Preparation and Analysis of the Peripheral Nervous System

Toxicologic Pathology, 39: 66-72, 2011 Copyright # 2011 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623310387618 Preparation and Analysis of the Peripheral Nervous System BERNARD S. JORTNER ABSTRACT This article is from a presentation at the 2010 STP/IFSTP Symposium on Neuropathology. The organization and basic structure of the peripheral nervous system is reviewed. Examples of toxicant-induced peripheral nerve injury such as neuronopathy, axonopathy, and myelinapathy are discussed, as are contemporary methods for examination of these tissues. Keywords: neuropathology; toxicologic pathology; peripheral nerve. The intent of this article is to review the organization of the peripheral nervous system, demonstrate basic pathological reactions in these tissues, and present contemporary methods for investigation of neurotoxic events in the peripheral nervous system. The focus is to relate this information to toxicologic pathology investigations. The peripheral nervous system is defined as those portions of the motor, primary sensory, and autonomic neurons that extend outside the central nervous system and are associated with Schwann cells or ganglionic satellite cells (Schaumburg et al. 1983). This system includes dorsal and ventral spinal roots, spinal and cranial nerves (except for the first and second cranial nerves), dorsal root and other sensory ganglia, sensory and motor terminals, and the bulk of the autonomic nervous system (Schaumburg et al. 1983). The concept of separate peripheral and central nervous systems is artificial, since cell bodies of motor neurons with peripherally directed axons lie within the central nervous system and some peripheral sensory neurons have extensive central projections. ORGANIZATION AND COMPONENTS OF THE PERIPHERAL NERVOUS SYSTEM Figure 1 provides a schematic view of the peripheral nervous system showing its major components and their relationship to the central nervous system. Both somatic and autonomic components are demonstrated, but for the remainder of this article, I will largely focus on the myelinated fibers of the somatic system, which includes both motor, with cell bodies in the ventral horn of the spinal cord and some cranial nerve nuclei, and sensory, which has cell bodies in dorsal root ganglia and some cranial nerve ganglia. The organization of a peripheral nerve as seen in crosssection is demonstrated in Figures 2 and 3, which show its components, including the connective tissue sheaths, the outer epineurium, and the specialized perineurial barrier. The latter is a multilayered structure with tight junctions between cells, Address correspondence to: Bernard S. Jortner, Virginia Tech Virginia- Maryland Regional College of Veterinary Medicine, Laboratory for Neurotoxicity Studies, Blacksburg, VA 24061-0442; e-mail: bjortner@vt.edu. forming a diffusion barrier isolating the interior of the fascicles. Within the perineurium lie the fascicles, which contain the nerve fibers and supporting endoneurium. The nerve fibers are encompassed by a series of Schwann cells, forming the myelin sheath segments in myelinated fibers, separated by nodes of Ranvier. Multiple unmyelinated fibers are enfolded by the cytoplasm of a single Schwann cell. An example of a peripheral ganglion, in this case a dorsal root (or spinal) ganglion, is provided in Figure 4. This illustration demonstrates the variable sized neurons, their supporting satellite cells and the ganglionic capsule. TOXIN-INDUCED PATHOLOGIC REACTIONS OF PERIPHERAL NERVES There are three basic pathologic reactions of peripheral nerves induced by toxic agents: neuronopathy, in which the injury is concentrated on the neuronal cell body; axonopathy, in which the axon is the major site of injury expression; and myelinopathy, in which the myelin sheath or Schwann cell is involved (Figure 5). Toxicant-induced examples of these reactions follow. Neuronopathy is a condition in which the primary insult is in the neuronal cell body. An example of neuronopathy is pyridoxine (vitamin B 6 ) toxicity, in which toxic doses of this vitamin elicit injury to the soma of larger neurons of primary sensory ganglia, such as dorsal root ganglia. This process leads to progressive injury to the neuron, resulting in necrosis (Figure 6). A consequence is the inability of the injured neuronal cell body to maintain its long axon. Thus, there is axonal degeneration progressing to myelinated fiber degeneration, initially more manifest distally (Jortner 2000). In severe or prolonged intoxication, proximal fiber levels are affected, such as dorsal spinal roots (Figure 7). Central branches from affected neurons, such as in the gracile fasciculus, are also affected. If the neurons are severely injured or necrotic, then peripheral nerve regeneration is not possible (Figure 8). The mechanism of pyridoxine neuronotoxicity is not fully understood, but it is thought that high circulating levels of pyridoxine might have a direct toxic effect on neurons of the peripheral sensory ganglia (Jortner 2000), which may relate to the presence of a lower ( leaky ) blood nerve barrier in such regions (Olsson 1984). The more complete blood brain barrier in the 66

Vol. 39, No. 1, 2011 PERIPHERAL NERVOUS SYSTEM 67 FIGURE 1. The principal components of the peripheral nervous system, and the cellular organization of peripheral nerves. From Schaumburg et al. 1983, with permission. central nervous system, along with a saturable pyridoxine transport system, protects neurons in those regions from high blood levels of pyridoxine (Schaumburg 2000). Axonopathy is a condition in which the toxicant elicits its major injurious effect on the axon, which degenerates, followed quickly by the myelin sheath (which needs an intact axon to survive). This reaction mimics that of physical transection of a fiber, and hence it is termed Wallerian-type degeneration. An example of this degeneration is the delayed neurotoxicity of certain organophosphates. These are compounds such as phenyl saligeninphosphate, thetoxicmetaboliteoftri-ortho-tolyl phosphate, and mipafox, both of which can quickly (in 24 48 hours)

68 JORTNER TOXICOLOGIC PATHOLOGY FIGURE 2. Diagram of peripheral nerve in cross section, showing three fascicles of myelinated and unmyelinated nerve fibers, each surrounded by a perineurial sheath. A connective and adipose tissue covering, the epineurium, enwraps this sheath. The relationship of Schwann cell, the myelin sheath it generates, and the associated axon are also demonstrated. From Schaumburg et al. 1983, with permission. inhibit the neuronal enzyme neurotoxic esterase. There also is molecular rearrangement of the inhibited enzyme, termed aging. A degree of inhibition of 70% or more after a single dose will result in axonopathy progressing to myelinated fiber breakdown some 7 10 days after dosing in the chicken, a standard test animal for this neuropathy (Figures 9, 10, and 11) (Ehrich and Jortner 2010). This process preferentially affects larger fibers, and again it is more prominent distally. Both central and peripheral nerve fibers can be affected. With appropriate exposures, there can be progression proximally, producing a dying-back neuropathy (this term is often employed to indicate disease in peripheral nerve). Since the neuronal cell body giving rise to the fiber is intact, axonal regeneration is seen in peripheral nerve, but not in the spinal cord or brain. The mechanism of the neuropathy is not clearly understood, nor is there a demonstrated mechanistic relationship between the neurotoxic esterase inhibition and nerve fiber degeneration. Myelinopathy is a state in which degeneration of myelin is the primary lesion. The degeneration may be related to an attack on the sheath or on the Schwann cell, which forms and maintains it. The model I have chosen to illustrate this process is tellurium neurotoxicity, in which neonatal rats are fed a diet containing 1 1.5% elemental tellurium from days one through seven, a period of active myelination (Bouldin et al. 1989). Thus, Schwann cells that form the larger myelin internodes are affected, with tellurium-mediated cellular injury and inhibition of squalene epoxidase, the obligate enzyme in cholesterol synthesis (Figure 12). The major metabolite responsible for this process is the tellurite ion. Diminished cholesterol, a major myelin lipid, not only halts ongoing myelination, but it also destabilizes the already formed myelin sheaths, leading to their segmental breakdown, along with endoneurial edema (Bouldin et al. 1989) (Figure 12). The demyelinating effect of tellurium is transient, presumably related to growth-associated demand for myelin synthesis. After day seven, even with continuation of the tellurium-containing diet, there is remyelination of the demyelinated axons (Figure 12). SAMPLING, FIXATION, AND TISSUE PROCESSING FOR PERIPHERAL NERVES AND GANGLIA In general, when selecting peripheral nerves for study, it is important to choose specimens representing various parts of the peripheral nervous system. It is of particular importance to obtain samples from proximal and distal nerve levels, since axonopathies and neuronopathies will often affect more distal segments earlier and more severely than proximal regions. It is also useful to examine peripheral ganglia, such as the dorsal root or trigeminal ganglion. The basic concerns for fixation of tissue from the peripheral nervous system are the same as for the brain and spinal cord, namely that it be rapid and complete and that it avoid tissue distortion. Perfusion fixation is preferred, and procedures for this method have been published (Fix and Garman 2000; Hancock et al. 2004). Fixatives for perfusion vary, depending on the methods of study to be used. For morphology that includes electron microscopy, 2.5 3% glutaraldehyde is preferred, but this method is not suitable if immunohistochemistry is also anticipated, since this fixative interferes with antigen detection. Paraformaldehyde used in concentrations of 1 4% is better for immunohistochemistry, but it does not preserve myelin very well. The addition of a small amount of glutaraldehyde (0.1 0.2%) will help preserve the myelin without interfering with the immunostaining. For perfusion-fixation, the solutions should be administered at room temperature, and following successful whole-body perfusion, it is useful to refrigerate and hold the carcass for several hours prior to dissection (Fix and Garman 2000). More details concerning fixatives used for the nervous system, including issues of buffering and osmolarity, are dealt with by Fix and Garman (2000) and Hancock et al. (2004).

Vol. 39, No. 1, 2011 PERIPHERAL NERVOUS SYSTEM 69 FIGURE 3. Cross-section of a rat sural nerve, showing the epineurium (*) and perineurium (arrow). The latter encloses the endoneurial space containing myelinated (surrounded by dark blue stained myelin sheaths) and unmyelinated (clusters of small unstained axons [arrowhead]) fibers. Toluidine blue and safranin stain. FIGURE 4. Longitudinal section of a rat dorsal root ganglion with associated nerve roots. Note the presence of large light (black arrows) and small dark (white arrow) neurons. Dorsal root fibers course through the ganglion, and the ventral root (*) lies adjacent to it. Toluidine blue and safranin stain. In instances in which perfusion is not possible, immersionfixation will suffice to yield adequately fixed segments of peripheral nerve. Although this method is less optimal than perfusion-fixation, it does provide useful material for evaluation and is used for nerve biopsy specimens (Kasukurthi et al. 2009; King 1999). For immersion-fixation in animal necropsy studies, an indicated segment of the nerve should be quickly removed after euthanasia, gently adhered to an index card in an elongated state, and immersed (while still attached to the card) into the fixative. Since the perineurium provides a diffusion barrier, and fixatives, especially glutaraldehyde, penetrate slowly, larger nerves should be longitudinally sectioned to reduce the thickness. A sharp blade is needed for this step, to reduce crush FIGURE 5. Drawing of a neuron contributing an axon to peripheral nerve. Primary sites of toxicant-induced injury are in the neuronal cell body (neuronopathies), the myelin sheath or Schwann cell (myelinopathies), or on the axon (axonopathies). From Jortner 2000, with permission. artifact. Immersion-fixatives are the same as those noted above, or alternatively 10% neutral buffered formalin. Peripheral nerve segments should be immersed in the fixative for 10 12 hours. Formalin fixation may be done at room temperature, but samples immersed in glutaraldehye or paraformaldehyde should be held at refrigerator temperature (Hancock et al. 2004). It should be noted that the lipids of myelin are not well preserved by formalin and are thus dissolved by xylene during paraffin embedding (King 1999). This process produces the neurokeratin artifact, which is manifest by a herringbone appearance of the myelin sheath, best appreciated in longitudinal sections (Midroni and Bilbao 1995). Any of the above fixative procedures can be used for epoxy resin embedding, which provides optimal specimens for light microscopy. For epoxy resin embedding, fixed segments of nerves are dissected free of any attached tissue. The ends of the nerve, where it has been handled in the fresh state, are trimmed away using a sharp blade. The samples are then post-fixed in

70 JORTNER TOXICOLOGIC PATHOLOGY FIGURE 6. Injured neurons of dorsal root ganglia from a rat exposed to 600 mg/kg pyridoxine twice a day for four days, with a two-day recovery period. A shows a severely injured neuron with a pale-stained eccentric nucleus and somal cytoplasm containing blue-staining material, which likely includes autophagosomes, dense bodies, and altered mitochondria. In B, neuronal necrosis has occurred, and the neuron is being phagocytized by satellite cells. Toluidine blue and safranin stain. FIGURE 8. Cross-section of the sural nerve from a pyridoxine-treated rat dosed as in Figure 6, with a forty-day recovery period. There is a failure of axonal/fiber regeneration in large regions of the nerve, although pale staining bands of Büngner (arrow) are seen. Toluidine blue and safranin stain. FIGURE 7. Spinal roots from pryidoxine-treated rat in Figure 6. There is marked myelinated fiber degeneration in the dorsal root (above) related to pyridoxine-induced neuronal injury in dorsal root ganglia, whereas fibers in the ventral (motor) root (below) are spared. Toluidine blue and safranin stain. 1% osmium tetroxide for two hours at room temperature, embedded in epoxy resin in cross and longitudinal orientation, and then sectioned at 1-mm thickness and stained with toluidine blue. As seen in Figures 3, 4, 6, 7, 8, 10 in this article, these processing conditions provide optimal resolution for light microscopic examination and are the basis of contemporary peripheral nerve histopathology. The clarity of myelin sheaths and axons in such preparations allows measurement and counts of these structures, providing quantitative data for analysis. Properly fixed nerve so embedded can also be further processed and used for transmission electron microscopy.

Vol. 39, No. 1, 2011 PERIPHERAL NERVOUS SYSTEM 71 FIGURE 9. Teased peripheral nerve fiber preparation from a hen with organophosphate-induced delayed neurotoxicity fourteen days after toxicant exposure, showing one myelinated fiber undergoing Wallerian-type degeneration (arrowhead). The adjacent fiber is intact and demonstrates a node of Ranvier (arrow). Osmium tetroxide stain. From Jortner 2000, with permission. FIGURE 10. Cross-section of a region of peripheral nerve from a chicken exposed to a neurotoxic dose of mipafox. Several stages of axonal injury and its progression to myelinated fiber degeneration are shown (arrows). Toluidine blue and safranin stain. It must be noted that paraffin embedding, sectioning in the cross and longitudinal planes, and staining with hematoxylin and eosin supplemented by special stains and immunohistochemistry to delineate components of peripheral nerve (myelin, axons, Schwann cells) provides useful material for study. Longitudinal sections of paraffin-embedded nerve segments are particularly helpful, since degenerating fibers are easier to see in such preparations. However, sections of paraffin-embedded nerve do not provide the resolution of epoxy resin embedded sections. Special procedures for pathological evaluation of peripheral nerve include morphometry and nerve fiber teasing (King 1999). There is a normal relationship between axon diameter and myelin sheath thickness, which is altered in some disease states. Cross-sections of well-preserved, well-prepared sections of epoxy resin embedded nerves can be used to measure size and concentration of axons and myelinated fibers. This process includes determination of the g ratio, which is the ratio of axon diameter to total fiber diameter and ranges between 0.5 and FIGURE 11. Electron micrograph of a cross-sectioned peripheral nerve myelinated fiber from a chicken with organophosphateinduced delayed neurotoxicity. The axon is distended with masses of abnormal (swollen) mitochondria, dense bodies, and a central collection of neurofilaments. Original magnification 7,200. 0.7 for most myelinated fibers. These data can be expressed graphically and are useful in detecting subtle changes in demyelination, loss of fibers of certain size, and axonal atrophy, as may be seen in peripheral neuropathies. In myelinated nerve fiber teasing, nerves fixed as noted above and post-fixed with osmium tetroxide are infiltrated by cedarwood oil, glycerin, or unpolymerized epoxy resin and used for study of individual myelinated nerve fibers (King 1999; Krinke et al. 2000), which is accomplished by tearing the perineurium and carefully teasing out individual fibers using needles and a stereomicroscope. The teased fibers are aligned on a slide for microscopic study. Since the osmium stains the myelin, the length of myelin internodes, width of the nodes of

72 JORTNER TOXICOLOGIC PATHOLOGY FIGURE 12. Cross-sections of sciatic nerve fibers from neonatal rats fed a diet containing 1.25% elemental tellurium (specimens provided by Dr. Jeffrey Goodrum, University of North Carolina Chapel Hill). A. Early changes of cytoplasmic lipid droplet and membranous inclusions (arrow) in a Schwann cell of an animal fed the tellurium diet for five days. The myelin sheath is intact at this stage. B. A fully demyelinated fiber with myelin debris contained within a Schwann cell, as is the demyelinated axon (arrow). C. On day 9, a remyelinating axon is characterized by a thin myelin sheath. Original magnifications: A: 10,000, B: 7,200, C: 14,000. A. is reproduced from Jortner 2000, with permission. Ranvier, sequence of demyelination, and axonal degeneration can be evaluated (Figure 9). Other stains, such as Sudan black staining can also be used to demonstrate myelin sheaths, especially when glycerin is used for infiltration (Krinke et al. 2000). Within the past twenty years, human neurology has seen increasing use of sections of skin-punch biopsy specimens immunostained with the pan-axonal marker protein gene product 9.5 (PGP 9.5) to demonstrate small-caliber, unmyelinated, nociceptor C fibers terminating in the epidermis (Griffin et al. 2001). This approach has been used to study spatiotemporal changes in this epidermal innervation in a number of diseases, since it allows physicians to evaluate progression of fiber loss and regeneration of this population of fibers in a patient. This approach has recently been used to investigate neurotoxic effects of chemotherapeutic agents such as paclitaxel and cisplatin in studies using the rat footpad (Lauria et al. 2005). It would seem to have value for sequential in-life assessment of toxicant-induced terminal fiber degeneration. REFERENCES Bouldin, T. W., Earnhardt, T. S., Goines, N. D., and Goodrum, J. (1989). Temporal relationship of blood-nerve-barrier breakdown to metabolic and morphologic alterations of tellurium neuropathy. Neurotoxicology 10, 79 90. Ehrich, M., and Jortner, B. S. (2010). Organophosphorus induced delayed neuropathy. In Hayes Handbook of Pesticide Toxicology, 3 rd ed (R. Krieger, ed). Elsevier, Amsterdam, pp. 1479 504. Fix, A. S., and Garman, R. H. (2000). Practical aspects of neuropathology: A technical guide for working with the nervous system. Toxicol Pathol 28, 122 31. Griffin, J. W., McArthur, J. C., and Polydefkis, M. (2001). Assessment of cutaneous innervation by skin biopsies. Curr Opin Neurol 14, 655 59. Hancock, S. K., Hinckley, J., Ehrich, M., and Jortner, B. S. (2004). Morphological measurement of neurotoxic injury in the peripheral nervous system. Preparation of material for light and electron microscopy. In Current Protocols in Toxicology, vol. 2, Suppl. 22 (L. E. Costa, E. Hodgson, D. J. Reed, eds.), John Wiley, Hoboken, NJ, pp. 12.12.1 12.12.17. Jortner, B. S. (2000). Mechanisms of toxic injury in the peripheral nervous system: neuropathologic considerations. Toxicol Pathol 28, 54 69. Kasukurthi, R., Brenner, M. J., Moore, A. M., Moradzadeh, A., Ray, W. Z., Santosa, K. B., Mackinnon, S. E., and Hunter, D. A. (2009). Transcardial perfusion versus immersion fixation of peripheral nerve regeneration. J Neurosci Meth 184, 303 9. King, R. H. M. (1999). Atlas of Peripheral Nerve Pathology. Arnold Publishing, London. Krinke, G. J., Vidotto, N., and Weber, E. (2000). Teased-fiber technique for peripheral myelinated nerves: Methodology and interpretation. Toxicol Pathol 28, 113 21. Lauria, G., Lombardi, R., Borgna, M., Penza, P., Bianchi, R., Savino, C., Canta, A., Nicolini, G., Marmiroli, P., and Cavaletti, G. (2005). Intraepidermal nerve fiber density in rat foot pad: Neuropathologic-neurophysiologic correlation. J Periph Nerv Syst 10, 202 8. Midroni, G., and Bilbao, J. M. (1995). Biopsy Diagnosis of Peripheral Neuropathy. Butterworth-Heinemann, Boston, MA. Olsson, Y. (1984). Vascular permeability in the peripheral nervous system. In Peripheral Neuropathy (P. J. Dyck, P. K. Thomas, E. H. Lambert, and R. Bunge, eds). W. B. Saunders, Philadelphia, PA, pp. 579 97. Schaumburg, H. H. (2000) Pyridoxine. In: Experimental and Clinical Neurotoxicology (P. S. Spencer, H. H. Schaumburg, and A. C. Ludolph, eds). Oxford University Press, New York, pp. 1044 47. Schaumburg, H. H., Berger, A. R., and Thomas, P. K. (1983). Disorders of Peripheral Nerves, 2 nd ed. F. A. Davis Co., Philadelphia. For reprints and permissions queries, please visit SAGE s Web site at http://www.sagepub.com/journalspermissions.nav.