demonstrating bilaterally asymmetric MS plaques indicated by multifocal, randomly distributed, locally extensive demyelination (total loss of blue [LFB] myelin staining). Processing conditions: formalin fixation by immersion, paraffin embedding, Luxol fast blue with unspecified counterstain (consistent with periodic acid–Schiff [PAS]). [This figure is reproduced from RA Multz and JT Ahrendsen, “CNS myelin disorders: Multiple sclerosis,” 2023,²⁰ https://www.pathologyoutlines.com/topic/cnsmultiplesclerosis.html, by courtesy of PathologyOutlines.com.].

Figure 7.

Distinct patterns of toxicant-induced white matter vacuolation resulting from intramyelinic edema. Such lesions are particularly prominent in major white matter tracts (shown here in the deep cerebellum) and cerebrum. Findings usually are bilaterally symmetrical. The pathogenesis is progressive intramyelinic accumulation of fluid, which may be seen at the light microscopic level to be clefts (laminar separation) in the myelin sheaths (lower left panel) or vacuoles (lower right panel). Toxicants (unspecified doses): left panels = acute exposure to triethyltin (TET); right panels = subchronic exposure to vigabatrin (an anti-epileptic GABAergic drug). Species: rat (unspecified strain). Processing conditions: formalin fixation by intravascular perfusion, paraffin embedding, H&E staining. [This figure is reproduced with a modified legend from B Bolon et al,⁵ “Nervous system,” in Haschek and Rousseaux’s Handbook of Toxicologic Pathology, 3^rd ed., Vol 3 (Haschek WM, Rousseaux CG, Wallig MA, eds), San Diego, Academic Press, 2013:2005-2093, by courtesy of Elsevier.]

Conclusions

Investigation of myelin integrity is a key element of evaluating the nervous system for test article-related toxicity. The choice of myelin method depends on such parameters as the cost, institutional preference, intended processing protocol (fixation and embedding options), and study aims. In general, routine FFPE processing with H&E staining is an acceptable approach for the initial microscopic screening of myelin in routine animal toxicity studies. Other methods for FFPE tissues including colored dyes (eg, LFB, TB) and IHC assays to detect myelin proteins (eg, MBP, PMP22, S100) may be used to specifically highlight myelin when the test article is intended to interact with a molecular target in the nervous system or neurotoxicity to any neural component (neurons, glia, and/or myelin) is anticipated. Special processing (osmium post-fixation, hard plastic embedding, and TB staining) provides exquisite structural preservation of myelin but is avoided for routine toxicity studies (except where necessitated in a regulatory guideline) due to its expense and low throughput. Care is needed when interpreting myelin stains since genuine effects on myelin may be confused with certain processing artifacts. Consultation with experienced histotechnologists may be warranted in designing microscopic evaluation of myelin and is essential if unexpected staining properties require troubleshooting to provide optimal myelin staining.

Footnotes

Acknowledgements

The authors gratefully thank Ms Beth Mahler for assistance in optimizing the figures and Mr Damien Laudier (of Laudier Histology) for discussions regarding myelin staining options for plastic-embedded material.

Author Contributions

The authors are solely responsible for the contents and crafting of this paper.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

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

ORCID iDs

Brad Bolon

Amber Moser

Elizabeth Chlipala

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