Technical Guide for Nervous System Sampling of the Cynomolgus Monkey for General Toxicity Studies

Abstract

For general toxicity studies, a technique was designed to consistently sample the most important neuroanatomic regions of the brain, spinal cord, and peripheral nerve of cynomolgus monkeys using a limited number of blocks and slides. Using the most rostral portion of the pons as a landmark, the entire fixed brain was cut dorsoventrally into cross-sectional slabs 4 mm in thickness. For microscopic evaluation, six blocks of the brain at the levels of the frontal pole, anterior commissure, rostral thalamus, caudal thalamus, middle cerebellum with brainstem, and occipital lobe were trimmed to fit in standard tissue cassettes. Cross- and oblique sections of the spinal cord including the dorsal root ganglion and dorsal and ventral nerve roots were obtained at the levels of C1–C4, T10–T12, and L1–L4. Cross- and longitudinal sections of the sciatic nerve were also obtained. This technique offers a consistent and reliable method to routinely sample most of the important regions of the central and peripheral nervous system of monkeys using ten blocks. This method is readily adaptable to other species of nonhuman primates, dogs, and minipigs and can be quickly learned by the technicians performing the trimming procedures.

Introduction

Evaluation of the brain, spinal cord, and nerves is important in general toxicity studies and in evaluation of animal models of neurodegenerative diseases and of brain or spinal cord injury. There is great variation in methods used to sample the brain for routine examination in toxicity studies and few references to guide toxicologic pathologists and technical staff in selection of appropriate regions for examination. Brain atlases of the rhesus monkey (Paxinos et al. 2000; Saleem and Logothetis 2007) are helpful in identifying brain regions in cynomolgus monkeys. In addition, Szabo and Cowan published in 1984 a stereotaxic atlas of the brain of the cynomolgus monkey. No atlas of the spinal cord anatomy in any macaque species is available; however, human (Alloway and Pritchard 2007; Crosby et al. 1962; Hendelman 2005) or domestic mammal (King 1999) textbooks generally may be used to define the neuroanatomy of the macaque spinal cord. It is recommended that a list of important brain regions be examined in general toxicity studies (Table 1). In this paper, a procedure for routine and consistent sampling of specific important neuroanatomic areas of the brain, spinal cord, and sciatic nerve of the cynomolgus monkey (Macaca fascicularis) is described. All procedures were approved by the animal care and use committee in accordance with federal regulations and the Guide for the Care and Use of Laboratory Animals.

Table 1.

Neuroanatomic Areas that Should Be Examined Microscopically in Standard Toxicity Studies

Neuroanatomic areas	Subneuroanatomic areas
Cerebral cortex	Cingulate, motor, sensory entorhinal, piriform, and primary visual cortex
Rostral subcortical brain regions	Basal forebrain/substantia innominata and septal nuclei
Basal ganglia	Caudate, putamen, globus pallidus, substantia nigra, and claustrum
Major white matter tracts	Corpus callosum, internal and external capsule, pyramids
Amygdala
Thalamus	Rostral and caudal
Hypothalamus
Hippocampus	Dentate gyrus, hippocampus proper, and subiculum
Colliculi	Anterior (superior) and/or posterior (inferior)
Geniculate	Medial and/or lateral
Midbrain	Aqueduct, reticular formation, raphe nuclei red nuclei, cerebral peduncles
Cerebellum	Including the deep cerebellar nuclei
Pons
Medulla oblongata

Table 2.

Neuroanatomic Areas Included in the Brain Section of the Frontal Pole (Figure 4)

Piriform cortex (Pir): The piriform cortex processes olfactory information. It receives axons from the olfactory nerve/bulb and sends projections to multiple hypothalamic nuclei, thalamic nuclei, the hippocampus, and the amygdala.

Prefrontal cortex: Regions of the cerebral cortex within the frontal pole section include the supplementary (e.g., 6 M) and premotor cortex (ProM) that control voluntary movements, the anterior aspect of the olfactory nuclei (processing olfactory signals), and the orbital frontal cortex, which is involved in emotion and reward.

Caudate nuclei (Cd): These nuclei facilitate learning and memory, especially with regard to feedback processing. They receive axonal inputs from the cerebral cortex and substantia nigra and project to the globus pallidus.

Putamen (Pu): The caudate nucleus and putamen form the striatum. The main function of these two nuclei is to regulate movement and influence various types of learning. The putamen and the caudate nucleus receive axonal inputs from the cerebral cortex and substantia nigra and send inputs to the globus pallidus.

Corpus callosum (cc): Composed of millions of axons that connect the left and right cerebral hemispheres, the corpus callosum consists of white matter tracts that facilitate interhemispheric communication.

Internal capsule (ic): Composed of ascending and descending axons that separate the caudate nuclei and thalamus from the putamen and globus pallidus. These axons run between the cerebral cortex and the pyramids of the medulla.

External capsule (ec): Composed of cholinergic fibers that run from the basal forebrain to the cerebral cortex, this white matter tract passes lateral to the putamen and globus pallidus and joins the internal capsule along the inferior aspect of these nuclei.

Claustrum (Cl): Although the function of the claustrum is not completely known, it is believed to play an important role in the integration of actions so that individuals are able to convert multiple types of information into a single perception. The claustrum has afferent and efferent connections to and from the primary motor, premotor, auditory and visual cortices and perhaps the hippocampus, amygdalae, and caudate nuclei.

The objective of this paper is to provide guidance on how to perform the evaluation of the central and peripheral system of cynomolgus monkeys for general toxicity studies, in addition to providing a brief review of the neuroanatomic regions present within the sections included in this technique. These methods have been illustrated in a CD-ROM presentation that is available from the authors (Pardo et al. 2010).

The Brain

General Descriptive Anatomy of the Brain of the Cynomolgus Monkey

The brain of the cynomolgus monkey weighs, on average, 74 g, ranging from 62 to 85 g (historical organ weight data from Pfizer, Inc., Groton, CT). This weight represents an average for cynomolgus monkeys of 2.5–5.0 years of age (the typical age selected for general toxicity studies). Brain weights can vary with age and sex of the animals. Brain weights of male and older animals are greater than those of female and younger animals. In contrast to dogs and rodents, the brain of the cynomolgus monkey has a relatively larger and more sharply pointed frontal pole, smaller olfactory lobes, narrow lateral and third ventricles, prominent gyri and sulci corresponding to a well-developed cerebral cortex, and extension of the occipital cortex over the brainstem and cerebellum. In addition, the horizontal plane of the brainstem is oriented at approximately 45° to the horizontal plane of the hemispheres, resulting in a wedge-shaped midbrain. Compared to humans, cynomolgus monkeys have a proportionally smaller frontal lobe, a wider interthalamic adhesion bridging the third ventricle, and a brainstem that is positioned at a less acute angle.

Collection, Sampling, and Sectioning of the Cynomolgus Monkey Brain

For general toxicity studies, the technique described below is recommended to consistently sample most of the important neuroanatomic regions of the brain using a limited number of blocks and slides. This technique is very simple and requires minimal neuroanatomic knowledge. Additional regions of interest can be easily added.

The brain is obtained by removing the skin, connective tissue, and muscle from the superior aspect of the skull (calvarium) that extend from the top of the orbits and ears to the atlanto-occipital area. The exposed calvarium is removed using a bone saw (Lipshaw Autopsy Saw Model #100000, 1.5 amps, 115 volts AC, Hz50/60, Pittsburgh, PA or Stryker Autopsy Saw, Model 810, 120V-60 Hz 2.0A, Kalamazoo, MI), being careful not to damage the brain parenchyma. The falx cerebri, a strong, arched fold of dura mater that descends vertically in the longitudinal fissure between the cerebral hemispheres, is removed at the midline, so that the brain can removed from the skull. Note that the pituitary gland will not come out with the brain, and it must be removed from within the sella turcica. Because the olfactory bulbs are small and embedded in the skull, collection of the olfactory tracts and bulbs requires care during the dissection of the brain. The brain is fixed in 10% neutral buffered formalin for at least forty-eight hours before trimming at a volume of about 10:1 to 20:1 (formalin: tissue).

Using the most rostral portion of the pons as a landmark, the entire fixed brain can be held firmly and cut (Tissue Slicer Blade, Thomas Scientific, Swedesboro, NJ, catalog #6727C18) dorsoventrally at a 45° angle (Figures 1A and 1B). A rectangular slicing matrix tool (fabricated in house) that is 15 cm in length and 8 cm in width and has two outer raised edges that are 1 cm in width and 4 mm in height is recommended to facilitate the brain slicing (Figure 2A) into multiple cross-sectional slabs 4 mm in thickness (Figure 2B). There are approximately fourteen cross-sections of the brain using this rectangular slicing matrix tool. All sections of brain are 4 mm in thickness with the exception of cross-sections 1 and 14, which are thicker because they are the rostral and caudal regions of the brain (Figure 3). Please note that the rostral aspect of each slice will be embedded downward in the paraffin boat and will be the side to be sectioned.

Figure 1.

(A) Using the most rostral portion of the pons as landmark (arrow), the fixed brain is placed with the superior surface down and at approximately 45° angle. (B) The brain is sliced once vertically in cross-section at the level of the most rostral portion of the pons.

Figure 2.

(A) A rectangular slicing matrix tool (fabricated in house).15 cm in length, 8 cm in width, with two outer raised edges that are 1 cm in width and 4 mm higher than the central trough, is used to slice the entire brain into cross-sectional slabs 4 mm in thickness. Fixed brain is cut with a Tissue Slicer Blade (Thomas Scientific, Swedesboro, NJ, catalog #6727C18). (B) The rostral half of the brain is placed cut surface down on the rectangular slicing matrix and is sectioned in multiple slices that are 4 mm thick. The caudal half of the brain is also sectioned in an identical manner.

Figure 3.

The fixed brain is sliced into fourteen cross-sections of 4 mm in thickness at periodic intervals. Cross-sections 2, 5, 7, 9, and 12 represent the frontal pole; anterior commissure; rostral thalamus; caudal thalamus; and occipital lobe, cerebellum, and brainstem, respectively. Please note that the rostral aspect of each slice is shown in this photo and it will be embedded downward in the paraffin boat to be the side to be sectioned.

For microscopic evaluation, six blocks of the brain at the levels of the frontal pole, anterior commissure, rostral thalamus, caudal thalamus, middle of the cerebellum with brainstem, and occipital lobe are trimmed to fit into standard tissue cassettes and slides (1 × 3-in., VWR Micro Slides Superfrost, White, Clipped corner for Leica IP Ink Jet Printer, Catalog #89078-500, VWR International, LLC, Radnor, PA). These sections are obtained unilaterally with the exception of the cerebellar/brainstem section, which is a full coronal slice. Sections of the cerebrum are taken unilaterally to maximize the numbers of neuroanatomic regions that are sampled, and since toxicologically induced lesions are typically bilateral in localization (Figure 3). However, contralateral slices of the sectioned brain should be preserved in formalin for future sampling. Although this technique provides a very consistent way to sample the brain, comparable sections from different animals will not be identical because of the small size of some neuroanatomic areas. The lack of a high degree of section homology is not considered to represent a problem in general toxicity studies, because having highly homologous sections results in the exclusion of some neuroanatomic regions from all of the sampled brains.

The neuroanatomic areas generally present at each level of the sectioned brain are described below. Selected sections of brain at the levels of the frontal pole, anterior commissure, rostral thalamus, caudal thalamus, middle of the cerebellum with brainstem, and occipital lobe can be processed and stained for light microscopic examination (Figures 4–9). In addition, Tables 2–7 briefly describe the functions, neural connections, and principal neurotransmitters present in a variety of important neuroanatomic areas within selected brain sections.

Figure 4. Hematoxylin and eosin cross section of the cynomolgus monkey brain at the level of the frontal pole (cross-section 2): 6 M, accessory motor cortex; cc, corpus callosum; Cd, caudate nucleus; Cl, claustrum; ec, external capsule; ic, internal capsule; Lv, lateral ventricle; Pir, piriform cortex; ProM, premotor cortex; Pu, putamen.

Figure 5. Hematoxylin and eosin cross section of the cynomolgus monkey brain at the level of the anterior commissure (cross-section 5): acp, anterior commissure; B, basal forebrain region; cc, corpus callosum; Cd, caudate nucleus; CG, cingulate cortex; Cl, claustrum; opc, optical chiasm; ec, external capsule; EGP, external globus pallidus; ER, entorhinal cortex; HT, hypothalamus; ic, internal capsule; IC, insular cortex; lf, lateral fissure; Pu, putamen; RA, rostral amygdala; rf, rhinal fissure; Rt, rostral thalamus; TC, temporal cortex; VP, ventral pallidum.

Figure 6. Hematoxylin and eosin cross-section of the cynomolgus monkey brain at the level of the rostral thalamus (cross-section 7): cc, corpus callosum; Cd, caudate nucleus; CG, cingulate cortex; cp, cerebral peduncle; ec, external capsule; ER, entorhinal cortex; GP, globus pallidus; Hip, hippocampus; ic, internal capsule; IC, insular cortex; lf, lateral fissure; LG, lateral geniculate; Lv, lateral ventricle; opt, optic tract; Pu, putamen; rf, rhinal fissure; SN, substantia nigra; T, thalamus; TC, temporal cortex.

Figure 7. Hematoxylin and eosin cross-section of the cynomolgus monkey brain at the level of the caudal thalamus (cross-section 9): Aq, aqueduct (mesencephalic aqueduct or third ventricle); cc, corpus callosum; CG, cingulate cortex; DpMe, deep mesencephalic nuclei; eml, external medullary lamina; ER, entorhinal cortex; Hip, hippocampus; IC, insular cortex; Lv, lateral ventricle; PC, parietal cortex; Pi, pineal gland; PnO, pontine nuclei; pons, pons; Pul, pulvinar nucleus; Rn, raphe nuclei; Rt, reticular thalamic nuclei; Sc, superior colliculus; TC, temporal cortex.

Figure 8. Hematoxylin and eosin cross-section of the cynomolgus monkey cerebellum (cross-section 13): DNC, deep cerebellar nuclei; ON, olivary nuclei; Py, pyramidal tracts; V4, fourth ventricle; Ve, vestibular nuclei.

Figure 9. Hematoxylin and eosin cross-section of the cynomolgus monkey brainstem (cross-section 13): cf, calcarine fissure; MTV5, secondary visual cortex (medial temporal area); V1, primary visual cortex; V2, V3, V4, secondary visual cortex.

Figure 10.

(A) The fixed spinal cord is sectioned coronally (cross-section) at the level of C1–C2. (B) The fixed spinal cord is sectioned obliquely at the level of T11–T12. (C) Cross- and oblique-fixed spinal cord sections at the level of C1–C2. Please note that the rostral aspect of each slice is shown in this photo, and it will be embedded downward in the paraffin boat to be the side to be sectioned.

Figure 11.

Hematoxylin and eosin cross–section of the cynomolgus money spinal cord at the level of C1–C2: ACT, anterior corticospinal tract; ALS, anteriolateral system; AST, anterior spinocerebellar tract; FC, fasciculus cuneatus; FDL, fasciculus dorsolateralis; FG, fasciculus gracilis; I, lamina 1; II, lamina II; III, lamina III; IV, lamina IV; V, lamina V; VI, lamina VI; VII, lamina VII; VIII, lamina VIII; IX, lamina IX; X, lamina X. Ascending tracts (right): MLF, medial longitudinal fasciculus; PST, posterior spinocerebellar tract. Descending tracts (left): DF, dorsal funiculi; DIS, dorsal intermediate septa; DH, dorsal horn; DLS, dorsolateral sulcus; DMS, dorsal median septum; DNR, dorsal nerve roots; IF, interfascicularis; LCT, lateral corticospinal tract; LH, lateral horn; LVT, lateral vestibulospinal tract; MRT, medullary (lateral) reticulospinal tract; P, pontine (medial) reticulospinal tract; RB, rubrospinal tract; SM, septomarginalis; VF, ventral funiculi; VH, ventral horn; VLS, ventrolateral sulcus; VMF, ventral median fissure.

Figure 12.

Hematoxylin and eosin cross-section of the cynomolgus money spinal cord at the level of T11–12: I, lamina I; II, lamina II; III, lamina III; IV, lamina IV; V, lamina V; VII, lamina VII; VIII, lamina VIII; IX, lamina IX; X, lamina X; NC, dorsal nucleus of Clarke. Ascending tracts (right): ALS, anteriolateral system; AST, anterior spinocerebellar tract; FC, fasciculus cuneatus; FDL, fasciculus dorsolateralis; FG, fasciculus gracilis; PST, posterior spinocerebellar tract. Descending tracts (left): CN, central canal; DF, dorsal funiculi; DH, dorsal horn; DIS, dorsal intermediate septa; DLS, dorsolateral sulcus; DMS, dorsal median septum; DNR, dorsal nerve roots; IF, interfascicularis; LCT, lateral corticospinal tract; LF, lateral funiculi; LVT, lateral vestibulospinal tract; MRT, medullary (lateral) reticulospinal tract; P, pontine (medial) reticulospinal tract; RB, rubrospinal tract; SM, septomarginalis; VF, ventral funiculi; VH, ventral horn; VLS, ventrolateral sulcus; VMF, ventral median fissure.

Figure 13.

Hematoxylin and eosin cross-section of the cynomolgus monkey spinal cord at the level of L3–L4: I, lamina 1; II, lamina II; III, lamina III; IV, lamina IV; V, lamina V; VI, lamina VI; VII, lamina VII; VIII, lamina VIII; IXL, lamina IX lateral; IXM, lamina IX medial; X, lamina X. Ascending tracts (right): ALS, anteriolateral system; AST, anterior spinocerebellar tract; FG, fasciculus gracilis; FDL, fasciculus dorsolateralis; PST, posterior spinocerebellar tract. Descending tracts (left): DF, dorsal funiculi; DH, dorsal horn; DIS, dorsal intermediate septa; DLS, dorsolateral sulcus; DMS, dorsal median septum; DNR, dorsal nerve roots; LCT, lateral corticospinal tract; LF, lateral funiculi; LH, lateral horn; LVT, lateral vestibulospinal tract; MRT, medullary (lateral) reticulospinal tract; P, pontine (medial) reticulospinal tract; RB, rubrospinal tract; SM, septomarginalis; VCT, ventral corticospinal tract; VF, ventral funiculi; VH, ventral horn; VLS, ventrolateral sulcus; VMF, ventral median fissure; VNR, ventral nerve roots; VT, vestibulospinal tract.

Table 3.

Neuroanatomic Areas Included in the Brain Section at the Level of the Anterior Commissure (Figure 5)

Anterior commissure (acp): This white matter structure consists of bundles of nerve fibers that cross the midline of the brain, connecting the two cerebral hemispheres. Specifically, it connects the left and right temporal lobes and amygdalae (RA), contains decussating fibers projecting from the olfactory tracts to the opposite side of the olfactory cortex, and forms part of the neospinothalamic tract for pain perception.

Basal forebrain region (B): This region is associated with motor control (including eye movement) and learning and also plays an important role in motivation through connections to the nuclei accumbens. It is connected to the striatum (caudate nuclei and putamina), globus pallidus, substantia nigra, and subthalamic nuclei.

Basal nuclei (of Meynert): The basal nuclei are groups of cholinergic neurons within the substantia innominata. Projections from the basal nuclei, as well as projections from the septal nuclei, interconnect with the neocortex, hippocampus, and amygdalae and are responsible for general regulation of forebrain activity.

Globus pallidus (GP): The globus pallidus is composed of both the dorsal and ventral pallidus. The dorsal pallidus is divided into the internal and external pallidus by the medial medullary lamina. The internal pallidus contains GABAergic neurons that send their axons to the dorsal thalamus, the centromedian complex, substantia nigra and the pedunculopontine complex. Fibers from the external pallidus are the main regulators of the basal ganglia system. The ventral pallidum is involved in the regulation of motivation, behavior, and emotions. The globus pallidus receives input from the temporal lobes and the hippocampus via the ventral striatum and transmits this information to the medial dorsal and ventral anterior nuclei of the thalamus.

Septal nuclei: As part of the limbic system, the septal nuclei have connections from and to the olfactory bulb, hippocampus, amygdala, hypothalamus, midbrain, habenula, cingulate gyrus, and thalamus. The septal nuclei, along with the nucleus accumbens, play a role in reward and reinforcement. (Note that the septal nuclei are only poorly visualized along the medial margin of the section in Figure 5 but are present in the frontal pole section shown as Figure 4, as well as in Figure 3, block 5).

Amygdalae (RA): The amygdala is part of the limbic system and contributes to processing and memory of emotional reactions, playing an important role in anxiety and depression. It is composed of a least twelve nuclei that perform different functions. The amygdala sends inputs to the hypothalamus, thalamic reticular nucleus, trigeminal nerve nuclei, facial nerve nucleus, ventral tegmental area, locus coeruleus, hippocampus, and laterodorsal tegmental nucleus. It receives inputs from the olfactory bulb and olfactory cortex to process the sense of smell.

Thalamus (T, rostral thalamus Rt, Pulvinary nuclei-Pul, reticular thalamic nuclei-Rt): The thalamus, which is often referred to as “the gateway to the cortex,” contains nineteen different association or relay nuclei that pass information to the cortex from the spinal cord, brainstem, cerebellum, and basal ganglia (as well as from other regions of the cerebral cortex). The thalamus, therefore, functions to relay sensation, special sense stimuli (excluding olfaction), and motor signals to the cerebral cortex and is also involved in the regulation of consciousness, sleep, and alertness. Because of its complexity, only a few of the specific thalamic nuclei will be mentioned here. For example, the anterior thalamic nucleus is a key link between the limbic system and the cortex. The lateral geniculate nuclei receive input from the retina and relay information to the primary occipital visual cortex V1. The medial geniculate nuclei receive input from the inferior colliculus and send outputs to the primary auditory cortex. The ventral posterior nuclei (both lateral and medial) play key roles in somesthesis, relaying touch and proprioceptive information from the dorsal columns and trigeminal nuclei to the primary somatosensory cortex. Thalamic nuclei have strong reciprocal connections with the cerebral cortex, forming thalamo-cortico-thalamic circuits that are believed to be involved with consciousness. In addition, the thalamus has connections with the cerebellum for controlling motor functions.

Hypothalamus (HT): Multiple small hypothalamic nuclei (medial preoptic, paraventricular, anterior hypothalamic, suprachiasmatic, laterial preoptic, lateral, dorsomedial hypothalamic, ventromedial, arcuate, lateral tuberal, posterior, and mammillary nuclei) link the nervous system to the endocrine system via the pituitary gland (hypophysis).

Arcuate nucleus (included in HT): The arcuate nucleus is located in the periventricular area of the tuberal hypothalamus and contains many neurons that produce releasing and inhibiting hormones for control of the adenohypohysis and neurons important for the control of feeding behavior (prolactin, growth hormone-releasing hormone, neuropeptide Y, agouti-related protein, dopamine, GABA, and ghrelin). This nucleus is also bordered by specialized ependymal cells called tanycytes, which may also play a role in regulating neuroendocrine function.

Insular cortex (IC): The function of the insular cortex is linked to emotion and regulation of the body’s homeostasis. These functions include perception, motor control, self-awareness, cognitive functioning, and species (interpersonal in humans) experience. The insular cortex receives direct projections from the ventral medial nucleus of the thalamus and amygdala, sends projections to the amygdala and connects with the secondary primary sensory cortex. In addition, it receives inputs from spinothalamically activated ventral thalamic nuclei.

Cingulate cortex (CG): The cingulate cortex is dorsal to the corpus callosum and is part of the limbic system, which is involved with formation and processing of emotions, learning, and memory. It is also important for executive function and respiratory control. The rostral (anterior) and the caudal (posterior) portions of the cingulate cortex are related to the amygdala and hippocampus, respectively.

Entorhinal cortex (ER): The entorhinal cortex covers the anterior part of the parahippocampal gyrus and receives afferent inputs from the amygdala, olfactory bulb, limbic areas, and neocortex. The entorhinal cortex sends most of its efferent outputs to the hippocampus and plays an important role in spatial memories, including memory formation and consolidation of memories during sleep.

Sensory parietal cortex (PC): This region integrates sensory information to determine spatial awareness and navigation and permits the animal to map objects perceived visually into body coordinate positions. It receives afferent inputs from the visual, somatosensory, auditory, and vestibular areas.

Temporal cortex (TC): The temporal cortex is involved in auditory perception through connections to the primary auditory cortex. It is also important for the processing of semantics in both speech and vision. The temporal lobe connects with the hippocampus playing a role in the formation of long-term memories.

Optic chiasm (opc): This tract is composed of partially crossed optic nerve fibers and sends inputs to the lateral geniculate nuclei in the thalamus.

Table 4.

Neuroanatomic Areas Included in the Brain Section at the Level of the Rostral Thalamus (Figure 6)

Hippocampus (Hip): The hippocampus is a specialized cortical area integrated in the temporal lobe and belongs to the limbic system, playing important roles in long-term memory and spatial navigation. It is composed of different neuronal types that are organized into layers. The hippocampus is composed of the dentate gyrus, hippocampus proper (CA1, CA2, CA3, and CA4), and subiculum. The dentate gyrus receives afferent input from the entorhinal cortex and sends axons to the hippocampal pyramidal cells. The hippocampus proper (or Ammon’s horn) receives afferent inputs from the dentate gyrus and septal nuclei and sends efferent inputs to the subiculum and septal nuclei. The subiculum is a transitional area between the hippocampus proper and entorhinal cortex and represents the main output of the hippocampus.

Substantia nigra (SN): The substantia nigra is composed of two parts, the compact part (pars compacta), containing closely packed, pigmented dopaminergic neurons (containing neuromelanin) that project to the striatum, and a reticular part (pars reticularis), containing more loosely arranged neurons, receiving inputs from the striatum and projecting to the thalamus. The substantia nigra plays important roles in reward, addiction, and movement.

Cerebral peduncle (cp): The cerebral peduncle is composed of dense white matter tracts made up of tightly packed corticospinal, corticobulbar, and corticopontine axons that travel along the base of the midbrain.

Lateral geniculate nucleus (LG): The lateral geniculate nucleus is a very important thalamic nucleus involved in vision. It receives afferent axons from the retina via the optic tract and sends efferent axons to the primary visual cortex in the occipital lobe.

Optic tracts (opt): The optic tracts are composed of axons of retinal ganglion cells originating in the temporal half of the ipsilateral retina and the nasal half of the contralateral retina and connect with the lateral geniculate nucleus, superior colliculus, and other visual areas.

Table 5.

Neuroanatomic Areas Included in the Brain Section at the Level of the Caudal Thalamus (Figure 7)

Pons: The pons is a part of the brainstem that is rostrally and caudally connected to the midbrain and medulla, respectively. The pons contains gray and white matter connections between the cerebrum and cerebellum.

The pontine nucleus (pnO): The pontine nucleus is located in the pons and receives terminal afferent axons from the corticopontine fibers. Efferent axons from the pontine nuclei project across the midline into the cerebellum via the middle cerebellar peduncle.

Rostral (superior) colliculus (SC): The rostral colliculus receives afferent axons from the retina and visual cortex and sends efferent axons to the pulvinar nucleus and other thalamic nuclei in order to play an important role in directing visual attention and controlling eye movements.

Table 6.

Neuroanatomic Area Included in the Section at the Level of the Middle of the Cerebellum and Brainstem (Figure 8)

Cerebellum: The cerebellar cortex receives most of its inputs from extracerebellar sources such as the vestibular nerve, spinal cord, reticular formation, olivary nucleus, and cerebral cortex (via the pontine nuclei). Its primary output (via the Purkinje neurons) is to the deep cerebellar nuclei. The cerebellum plays an important role in motor control such as coordination and precision, but it is also involved in cognitive functions of attention and language.

Deep cerebellar nuclei (DCN): The deep cerebellar nuclei are composed of the three nuclei, the fastigial, interposed, and dentate nuclei. The fastigial nucleus, which is the most medially located cerebellar nucleus, contains afferent axons from the cerebellar vermis and sends efferent axons to the vestibular nuclei and reticular formation. The interposed nucleus is located between the dentate and fastigial nuclei and has two distinct subdivisions, the globose and emboliform nuclei. Both subdivisions receive inputs from the cerebellar cortex of the medial hemisphere and project (via the cerebellar peduncles) to the red nucleus and ventrolateral nucleus of the thalamus. The dentate nucleus receives inputs from the cerebellar cortex and sends (via the cerebellar peduncles) outputs to the red nucleus, inferior olivary nucleus, and thalamus.

Medulla oblongata: The medulla oblongata is part of the brainstem and contains nuclei that control autonomic functions such as respiratory, cardiovascular, and visceral activities and also relays signals between the brain and spinal cord. Sensory and motor tracts of the central nervous system run rostrally and caudally through the medulla oblongata.

Table 7.

Neuroanatomic Area Included in the Section at the Level of the Occipital Lobe (Figure 9)

Primary and secondary visual cortex (V1, V2, V3, V4 and MTV5): The visual cortex is composed of six functionally distinct regions within the occipital lobe. The primary visual cortex (V1) receives information directly from the lateral geniculate nucleus and processes information about position and movement of objects and pattern recognition. The secondary visual cortex contains connections from the primary visual cortex to regions V2, V3, V4, and V5. The secondary visual cortex is responsible for transmitting messages with respect to motion, shape, and position via intracortical pathways.

The Spinal Cord

General Descriptive Anatomy of the Cynomolgus Monkey Spinal Cord

The spinal cord in cynomolgus monkeys, as in humans, is composed of eight cervical, twelve thoracic, five lumbar, and five sacral segments, and one caudal (coccygeal) segment with their respective paired nerve roots. The spinal cord has two intumescences (one at each of the C5–T1 and L4–S2 levels) that correspond to larger numbers of neurons that innervate the skin, joints, and muscles of the limbs. The cervical nerves (C1–C7) emerge from the vertebral column rostral to the vertebra with the same number, whereas nerve C8 emerges rostral to the first thoracic vertebra. The remaining spinal nerves emerge caudal to the corresponding vertebrae.

The spinal cord in cynomolgus monkeys consists of central gray matter covered by white matter and surrounded by meninges (the dura mater, arachnoid, and pia mater). The center of the spinal cord holds the gray matter that contains symmetrical pairs of dorsal (posterior) and ventral (anterior) horns. The different levels of the spinal cord can be distinguished based on size, shape, and relative proportions of the white and gray matter.

Groups of myelinated axons that transmit sensory or motor information along the length of the spinal cord are located within three pairs of funiculi within the white matter. Within each funiculus, related groups of axons are bundled into tracts or fascicles that convey similar types of information. The posterior (dorsal) funiculi (medial to the dorsal root entry zones) contain fiber bundles that transmit somatosensory information to the brain. The lateral (between the dorsal root entry and the ventral root exit zones) and anterior (ventral; medial to the ventral root ext zones) funiculi have both ascending (sensory) and descending (motor) nerve tracts.

There are three types of fiber tracts: intersegmental, ascending, and descending. Intersegmental tracts project rostrally or caudally in the propriospinal tract that surrounds the outer edge of the central gray region, and these projections coordinate the different muscle groups that mediate spinal reflexes. The ascending tracts convey sensory information to the thalamus, cerebellum, or brainstem. Descending projections enable the cerebral cortex and brainstem to communicate with the spinal cord so that the brain can execute motor commands or modulate sensory transmission.

Cervical Levels

Cross-sections of the cervical cord have an oval shape. The funiculi in the cervical sections are large and are formed by the large number of ascending sensory tracts from the animal’s body and the descending motor tracts innervating groups of motor neurons. In addition, the anterior (ventral) horns are enlarged in sections C5–C8, because they contain additional motor neurons that innervate the upper limbs.

Thoracic Levels

Cross-sections of the thoracic cord are smaller than those of the cervical and lumbar cord, because the central gray region requires fewer neurons (which innervate the intercostal muscles of the trunk). Within the small lateral horns that protrude into the lateral funiculi, there is the intermediolateral cell column, which is composed of preganglionic sympathetic neurons of the autonomic system. All thoracic segments contain the dorsal nucleus of Clarke, which is located at the base of the dorsal horn and contains larger neurons. These nuclei are more prominent in the T10–T12 segments. In addition, the fasciculus cuneatus is present from T1–T6.

Lumbar Levels

Cross-sections of the lumbar cord have larger horns with a relatively smaller portion of white matter. The fasciculus gracilis is relatively narrowed compared to the thoracic cord. L1 and L2 have prominent lateral horns and dorsal nuclei of Clarke, but these structures are absent in L3–L5. The larger size of the anterior (ventral) horn is a result of the addition of motor neurons that innervate the muscles of the lower limbs.

Sacral and Coccygeal Levels

Sacral cord sections are characterized by small amounts of white matter and a relatively large proportion of gray matter. They have a thick gray commissure in the center of the cord. Caudal sections of the sacral cord become progressively thinner. The caudal segment is similar to sacral segments, but it is smaller in diameter.

Collection, Sampling, and Sectioning of the Cynomolgus Monkey Spinal Cord

Because some nuclei and fiber tracts are found only in certain levels of the spinal cord, at least one cross-section at the levels of the cervical region (near the brainstem), caudal thoracic region, and lumbar region should be sectioned and examined. The corresponding dorsal root ganglion and nerve roots should also be collected. The inclusion of a cervical cord section supports the evaluation of the distal portions of ascending tracts that pass through to the brainstem. Examination of lumbar sections provides for the analysis of the distal portions of descending tracts of the spinal cord. In a number of types of nerve fiber degeneration, the distal portions of the nerves may be the first to show morphologic changes detectable via light microscopic examination.

Some examples of specific nuclei and tracts occurring in cervical and lumbar cord are:

1. The intermediate septum is only present in the cervical and upper thoracic segments (C1–T6)

2. The fasciculus cuneatus is only present in the C1–T6 level sections.

3. The tectospinal tract is only present from C1–C4.

4. The sympathetic preganglionic neurons are located in the lateral horn of the spinal cord between segments T1 and L3. (Note that if parasympathetic alterations are suspected, sections of S2–S4 should be obtained.)

5. The medial longitudinal fasciculus is present only in sections of cervical cord.

An efficient method for sectioning the spinal cord for general toxicity studies in cynomolgus monkeys can be accomplished by performing a total or partial laminectomy. After removing the skin, spinal muscles, and adipose and connective tissues, the dorsal and transverse processes of the vertebrae can be removed using a bone saw or rongeur (World Precision Instruments, Sarasota, FL), being careful not to damage the cord. The entire cord (complete laminectomy) or segments at the levels of C1–C4, T10–T12, and L1–L4 (partial laminectomy) can be removed, along with the associated spinal nerve roots and corresponding dorsal root ganglion. The meninges can be used to stabilize and exert traction on the spinal cord during removal. Doing so will prevent mechanical destruction of the spinal cord parenchyma. Cross-sections of the spinal cord provide neuroanatomic orientation (Figure 10A). Oblique sections of the spinal cord (Figure 10B) provide an analysis of the funiculi in a semilongitudinal fashion, and lateral or longitudinal sections will improve optimal evaluation of nerve fiber degeneration. One single cross-section of each segment (C1–C4, T10–T12, and L1–L4) placed in one cassette, including the nerve roots with dorsal root ganglion (if possible), can provide an adequate analysis of most neural pathways of the spinal cord (Figure 10C). For general toxicity studies, staining of selected sections of cord can be limited to hematoxylin and eosin (H&E), although special chemical and immunohistochemical stains such as Luxol fast blue, glial fibrillary acidic protein, Iba1, calbindin, and silver techniques such as Bodian’s or Bielschowsky’s (to name just a few) can be performed to better characterize findings observed by routine light microscopic examination. Figures 11–13 depict neuroanatomic areas in the cervical, thoracic, and lumbar cord segments. Tables 8 and 9 briefly describe functions and neuroanatomy of the spinal cord segments shown in Figures 11–13.

Figure 14.

Longitudinal and cross-sections of the sciatic nerve placed in the cassette.

Table 8.

Neuroanatomic Areas Included in the Gray Matter of the Spinal Cord (Figures 11, 12, and 13)

Dorsal (posterior) horn anatomy (DH): The dorsal horn is primarily sensory in function and receives most of its inputs from afferent fibers that enter the cord via the dorsal nerve roots. Many of these incoming fibers send bifurcating axons rostrally and caudally through Lissauer’s tract before entering the dorsal horn. After leaving Lissauer’s tract, the small-diameter nociceptive fibers enter the lateral aspect and the wider cutaneous fibers enter the medial aspect of the posterior horn, respectively. The posterior horn is composed of five or six layers that vary in thickness, neuronal composition, and anatomic connections. Rexed lamina I or dorsomarginal nucleus (I): This nucleus is present in all spinal levels. Nociceptive information. Rexed lamina II or substantia gelatinosa (II): This nucleus is present in all spinal levels. Cutaneous and nociceptive information. Importantly, these neurons control or “gate” the efficacy of transmission between peripheral nerve fibers and the projection neurons. Rexed lamina III, IV, or proper sensory nucleus (III and IV): These nuclei are present in all spinal levels. Mainly cutaneous information (a few nociceptive neurons). Lamina IV contains multipolar neurons. Rexed lamina V (V): This nucleus is present in all spinal levels. Nociceptive and cutaneous information. Rexed lamina VI (VI): This nucleus is present only in the cervical and lumbar enlargements of the cord, where it receives input from the Golgi tendon organs, muscle spindles, and nociceptive free nerve endings. The lateral part receives descending projections from the motor cerebral cortex.
Lateral horn (LH): This region contains several nuclei that lie in different parts of the Rexed lamina VII and receive afferent connections from the primary sensory fibers, interconnections from other parts of the spinal cord, and descending inputs from the cerebral cortex and other areas of the brain. Dorsal nucleus of Clarke (VII): This nucleus is present in spinal levels from T1 (C8)-L3. This nucleus is a major relay center for unconscious proprioception. Axons from this nucleus go through the spinocerebellar tract to the cerebellar cortex. Intermediomedial nucleus (VII): This nucleus is present in all spinal levels. This nucleus sends local projections to the intermediolateral nucleus, and some neurons receive inputs from visceral and somatic afferent fibers and send their axons into the anterolateral system. It is responsible for referred pain. Intermediolateral nucleus (VII): This nucleus is present in spinal levels T1-L3. This nucleus is composed of cholinergic preganglionic cells.
Ventral (anterior) horn (VH): The ventral (anterior) horn contains multiple large and small neurons associated with voluntary and reflexive movements, and it receives descending input from the motor brain regions including the cerebral cortex, red nucleus, and the reticular formation. Neurons in these regions receive sensory information from the peripheral receptors either directly from the dorsal nerve roots or indirectly via intersegmental pathways that originate from neurons in the posterior horn. This horn is composed of the following laminae: Rexed lamina VIII (VIII): This nucleus is present in all spinal levels. This lamina receives descending input from the brainstem that is important for modulating muscle tone. Rexed lamina IX (IX): This nucleus is composed mostly of alpha motor neurons that innervate the skeletal muscle. These are large, multipolar neurons that innervate long, extrafusal fibers of the peripheral muscle and release acetylcholine from their synaptic terminals at the neuromuscular junction. The alpha motor neurons are called “final common pathway neurons,” because motor commands must converge onto the alpha neurons. In addition, this lamina contains small gamma motor neurons that innervate specialized receptors of the muscle spindle and release acetylcholine at their synapses to cause contraction of the short intrafusal fibers. One of the most important nuclei in the spinal cord is the phrenic nucleus of C3-C5 that innervates the diaphragm to regulate inspiration together with the intercostal muscles by integrating descending projections from the motor cortex (voluntary control) and caudal brainstem (involuntary autonomic control).

Table 9.

Ascending and Descending Tracts in the White Matter of the Spinal Cord

Ascending tracts: Ascending tracts are composed of groups of axons that transport somatosensory sensations (originating from a variety of mechanoreceptors and nerve endings of the skin, joints, and muscles) from the spinal cord to the brain with the objective of conscious perception. Interestingly, most afferent fibers entering the spinal cord have collaterals that distribute sensory information for a given modality to several spinal tracts so that damage to a sensory tract may produce only temporary sensory loss. The ascending tracts are composed of the following: Fasciculus gracilis (FG): This fasciculus is located in the posterior funiculus and ascends through the entire spinal cord, terminating in the nucleus gracilis at the caudal end of the medulla oblongata. The primary afferent fibers from first-order neurons are present in the dorsal root ganglia with axons extending from the foot, leg, and lower trunk; however, fibers from second-order neurons that originate from the dorsal horn also ascend though these fasciculi. Fasciculus cuneatus (FC): This fasciculus is also located in the dorsal funiculus, but it is present only from C1 to T6. It terminates in the nucleus cuneatus at the caudal end of the medulla oblongata, adjacent to the nucleus gracilis. The primary afferent inputs (from first-order neurons) are present in the dorsal root ganglia, with axons originating from the upper trunk, arm, shoulder, neck, and posterior head region; however, fibers from the second-order neurons also originate from the dorsal horn. The fibers of the fasciculi gracilis and cuneatus run ipsilateral to their site of origin. Spinothalamic tracts (part of the anterolateral system-ALS): These tracts are composed of ascending fibers located in the dorsal aspect of the lateral funiculi. These fibers originate from neurons in laminae I, IV, and V of the dorsal horn and convey sensations of pain and temperature to the ventral posterolateral nucleus of the thalamus. Spinomesencephalic tract (part of the anterolateral system-ALS): These tracts are composed of ascending fibers located in the dorsal aspect of the lateral funiculi. These fibers originate from neurons in laminae I, IV, and V of the dorsal horn and terminate in the periaqueductal gray area. The function of this tract is to stimulate the periaqueductal neurons to produce high amounts of endorphin to regulate pain sensations to the limbs. Spinotectal tract (part of the anterolateral system-ALS): These tracts are composed of ascending fibers located in the dorsal aspect of the lateral funiculi. These fibers originate from neurons in laminae I, IV, and V of the dorsal horn and terminate in the superior colliculus, which is involved in controlling eye and head movement during changes in visual attention. Therefore, this tract functions to direct visual attention to body regions that experience intense somatosensory stimulation. Cuneocerebellar tract (part of the anterolateral system-ALS): This tract is composed of fibers from the arm. These fibers enter the ipsilateral aspect of the fasciculus cuneatus and ascend to the medulla to terminate on the lateral cuneate nucleus. This tract merges with the inferior cerebellar peduncle before terminating in the ipsilateral vermis of the cerebellum to control the proprioceptive information coming from the arms. Anterior (ventral) spinocerebellar tract (AST): This tract originates from cells scattered throughout laminae V, VI, and VII from L1 to the coccygeal region; it receives input from the Golgi tendon organ fibers, several mechanoreceptor fibers, and descending spinal tracts and crosses the midline twice (entering to the cerebellum throughout the superior cerebellar peduncle before crossing the midline again to innervate the ipsilateral vermis of the cerebellum). This tract transmits proprioceptive information from the lower-limb muscles and their joints and lower body to the cerebellum, which controls posture and coordinates fine movements. Posterior (dorsal) spinocerebellar tract (PST): This tract originates from the neurons in the dorsal nucleus of Clarke (lamina VII) and extends from segments C8 to L2, merging with the inferior cerebellar peduncle in the brainstem to terminate in the ipsilateral vermis of the cerebellum. This tract receives input from the muscle spindles, Golgi tendon organs, encapsulated joint endings, and cutaneous mechanoreceptors in the leg and lower trunk. This tract transmits proprioceptive information from the lower limb muscles and their joints and lower body to the cerebellum, which controls posture and coordinates fine movements.
Descending tracts: Multiple brain regions send descending projections to the spinal cord to execute skeletal movements, regulate anatomic functions, or modulate the transmission of incoming sensory inputs. Descending tracts include the following: Corticospinal tract (lateral corticospinal tract [LCT] and anterior [ventral] corticospinal tract [ACT]): This is the major pathway controlling voluntary movements, and it is the only tract that transmits impulses from the cerebral cortex directly to the spinal cord. The corticospinal pathway originates in the motor regions of the frontal and parietal lobes, descends ipsilaterally through the internal capsule and crus cerebri, passes through the pyramidal tract decussation (most of the fibers), and travels through the lateral corticospinal tracts in the ventral aspect of the lateral funiculi. A few of the fibers remain uncrossed on the anterior surface of the spinal cord to form the anterior or ventral corticospinal tracts, which eventually cross the midline at the spinal cord segment in which they terminate. A lesser number of the fibers descend uncrossed within the lateral funiculi. These fibers terminate on interneurons that synapse with large alpha motor neutrons in the ventral horn. These tracts play a role in modulating flexor muscle tone and spinal reflexes. Rubrospinal tract (RT): This tract originates from the magnocellular neurons in the red nucleus and crosses the midline in the ventral tegmental decussation; descends the brainstem; enters the lateral funiculi, where it is partially intermingled with fibers of the lateral corticospinal tract; descends the entire length of the cord; and terminates in laminae V, VI, and VII. This tract augments the functions of the corticospinal tract by facilitating flexor movements. Tectospinal tract (TS): This tract is located in the ventral funiculus and originates in the deep layers of the superior colliculus, crosses the midline in the dorsal tegmental decussation, and descends through the anterior (ventral) funiculus to synapse with neurons in laminae VI, VII, and VIII of the spinal segments C1 to C4. This tract activates the motor neurons that control position of the head and neck after the rostral colliculus receives projections from the visual, auditory, and somatosensory systems. Reticulospinal tracts (medullary reticulospinal tract [lateral, MRT] and Pontine [medial, P]): There are both medial and lateral reticulospinal tracts located in the ventral and lateral funiculi, respectively. The lateral reticulospinal tract originates in the gigantocellular nuclei of the medullary reticular formation, and the medial reticulospinal tract originates in the oralis and caudalis nuclei of the pontine reticular formation. The projections from these two tracts descend the spinal cord and terminate in neurons of laminae VII, VIII, and IX. The function of these tracts is to facilitate or inhibit spinal circuits involved in reflexive or voluntary movements and in controlling spinal motor neurons. In addition, there are autonomic fibers that descend through this tract. Components of the reticulospinal tracts that project to sympathetic and parasympathetic centers of the spinal cord control cardiovascular, respiratory, gastrointestinal, and urinary systems, thus modulating the heart rate, respiratory rate, blood pressure, perspiration, and papillary dilation. Vestibulospinal tracts (LVT): These tracts originate in the medial and lateral vestibular nuclei and send projections to the spinal cord through the ventral and lateral funiculi, respectively. These are mainly uncrossed fiber projections that terminate in laminae VII and VIII and interact with neurons of lamina IX. The function of these tracts is to facilitate muscle stretch reflexes and enhance contraction of extensors. They also coordinate the movements of the eye, head, and limbs in response to angular or linear acceleration. Medial longitudinal fasciculus (MLF): The MLF originates in the vestibular nuclei and contains connections of the cranial nerves III, IV, and VI. The MLF carries information directing eyes movement and also descends into the cervical spinal cord to innervate some muscles of the neck. Fasciculi interfascicularis and septomarginalis (IF and S): These two small tracts enable sensory information to descend short distances within the cord in the cervical/thoracic (interfascicularis) and lumbar (septomarginalis) cord regions, respectively.

Functional neuroanatomy of the brain areas included in Tables 2–7 (Figures 4–9) were obtained from Afifi and Bergman 2005; Alloway and Pritchard 2007; Crosby et al. 1962; Hendelman 2005; and Nolte 2009.

Functional neuroanatomy of the spinal cord included in Tables 7 and 8 (Figures 10–12) were obtained from Alloway and Pritchard 2007; Crosby et al. 1962; Hendelman 2005; and King 1999.

The Peripheral Nerve

General Descriptive Anatomy of the Cynomolgus Monkey Peripheral Nerve

Peripheral nerves are responsible for afferent and efferent communication between the body and the central nervous system. The cell bodies of afferent fibers are located in the dorsal root ganglia and are referred to as pseudounipolar neurons. The efferent fibers of the ventral nerve roots originate from cell bodies located in the lateral or ventral horns of the spinal cord. After leaving the spinal cord, these efferent fibers merge with distal processes of the afferent fibers to form a mixed spinal nerve; however, efferent fibers originating from motor neurons in lamina IX proceed to skeletal muscle and terminate in neuromuscular junctions.

The brachial and sciatic nerves are formed by the spinal nerves C5–T1 and L4–S3, respectively, although variation in the origin of the brachial nerve was observed in one monkey by the first author. The brachial nerve is responsible for cutaneous and muscular innervation of the entire upper limb, with the exceptions of the trapezius and intercostobrachial muscles, which are innervated by the spinal accessory nerve and intercostobranchial nerves, respectively. The brachial nerve gives origin to the axillary, radial, and ulnar nerves, as well as to other important nerves. The sciatic nerve innervates the skin of the legs, muscles of the back of the thigh, and leg and foot muscles such as the biceps femoris, semitendinosus, semimembranous, and adductor magnus. The sciatic nerve branches to form the tibial and common peroneal nerves.

Collection, Sampling, and Sectioning of the Cynomolgus Monkey Peripheral Nerve

For most general toxicity studies, the evaluation of longitudinal and cross-sections of the sciatic nerve from one of the legs is adequate to examine the peripheral nerve system (Figure 14). The section can be taken at the mid-thigh level, just prior to the point of branching of the sciatic nerve into the tibial and peroneal nerves. The longitudinal sample should be ≥ 1 cm in length and fit in a standard cassette. It is highly recommended to mark with ink the proximal versus the distal aspect of the nerve to keep the nerve orientation at sectioning. Examination of the sciatic nerve of both legs would be recommended if local injection site trauma to the nerve is suspected. Additional peripheral nerves such as the tibial, femoral, sural, and brachial nerves may be collected if clinical signs suggest peripheral nerve dysfunction.

Conclusions

There is general agreement among neuropathologists that a greater degree of brain sampling is necessary to adequately perform safety evaluations. The technique described in this paper provides a consistent way to sample and evaluate the nervous system of cynomolgus monkeys in general toxicity studies. This method is readily adaptable to species other than nonhuman primates, such as dogs and minipigs, and this method can readily be learned by prosectors and histotechnicians with relatively basic training in neuroanatomy.