Abstract
The central (CNS) and peripheral (PNS) nervous systems of vertebrates represent divisions of a continuous, body-wide communication grid based on conserved principles of structural organization. Discrete neuroanatomic regions within this grid are associated with specific neural functions, so distinct patterns of neurological dysfunction (“problems”) can provide guidance regarding neural regions to evaluate beyond those in published sampling schemes or institutional standard operating procedures. Each neurological problem or syndrome (i.e., a group of in-life signs indicating that a given neuroanatomic region is damaged) is associated with a particular list of differential diagnoses and causes. Vulnerability of neural cells and tissues is influenced by intrinsic tissue properties (e.g., high metabolic rates of neurons, presence of blood:tissue barriers, degree of collateral vascular supply) and extrinsic factors (bone protuberances and connective tissue partitions impinging on neural surfaces, fluid flow patterns in the cerebroventricular system and meninges, etc.). In the toxicologic pathology setting, expansion (when warranted) of routine neural sampling protocols to collect additional anatomic regions correlated to a specific neurological problem improves the likelihood that a neuropathological evaluation will identify lesions and causes responsible for neurological conditions as well as detect findings related to potential test item-related neurotoxicity.
Keywords
This article is the companion piece to a presentation entitled “A Fandango of Form and Function: The Interplay between Clinical Neurology and Neuropathology Investigation” that was delivered by the first author (BB) as a keynote lecture at the fifth Cutting Edge Pathology Congress (August 28-31, 2024) in San Lorenzo de El Escorial (Madrid), Spain. The choice of “Fandango” to headline this “core concept” talk was deliberate, likening the name of the lively Spanish dance with (1) the intricate interweaving of neural cells and processes that is necessary to shape the control centers and circuits of the developing central (CNS) and peripheral (PNS) nervous systems as well as (2) the multifaceted battery of technically demanding functional (in-life) and structural (in-life or postmortem) tests required to assess neural form and function. Core functional assessments include screens like the functional observational battery (FOB48,52,86,93,142) and more rigorous evaluations such as comprehensive neurological examinations;37,82 electrophysiological testing (e.g., electroencephalography,3,4,117 electromyography,45,134 nerve conduction velocity,36,125 sensory evoked potentials32,42); and formal neurobehavioral testing (for cognitive, motor, and sensory functions81,92). Structural evaluations for neural tissues may include in-life procedures like noninvasive imaging 47 (e.g., computed tomography [CT], magnetic resonance imaging [MRI], positron emission tomography [PET]) and routine postmortem procedures16,19 like brain weights, macroscopic observations, microscopic examinations, and in some cases clinical pathology parameters such as leukocyte numbers or soluble biomarkers (e.g., cytokine or protein levels in cerebrospinal fluid [CSF] or plasma).8,124,130,132 Integrated functional and structural evaluation is essential in identifying lesions and mechanisms for neurological diseases in animal and human patients (the province of neurology and diagnostic neuropathology) as well as in evaluating the impact of novel substances on the health of individual neural organs and the nervous system as a whole (the realm of neurotoxicology and toxicologic neuropathology).
Compared to other organs and systems, the CNS and PNS are morphologically diverse. The CNS and various components of the PNS provide an uninterrupted, body-wide communication grid. The CNS consists of the brain and spinal cord while the PNS is partitioned between somatic (voluntary) and autonomic (involuntary) divisions. The somatic component of the PNS includes several clinically important cranial nerve (CN) ganglia (e.g., spiral [for CN VIII] and trigeminal for CN V]), many dorsal root ganglia (DRG), and their attached sensorimotor nerves. The autonomic portion of the PNS is segregated among three networks of ganglia and nerves regulating homeostatic (enteric and parasympathetic parts) and emergency response (sympathetic part) functions. The fundamental organization of the nervous system in terms of structure and function is conserved across all mammals including humans.25,27,29,34,37,43,67,70,97,126 Discrete regions in the CNS are defined by somatotopically arranged cell populations or nerve fiber tracts fulfilling distinct roles (where “somatotopic” means that the task fulfilled by an area is related to its connection to—and therefore the function of—a given body region). Details of comparative (i.e., interspecies) and correlative (i.e., site matched to function) neuroanatomy for the common nonclinical test animals9,25,37,82,126,128 and humans24,43,77 may be explored elsewhere. A firm grasp of correlative neuroanatomy is essential for investigating neurological problems or syndromes (where a “syndrome” is a group of in-life signs indicating that a particular neuroanatomic region is damaged) in both the clinical and toxicological testing settings.
This article reviews the relationship between principal vertebrate neuroanatomical regions and the particular patterns of functional abnormalities that indicate damage to these sites. Common neurological problems or syndromes are presented as text vignettes accompanied by a table that relates the neurological presentation and selected causes responsible for neural damage in that area. Selected figures are included for additional context relevant to core messages. The final text summation provides guidance regarding how a given constellation of in-life neurological signs may be employed in nonclinical studies to optimize neural tissue sampling, neuropathological evaluation, and translation of nonclinical data sets for human risk assessment.
A Brief Overview of Integrated Neurology/Neuropathology Assessment
Damage centered in a particular neural region (altered “form”) results in specific patterns of in-life clinical signs (aberrant “function”) that define distinct neurological problems that can be localized to particular neuroanatomic areas.22,37,82 Therefore, a primary task of neurologists and neuropathologists in addressing neurological problems is “lesion localization,” in which affected neural structures are identified that can explain the constellation of in-life signs. Alternatively, “lesion localization” may be framed based on the enhanced vulnerability of particularly sensitive neural cell populations (especially neurons) resulting from their specific attributes, such as high metabolic demands and residence in regions possessing limited collateral vascular supply. Lesion localization allows a list of differential diagnoses (potential causes) and pathogeneses for the problem to be ranked according to their relative likelihood. For this article, “lesion localization” will be used in the context of determining site(s) of neural cell/tissue damage rather than addressing cell type-specific susceptibility.
Lesion localization to define sites of neuroanatomic involvement relies in-life on the initial neurological examination with supplemental functional testing. Postmortem (or less often ante mortem) neuropathological evaluation may confirm lesion localization but is typically more critical for discerning the nature of the neural disease and discriminating among various causes and pathogeneses.
The neurological examination is an essential first step in investigating neurotoxicity. In the clinical setting, detailed neurological examinations are common for individual patients. In animal toxicity testing, truncated functional testing (e.g., an FOB) is generally used as a rapid screen as multiple animals must be assessed in a relatively short time frame, but formal neurological examinations may be performed when warranted (for nonrodent species) to better define potential neural target sites for reproducible patterns of in-life clinical signs. In both the clinical and toxicity testing settings, neurological examinations should be done systematically by trained and experienced examiners. Neurologists generally proceed from general to specific assessments, starting with the animal’s mental status and then assessing gait; posture; musculoskeletal tone (by palpation); postural reactions (conscious proprioception, hopping, etc.); reflexes (spinal and cranial nerves); and sensation (superficial and deep pain). This ordered approach allows the examiner to progressively localize the lesion from general structures (e.g., brain) to more specific areas (e.g., cerebral cortex). Results of the neurological examination may also imply involvement of particular neural cell populations (e.g., larger pyramidal neurons of the cerebral cortex).
As applied in a clinical (medical or veterinary medical) setting, the patient’s signalment (species, race/breed/strain, sex, age, etc.); history (signs, symptoms [for humans], diet, etc.); and results of the physical and neurological examinations determine the neurological as well as extra-neural problem(s). These factors would be considered together with other clinicopathologic features to localize the lesion site(s), define the list of differential diagnoses, select necessary tests to determine the cause(s), and establish a prognosis with or without treatment. Generally, causes that account for all (or most) neurological problems should receive highest priority, although multiple conditions and affected neural regions may ultimately be identified. In the clinical setting, neurotoxicity requires deliberate testing to show that patients have been exposed to a suspected toxicant at levels sufficient to produce the toxic syndrome. For a diagnostic neuropathological evaluation, neural samples are collected in accord with an institutional standard operating procedure (SOP) that is modified to specifically harvest any additional sites implicated by the spectrum of in-life neurological abnormalities observed for the patient.
In animal toxicity testing, a test item (alternatively: test article or test substance) administered at a given dose and frequency by a designated route is the expected cause for any neurological problem observed in life. Importantly, however, some neurological signs (especially behavioral abnormalities) may be present predosing, emphasizing the need to assess animals at baseline. Moreover, care is necessary to discriminate test item-related changes from confounding causes of in-life neurological signs (e.g., incidental background findings or damage produced by a neuro-invasive procedure such as direct injection into the CSF or neural tissue). With that said, the neuropathological evaluation in toxicity testing is focused on identifying and characterizing the neural and potentially extra-neural lesion location(s); nature (e.g., degeneration, inflammation, neoplasia); dose dependence; severity; and reproducibility of neural findings. Information on these attributes may be used to determine the underlying mechanisms responsible for such structural changes, which in turn may provide insights that can help in modifying test items to have more efficacy and/or less neurotoxicity. In animal toxicity testing, current approaches to CNS and PNS sampling of common mammalian species10-12,16,19,49,96,98-100,107 typically involve collection of samples focused on major neural regions with well-defined structure-function relationships and known sensitivities to neurotoxic agents. Additional neural sampling may be undertaken on a case-by-case basis if the constellation of in-life signs suggests that a region not subject to routine sampling might be damaged.
In both clinical and nonclinical toxicity settings, standardized approaches to neural sampling cannot survey all neuroanatomical areas found in vertebrates (e.g., 600+ defined brain nuclei, dozens of discrete neural tracts in the brain and spinal cord, myriad ganglia and nerves), nor do standard approaches guarantee evaluation of all regions linked to documented neurological syndromes. Accordingly, the presence of a particular neurological problem is an important consideration in devising further in-life testing to design a focused neural sampling plan that maximizes the likelihood that lesions (and causes) responsible for neural dysfunction will be detected. 20 A simple example of this concept relates to an animal with paraparesis and normal thoracic limb function, indicating that the lesion likely lies caudal to the second thoracic (T2) spinal cord segment. The next step in lesion localization would be to decide whether there are upper motor neuron (UMN) or lower motor neuron (LMN) signs (see discussion below) in the pelvic limbs, which would suggest involvement at T3-L3 or L4-S3 segments, respectively (varying somewhat with species). Further sensory testing (e.g., for the panniculus reflex and paraspinal hyperesthesia) can generally isolate T3-L3 lesions within three spinal cord segments, thereby allowing precise sampling; such testing is typically not performed in nonclinical studies unless the neurological syndrome occurs in multiple animals. Pathologists with skill in localizing neuroanatomic lesions using signs such as these will improve their ability to devise effective neural sampling schemes and identify neural lesions and their causes.
Lesion Localization for CNS-Centered Neurological Syndromes
Neurological syndromes affecting the CNS are based on involvement of one or more brain and/or spinal cord regions. Here, the CNS differs somewhat from other organs, such as the kidney or liver, which tend to be more uniform in composition and tissue organization. In this regard, syndromes due to brain lesions differ considerably depending on the area/region affected. These lesions typically affect a region broadly, as with the effect of lead poisoning or thiamine deficiency on cortical neurons in the cerebrum or diffuse white matter involvement due to certain toxicants like hexachlorophene and triethyltin. Nonetheless, toxicants can affect specific brain nuclei, with a classic example being necrosis of the globus pallidus and substantia nigra in horses that develop nigropallidal encephalomalacia following ingestion of yellow star thistle (Centaurea solstitialis).
Conceptually, neurological conditions arising in the CNS may be approached using various organizational principles. For CNS neurological problems in animals, approaches usually consider functional distinctions (e.g., motor vs sensory) or structural locations at the organ (e.g., brain vs spinal cord) or regional (e.g., forebrain vs cerebellum vs brainstem) levels. A successful neuropathological evaluation typically integrates both approaches.
Function-Oriented Analysis—Upper Motor Neuron (UMN) vs. Lower Motor Neuron (LMN) Involvement
Upper motor neurons in the brain gray matter (mainly cerebrum but also certain brainstem nuclei including the red nucleus in the midbrain) supply axons to LMNs in the spinal cord gray matter and the brainstem motor nuclei of certain cranial nerves. Damage to UMNs and LMNs produces distinct patterns of neurological signs (Figure 1).37,82
Anatomic locations and clinical signs associated with upper motor neuron (UMN) and lower motor neuron (LMN) lesions. [Image adapted from Kornegay, 1991 78 by Mr. Tim Vojt.]
Signals from UMNs provide voluntary control over LMNs and mainly facilitate contraction of flexor muscles and inhibit the activity of extensor muscles. Therefore, damage to UMN cell bodies or axons leads to partial (“paresis” [weakness]) or complete (“plegia” [paralysis]) loss of voluntary motor function. Given that LMN function remains intact, reflexes and muscle tone will be retained and may even be exaggerated (i.e., hyperreflexia, hypertonia) because of the selective loss of extensor muscle inhibition. Neurological dysfunction almost always develops caudal to the level of UMN involvement, with neurological signs evident contralateral (opposite side) or ipsilateral (same side) depending on whether local lesions are rostral or caudal to the site of axonal decussation as white matter tracts course to their LMN targets. Thus, lesions affecting the cerebral cortex or thalamus, together termed the forebrain, cause contralateral neurological dysfunction; brainstem involvement generally yields ipsilateral motor impairment accompanied by ipsilateral cranial nerve deficits; and involvement of the spinal cord white matter causes ipsilateral deficits. Muscle mass is largely retained in UMN-mediated conditions, with evidence of mild (disuse) atrophy over weeks to months.
Signals from LMNs pass down cranial and spinal nerves to innervate muscles and glands throughout the body. Signaling by LMNs is regulated in health by the activity of UMNs, but in the absence of UMN input the LMNs participate in locally controlled reflex arcs. Damage to LMN cell bodies (brainstem or spinal cord) or axons (nerve roots, cranial and somatic nerves) leads to paresis/plegia and is distinguished from UMN involvement by (1) hyporeflexia or areflexia, (2) reduced or absent muscle tone, and/or (3) severe (“neurogenic”) muscle atrophy within 7-10 days. Sustained lesions lead to loss of myofibers, with replacement by fat and connective tissue, leading to associated flexor contractures after weeks to months in severe cases. Lesions involving the brainstem or spinal cord usually cause bilateral functional abnormalities that may be asymmetric corresponding to the lesion side, while damage to nerves is usually evident as unilateral limb (monoparesis, monoplegia) or head (mastication [CN V] or facial muscle [CN VII]) dysfunction.
Function-Oriented Analysis—Segmental Versus Long Tract Sensory Involvement
With either UMN or LMN involvement, there will generally be concomitant sensory deficits that are categorized as either long tract (lesions affecting sensory axons as they ascend the spinal cord or brain stem) or segmental (neuronal cell body lesions [locally within the spinal cord intumescences or certain brainstem nuclei] or axonal involvement [in cranial or spinal nerves]). The most consequential sensory pathways for in-life clinical signs transmit pain (superficial and deep) and proprioception (conscious and subconscious) information. Sensory input for the body is collected at peripheral receptors and transmitted centrally along somatic nerves to the DRG, where somatosensory neuron cell bodies are located. Corresponding sensory input for selected cranial nerves is relayed to sensory neurons located in the brain. With brainstem, spinal cord, or cranial/spinal nerve involvement, sensory deficits will be ipsilateral to the level of the lesion. Notably, as with motor deficits, forebrain lesions cause contralateral clinical signs due to the decussation of ascending axons in the brainstem (described in more detail below).
Characteristic clinical signs produced by segmental lesions in PNS neuronal cell bodies or axons include (1) anesthesia (total sensory loss, due to lesions affecting the entire structure); (2) hypoesthesia (decreased sensation, reflecting damage to part of the structure); (3) hyperesthesia (increased sensitivity to sensory stimuli) including allodynia (perception of nonpainful stimuli as noxious); or (4) paresthesia (where burning, numbness, prickling, or tingling is experienced in a denervated area [with the potential for self-mutilation]). Segmental sensory signs develop at the level of the neural lesion. Loss of sensation may be localized to dermatomes (cutaneous tissues corresponding to each spinal nerve) by pricking the skin with a needle or gently compressing tissue with a hemostat. At the same time, evidence of hyperesthesia at the lesion site, produced as nerve roots or the associated spinal meninges are stimulated, may be detected through deep palpation of paraspinal muscles. The patterns of in-life neurological signs associated with segmental lesions affecting the spinal cord, spinal nerve roots, and/or DRG often occur in tandem with LMN motor signs.37,82
Neurological signs of long tract disease result from damage to ascending white matter tracts in the spinal cord or brainstem. Conscious proprioception travels in tracts of the dorsal and dorsolateral spinal cord to reach the contralateral somatosensory cerebral cortex. Subconscious proprioception projects in spinocerebellar tracts of the dorsolateral spinal cord to reach the ipsilateral cerebellum. Superficial pain ascends in the dorsolateral and lateral spinal cord to the contralateral somatosensory cerebral cortex. Deep pain is conveyed bilaterally to the brainstem (reticular formation), thalamus, and cerebral cortex. In-life signs for long tract lesions typically include initial so-called “sensory” ataxia (clumsy movements) with abnormal positioning of the paws (loss of conscious proprioception) and/or dysmetria (impaired motor control due to loss of spinocerebellar input), followed by anesthesia or hypoesthesia. Notably, distinct forms of ataxia may also occur with cerebellar and vestibular lesions, as discussed below.
Long tract sensory signs develop caudal to the level of the neural lesion and often accompany UMN motor signs.37,82 The deficits will be ipsilateral to spinal cord and cranial/spinal nerve lesions and contralateral to those in the forebrain because of the decussation of long tracts in the brainstem. While the side of sensory deficits may vary with long tract involvement in brainstem lesions, most will be ipsilateral.
Anatomy-Oriented Analysis—Brain
Neurological syndromes linked to the CNS have been established for multiple regions of the brain and spinal cord (Figure 2).37,82 Characteristic signs generally allow brain lesions to be localized to either the forebrain (encompassing the cerebrum and thalamus), brainstem, cerebellum, or vestibular system (Figure 3). Clinical syndromes associated with spinal cord lesions are discussed below. From a diagnostic perspective, the examiner should always strive to localize clinical signs to lesions affecting one region. Nonetheless, multifocal disease should be expected for wide-ranging patterns of clinical signs, for instance an animal exhibiting both seizures (indicating forebrain disease) and paraplegia (pointing to a second lesion caudal to the T2 spinal cord segment). Multifocal disease is more typical of conditions that widely involve the CNS or body (e.g., inflammation, metabolic disturbances, toxicity).
Areas of the brain and spinal cord to which lesions can be localized based on characteristic in-life neurological signs. The thalamus can be considered anatomically as related to both the forebrain (solid) and subcortical centers (dashed line), but signs related to this region are most commonly associated with forebrain lesions. [Image prepared by Mr. Tim Vojt.]
Characteristic constellations of neurological signs for localizing brain lesions to specific neuroanatomic regions and brain-related neurological syndromes. [Image adapted from Kornegay, 1991 78 by Mr. Tim Vojt.]
Forebrain syndrome
The forebrain refers to that area of the brain derived embryologically from the prosencephalon, which divides further during later development into the telencephalon (cerebrum) and diencephalon (thalamus). Lesions in the cerebrum or thalamus cause similar signs (Figure 3 and Table 1), typically including reduced responsiveness (depression, stupor [capable of arousal], or coma [not capable of arousal]); abnormal behavior such as head pressing or (in the case of limbic system [temporal lobe] lesions) aggressiveness, tail chasing, and so on; abnormal gait (compulsive walking or circling); visual impairment; and/or seizures. Localized (focal or locally extensive and asymmetrical) damage may cause movement abnormalities affecting one side of the body—often circling toward the lesion with contralateral postural reaction deficits—or unilateral sensory (auditory or visual) impairment.
Forebrain syndrome.
Visual impairment due to forebrain involvement must be distinguished from lesions affecting the ophthalmic system or peripheral neural structures that also contribute to the pupillary light reflexes (PLRs). In general, involvement of the neural retina, optic nerves (and/or chiasm), and optic tracts affects both vision and the PLRs while lesions localized to the lateral geniculate nucleus (the thalamic relay center for visual stimuli), optic radiation, and occipital (visual) cortex produce only visual deficits. These deficits will be predominantly contralateral because of the decussation of optic nerve fibers at the optic chiasm. Lesions of the brainstem and other floor structures in the cranial vault may alter PLRs but do not impact vision.
The most common differentials for forebrain syndrome are nontoxic entities. Inflammation (encephalitis or meningoencephalitis) is a principal cause of diffuse or multifocal forebrain involvement in all species. Encephalitides may result from pathogens such as bacteria or viruses, but idiopathic, likely immune-mediated causes have been increasingly described. Other causes of diffuse forebrain involvement include metabolic conditions (e.g., hepatic or uremic encephalopathy); nutritional deficiencies (e.g., polioencephalomalacia 119 ); environmental toxicants (e.g., heavy metals, mycotoxins, shellfish toxins, solvents); and selected neuroactive therapeutic agents (e.g., amino acid neurotransmitter [NT] receptor antagonists and catecholamine NT agonists).39,118 Physiological conditions that induce transient in-life signs of forebrain syndrome include hyperthermia (e.g., fever or heat stroke); hypoxia; and altered intraparenchymal levels of electrolytes (hypocalcemia, hypomagnesemia, hyponatremia) or glucose (hyperglycemia, hypoglycemia). Several of these conditions selectively involve certain neuron populations leading to laminar (primates) or pseudolaminar (nonprimate species) cerebrocortical neuronal necrosis. “Salt poisoning” (sodium ion toxicosis) is a particular forebrain syndrome that occurs when water supplies are frozen and animals rehydrate too quickly once access is restored. The condition occurs when idiogenic osmoles (intracellular organic molecules like glycine and sorbitol) are synthesized within neurons to restore intracellular osmolar equivalency in the face of systemic dehydration. When water is made available again, fluid moves intracellularly to reestablish this equivalency, leading to cell and organ swelling. Interestingly, these idiogenic osmoles are thought to be chemoattractant for eosinophils, thus accounting for the predominance of this cell type in the perivascular infiltrates that are a hallmark microscopic feature of “salt poisoning.” Repetitive or sustained seizures also may induce marked and widespread neuronal degeneration and necrosis that must be distinguished from other metabolic and toxic insults. 117
Subcortical syndrome (alternatively: diencephalic syndrome)
Lesions affecting subcortical centers may cause signs over and above those typically associated with forebrain involvement as outlined above. This syndrome is associated with damage to subcortical nuclei in the thalamus or more often the hypothalamus (Figure 3 and Table 2). Frequent additional neurological signs include altered function of central autonomic centers (as shown by anomalous eating patterns and thermoregulatory control) and brain-localized endocrine system disruption, leading to changes in hypothalamic production of various hormones that act in the pituitary gland (e.g., corticotropin-releasing hormone) or on peripheral organs (e.g., arginine vasopressin [antidiuretic hormone], which binds to receptors in renal tubular epithelium to limit water loss). These signs may occur without gait and postural aberrations. However, gait changes similar to those seen with cerebral lesions and contralateral postural reaction deficits may develop if subcortical lesions affect ascending/descending pathways in the internal capsule; in such cases, contralateral visual deficits may also occur via disruption of the optic tract located adjacent to the internal capsule. Vision may also be disrupted when a hypothalamic or pituitary gland lesion encroaches on the optic nerves (i.e., CN II) or optic chiasm. Visual disruption may be rapid when pituitary apoplexy occurs (in which acute hemorrhage, typically within a pituitary tumor, quickly encroaches on the optic chiasm). Auditory dysfunction may be seen if the medial geniculate nucleus (the thalamic relay center which processes acoustic information) is affected.
Subcortical syndrome.
The most common causes for subcortical syndrome are space-occupying masses in or around the thalamus or hypothalamus, most often pituitary gland tumors. In general, these lesions are not associated with toxicant exposure.
Cerebellar syndrome
Cerebellar syndrome is a relatively common, easily identifiable neurological condition (Figure 3 and Table 3). Lesions in adults are localized most often to the neuronal layers (molecular, Purkinje cell, and/or granule cell) of the cerebellar cortex and/or to the deep cerebellar nuclei (DCN). Signs are generally bilateral involving all limbs if lesions are diffuse and ipsilateral if damage is asymmetric. The chief neurological signs are ataxia (clumsy movements with incoordination), dysmetria (usually hypermetria [overstepping]), and tremor (often upon initiating movement [i.e., “intention” tremor]). Importantly, “pure” cerebellar ataxia as occurs with hypoplasia due to in utero viral infections (e.g., bovine viral diarrhea, classical swine fever, feline panleukopenia) does not affect attitude, conscious proprioception, or motor function. Ocular signs (ipsilateral to the site of damage), characterized by fine side-to-side eye oscillations and/or a reduced or absent menace response, occur infrequently for lesions affecting the vermis and some DCN. 116 Vestibular signs (contralateral to the involved area) may be seen if the floccular or nodular lobes (vestibulocerebellum) are involved, causing a head tilt and loss of balance (i.e., paradoxical vestibular syndrome in which the head tilt and other vestibular signs are on the opposite side of the lesion). 116 Mental status and vision are generally unaffected.
Cerebellar syndrome.
Common causes of this syndrome include developmental defects, inflammation (including diseases such as canine distemper that selectively affect the postnatal cerebellum and viral infections during gestation that impact the developing cerebellum), or space-occupying masses (usually abscesses or neoplasms in the caudal [posterior] fossa) compressing the cerebellum. Some toxic chemicals, heavy metals, and natural toxins have been linked to this syndrome.
Brainstem syndrome
Brainstem syndrome is a comparatively common constellation of neurological signs due to lesions affecting the midbrain, pons, and/or medulla oblongata (Figure 3 and Table 4). The usual pattern combines abnormal mental status due to reticular activating system (RAS) involvement, long tract sensory signs and UMN motor signs due to involvement of ascending and descending white matter tracts, and CN deficits.
Brainstem syndrome.
Two distinctive brainstem syndromes are seen, alone or together, with brain herniation. In the first form, rostrotentorial masses (e.g., tumors, hematomas, abscesses) or parenchymal swelling (edema) can displace the parahippocampal gyri of the temporal cortex caudally beneath the tentorium cerebelli (a tough dural fold that divides the rostral and caudal compartments of the cranial vault). In these instances, an initial forebrain syndrome is compounded by signs of brainstem involvement due to compression of the midbrain located within the tentorial incisure. In the second type, progression of the rostrotentorial brain herniation or the presence of a space-occupying mass in the caudal compartment drives the caudal cerebellar vermis through the foramen magnum, leading to compression of the medulla oblongata. Both these conditions lead to altered consciousness with long tract sensory and UMN motor signs. Herniation affecting the midbrain is commonly accompanied by pupillary dilation resulting from compression of the oculomotor nerve (CN III) against the skull floor, while herniation affecting the medulla oblongata is often associated with life-threatening apnea (intermittent cessation of breathing).
Common causes include inflammation (primarily viral or immune-mediated), due to the space-occupying effect of edema, and neoplasia. Chemicals and small molecule drugs may injure the brainstem following local (intrathecal135,136) or systemic23,101 delivery. In many cases, brainstem parenchyma is not damaged in isolation but is accompanied by effects in other CNS regions.60,103,122
Vestibular syndrome
Vestibular syndrome is characterized by another easily recognized spectrum of neurological signs (Figure 3 and Table 5). The “central” form results from lesions affecting the vestibular nuclei in the brainstem (rostral medulla oblongata) while the “peripheral” form is induced by damage to either CN VIII (vestibular division) or the vestibular apparatus (i.e., the inner ear). Both variants share multiple signs including circling, head tilt, loss of balance with falling and rolling (all ipsilateral to the involved side); nystagmus (involuntary, rapid, repetitive eye movements [“dancing eyes”]); and strabismus (eye misalignment [“crossed eyes”], typically ventrolateral). In contrast, certain signs are unique to each variant. For the central form, the most reliable distinguishing features are long tract sensory and UMN motor involvement, generally causing ipsilateral postural reaction deficits, sometimes with depression if the RAS is affected; the presence of nystagmus is less reliable and is often vertical. The peripheral form is typically characterized by horizontal, jerk nystagmus with the fast phase opposite the side of the lesion. Involvement of multiple cranial nerves (CNs) generally points to a central lesion, particularly for laterally placed masses at the cerebellopontine angle where CNs V, VII, and VIII are closely clustered. Notably, CNs IV (trochlear nerve) and VI (abducens nerve) exit the brainstem on the midline so are usually not affected by laterally placed masses. It is important to remember that the somatic portion of CN VII (which innervates facial muscles) and postsynaptic sympathetic nerve fibers innervating the eye both course through the middle ear, so facial muscle paralysis; Horner’s syndrome (featuring ipsilateral enophthalmos [inward displacement of the globe], miosis [pupillary constriction], and ptosis [drooping eyelid] 144 ); or both can be seen with peripheral vestibular lesions.
Vestibular syndrome.
Asterisks denote that movement is ipsilateral toward the side with the lesion.
Central vestibular syndrome most commonly results from inflammation or neoplasia, as with brainstem lesions more generally. Causes vary among species; for instance, encephalitis is more common in cats while neoplasia is more frequent in dogs. Peripheral vestibular syndrome usually reflects inflammation of the middle (otitis media) or inner (otitis interna) ear or arises as an idiopathic condition (presumably immune-mediated). Toxicants that have been associated with vestibular syndrome generally act peripherally by inducing hair cell (sensory epithelium) lesions in the vestibular labyrinth.35,44,74,113,138
Anatomy-Oriented Analysis—Spinal Cord
Neurological syndromes associated with the spinal cord have been defined for five spinal cord regions: cervical, cervicothoracic, thoracolumbar, lumbosacral, and caudal (Figure 2). Lesions in the first four divisions are typically localized based on the spectrum of UMN and LMN signs (Figure 4). The caudal division is added because of the potential for spinal cord segments or spinal nerve roots to be traumatized during intrathecal injections into the lumbar cistern. The formulas for the spinal cord divisions in the text and figures use the typical locations in small animals (cats and dogs). Precise locations will vary modestly from these formulas for different vertebrate species, 19 but the general principles stated here are applicable to other animal species.
Characteristic constellations of neurological signs for localizing spinal cord lesions to specific neuroanatomic regions (varying somewhat with the species) and spinal cord-related neurological syndromes. Abbreviations: C = cervical, L = lumbar, LMN = lower motor neuron, S = sacral, T = thoracic, UMN = upper motor neuron. [Image adapted from Kornegay, 1991 78 by Mr. Tim Vojt.]
As discussed below, lesion localization in the spinal cord is typically based on the presence (or absence) of UMN or LMN signs. This diagnostic approach implies that the lesion is sufficiently severe to cause paresis (paraplegia). With mild lesions that cause only hyperesthesia or ataxia, reflexes and muscle tone may remain normal. More generally, compressive spinal cord diseases cause a predictable sequence of dysfunction due to pressure effects on more heavily myelinated axons, beginning with ataxia and loss of conscious proprioception, followed by altered motor function (paresis), and finally loss of superficial and then deep pain sensations. When function is lost outside this sequence, for instance if paresis occurs prior to loss of conscious proprioception, noncompressive spinal cord or neuromuscular (primarily motor) diseases should be suspected.
Lesions in the cervical (C1-C5) spinal cord (and brain) cause UMN signs bilaterally in both the thoracic and pelvic limbs (i.e., tetraparesis, tetraplegia). Those at the cervicothoracic (C6-T2) intumescence cause LMN signs in the thoracic limbs and UMN signs in the pelvic limbs. Importantly, with lesions caudal to the T2 spinal cord segment, the thoracic limbs are normal (i.e., affected animals have paraparesis or paraplegia). Lesions affecting the T3-L3 spinal cord segments cause UMN signs in the pelvic limbs, whereas those at the lumbar intumescence (L4-S3 spinal cord segments) cause LMN signs in the pelvic limbs. Lesions in the T3-L3 areas can be further localized based on loss of the panniculus reflex caudally if superficial pain is lost or in the case of compressive or inflammatory lesions via evidence of hyperesthesia on deep palpation of the paraspinal muscles. While lesions affecting the spinal cord caudal to the L7 segment usually cause only minimal pelvic limb deficits (because only S1 contributes to the nerves of the pelvic limbs), affected individuals may have both fecal and urinary incontinence due to loss of somatic innervation to muscles of the urinary bladder and anal sphincters in tandem with loss of autonomic innervation to the wall musculature of the bladder and colon. Lesions of the caudal spinal cord segments cause only tail dysfunction.
Sites of spinal cord lesions can be further localized by identifying the distribution of reduced or absent pelvic limb reflexes and segmental sensory deficits. For example, L4-L6 damage affects the femoral nerve leading to altered patellar reflexes and sensation to medial digits. Damage at L6-S1 impacts the sciatic nerve yielding a decreased or absent flexion reflex and loss of sensation in the lateral digits. The distribution of neurogenic atrophy in regional muscles will follow this same pattern. Details regarding the conduct and interpretation of neurological tests for discriminating lesion localization may be gleaned from reference texts in veterinary neurology.37,82
Urinary incontinence commonly seen with spinal cord disease warrants brief discussion as a distinct neurological problem. Urinary bladder emptying is achieved through the long-routed (bladder to pons) micturition reflex whereby the smooth muscle of the bladder wall innervated by pelvic (autonomic) nerves is activated; therefore, bladder muscle function is not under voluntary control. In contrast, urinary continence (voluntary bladder control) is maintained by the external urethral sphincter, a skeletal muscle innervated by the pudendal nerves. The micturition reflex is usually lost at about the same time as voluntary motor function. Accordingly, ambulatory animals are typically continent.
Neurogenic incontinence occurs as two principal syndromes. Lesions between the pontine micturition center and the sacral spinal cord segments cause UMN urinary incontinence. Afferent sensory impulses conveying bladder distension do not reach the pontine micturition center or cerebrum. As a result, the micturition reflex and awareness of the need to urinate are lost. However, local reflex arcs mediated at the level of the pelvic nerves and sacral spinal cord may lead to at least partial albeit inappropriate emptying of the urinary bladder. Pudendal nerve innervation also remains intact, so external urethral sphincter tone is normal or even accentuated due to loss of UMN inhibition. For this reason, even though voluntary control of the external urethral sphincter is lost, the urinary bladder may be difficult to empty by manual compression.
Lesions involving the detrusor muscle (in the urinary bladder wall), pelvic nerves, or sacral spinal cord segments also interfere with the micturition reflex, but in this syndrome there is LMN dysfunction of the detrusor and external urethral sphincter. Because of these effects, the bladder may be markedly expanded but is usually easily expressed manually. This LMN involvement of the bladder sphincter can be inferred by the presence of a dilated anus, loss of the anal sphincter reflex, and reduced or absent perineal sensation.
Cervical syndrome
Cervical syndrome is associated with damage to segments C1-C5 (Figure 4 and Table 6). The main neurological signs are tetraparesis (tetraplegia) with normal to exaggerated/increased reflexes (hyperreflexia) and muscle tone (hypertonia) with only mild (disuse) atrophy due to disrupted connections with the UMNs in the cerebral cortex and brainstem (red nucleus); altered cervical mobility; and gait and postural abnormalities (spastic weakness or paralysis). Additional long tract signs (ataxia and loss of conscious proprioception) may occur due to involvement of ascending sensory tracts in the white matter of the dorsal (posterior) columns. Other signs may include altered respiration (if the damage is cranial to the origin of the phrenic nerve, which innervates the diaphragm) and urinary incontinence (due to loss of the long-routed micturition reflex).
Cervical syndrome.
Usual causes for cervical syndrome are nontoxic in nature and include developmental (vertebral and spinal cord) malformations; inflammation of the spinal cord (myelitis and/or meningitis); intervertebral disk disease; neoplasia; and trauma. Some toxic chemicals and plant toxins have been reported to induce spinal cord injury in this region.66,69,80,119,121
Cervicothoracic syndrome
Cervicothoracic syndrome reflects involvement of spinal cord segments C6-T2 (Figure 4 and Table 7), which form the cervical intumescence (i.e., a spinal cord enlargement resulting from increased gray matter mass needed to support the brachial plexus that gives rise to the thoracic limb nerves). The main neurological signs are tetraparesis (-plegia) with decreased postural reactions and LMN signs including depressed or absent reflexes (areflexia or hyporeflexia) and muscle tone (atonia or hypotonia). In contrast, the pelvic limbs exhibit normal or enhanced muscle tone and reflexes while maintaining normal or exhibiting only limited loss of muscle mass. Symmetrical spinal cord damage impacts limbs on both sides of the body axis while focal injury (commonly to the brachial plexus but sometimes the spinal cord) affects ipsilateral limbs only. Additional signs may include Horner’s syndrome (from damage to sympathetic neurons in the spinal cord, although involvement of the presynaptic sympathetic fibers in the sympathetic trunk within the brachial plexus or neck is a more common cause) and urinary incontinence.
Cervicothoracic syndrome.
Causes for this syndrome in animals are typically nontoxic entities that disrupt neurotransmission in the spinal cord, typically involving damage to the vertebrae or brachial plexuses. Inflammation (immune-mediated, pathogen-related [bacterial, protozoal (dog and horse), or viral], or occasionally idiopathic) and space-occupying masses are frequent diagnoses in the clinical setting. Trauma in this region also may produce this effect due to mechanical damage; causes include developmental malformations, herniated intervertebral disks, fractures, or intrathecal injections associated with the spinal cord, or spinal nerve root avulsions or traumatic injections affecting the brachial plexus. Some small molecule therapeutics may induce this syndrome following local 115 delivery (usually into the brachial plexus) or systemic 1 administration.
Central cord syndrome
Central cord syndrome results from lesions at the cervical or less often the cervicothoracic regions.110,111 In domestic dogs and cats, the thoracic limbs are affected more severely compared to the pelvic limbs due to the more medial location (i.e., closer to the central gray matter) of the corticospinal and spinothalamic tracts responsible for the neurological signs. This syndrome is seen with acute concussive (intervertebral disk herniation, trauma); neoplastic; or vascular (fibrocartilaginous emboli, ischemia) lesions. To our knowledge, the condition has not been described in other animal species nor attributed to toxic causes.
Thoracolumbar syndrome
Thoracolumbar syndrome results from damage to spinal cord region T3-L3 between the cervical intumescence cranially and the lumbar intumescence (i.e., the spinal cord enlargement that supplies the lumbosacral plexus) caudally (Figure 4 and Table 8). Neurological signs for this syndrome are limited to the pelvic limbs (parapareis, -plegia), with decreased postural reactions and normal to exaggerated reflexes and normal to increased muscle tone. The thoracic limbs are typically normal, although they may be hyperextended with concomitant paraplegia (i.e., a Schiff-Sherrington response) with some lesions. Urinary incontinence occurs due to loss of the micturition reflex, with an increase in bladder sphincter tone. With loss of superficial pain sensation, the panniculus reflex will be lost caudal to the lesion. Notably, deep pain is carried over polysynaptic, nonmyelinated fibers and is lost only with severe lesions, thus providing a key prognostic indicator.
Thoracolumbar syndrome.
Causes for this syndrome include developmental diseases such as hemivertebrae, local inflammation or neoplasia, intervertebral disk disease, non-compressive neurodegenerative diseases, and trauma. No toxicants consistently produce effects in this region.
Lumbosacral syndrome
Lumbosacral syndrome is associated with a range of clinical signs related to the spinal cord segments (typically L4-S3 depending on the species 19 ) forming the lumbar intumescence (Figure 4 and Table 9). Clinical signs involve the pelvic limbs, urinary bladder and anal sphincters, and tail while thoracic limb function is normal. Key characteristics are paraparesis (-plegia) with diminished or absent postural reactions and reduced to absent pelvic limb reflexes and muscle tone as well as associated (potentially pronounced) neurogenic muscle atrophy. Fecal and urinary incontinence due to paralysis of the anal and urinary bladder sphincters that are innervated by the pudendal nerve arising from the sacral spinal cord segments may occur. With severe lesions, the anal reflex and perianal sensation mediated by the pudendal nerve may be lost. Lesion localization in mature animals requires remembering that spinal cord segments in the intumescence lie cranial to their corresponding vertebrae.
Lumbosacral syndrome.
Common causes are nontoxic entities including degenerative conditions such as intervertebral disk disease, developmental malformations (spina bifida and other forms of spinal dysraphism), inflammation (often of the spinal nerve roots or vertebrae), space-occupying masses, and trauma. Iatrogenic trauma to the spinal cord and spinal nerve roots is a frequent finding induced by intrathecal catheterization of injection to deliver therapeutic molecules into the lumbar cistern.7,26,104,120
Caudal syndrome
Caudal syndrome (alternatively: coccygeal syndrome) generally is limited to animals with tails. Neurological signs are confined to pain on manipulating the tail with sensory and/or motor deficts affecting the tail, but with normal limb function.
Known causes of caudal syndrome are generally nontoxic entities, including developmental disorders (e.g., spina bifida with spinal cord dysraphism); inflammation (of nerve roots in the cauda equina or tail tissues); space-occupying masses (e.g., chordomas), and trauma (e.g., “amputation neuromas” [disorganized axonal regeneration], vertebral fractures or subluxations). Regional nerves may be injured by inadvertent delivery of certain therapeutic agents into perivascular soft tissues rather than into tail veins, but the damage generally reflects procedural trauma or the chemical properties of the material (e.g., acidic pH) rather than direct toxicity to neural tissues.
Lesion Localization for PNS-Centered Neurological Syndromes
Lesions in the PNS may be localized to a specific nerve if sensation is depressed in skin innervated by the nerve and LMN motor signs related to the nerve are present. Generalized neuromuscular diseases should be suspected if LMN signs are present in both the thoracic and pelvic limbs as well as in some cases where muscles of the face/head innervated by cranial nerves are affected. This group of diseases widely involves neuronal cell bodies (neuronopathies), peripheral nerves (neuropathies), neuromuscular junctions (junctionopathies), or skeletal muscles (myopathies). Speaking specifically to neuropathies, both sensory and motor fibers are typically affected in “mixed” neuropathies, although either motor or sensory fibers may be predominantly impacted. Signs of generalized LMN dysfunction include tetraparesis, hyporeflexia or areflexia, hypotonia, and neurogenic muscle atrophy that progresses over time.
Dysfunction of the PNS presents with various neurological signs depending on which neural region is affected (e.g., somatic versus autonomic) and which area is damaged (e.g., somatic ganglia and nerves versus neuromuscular junctions; autonomic parasympathetic versus sympathetic divisions). In-life signs in the PNS define two neurological syndromes, reflecting involvement of either somatic or autonomic ganglia and/or nerves.
Somatic Ganglia and Nerves
Neuropathic syndrome
Neuropathic syndrome results from damage to spinal (or less often cranial) ganglia and/or nerves, or the synapses at the neuromuscular junctions (Table 10). Key signs for the neural variant (resulting from ganglionic and/or nerve injury) include decreased or absent muscle tone or reflexes, weakness or LMN (flaccid) paralysis, and nerve-based (“neurogenic”) atrophy of the affected muscles if the insult is repeated or sustained. The mixture of motor and sensory signs varies depending on whether it is a “mixed” neuropathy (affecting both motor and sensory elements) or is localized to predominantly motor or sensory components. Autonomic signs (described below) and sensory deficits (e.g., cutaneous perception and/or proprioceptive deficits) may occur, but are variable and often limited when the somatic PNS is the main site of involvement. Neurological signs may be widespread or localized depending on the cause. Diffuse signs reflect substantial damage to multiple ganglia and/or nerves, produced by direct (e.g., intrathecal) or systemic exposure to high and/or sustained doses of adeno-associated virus (AAV) gene therapy vectors or cytotoxic antineoplastic agents. 5 Focal signs (localized to a particular body region served by one or a few closely associated ganglia and/or nerve trunks) result from space-occupying masses (usually inflammatory [abscess or granuloma] or neoplastic) or trauma (avulsion or transection).
Neuropathic syndrome.
Junctionopathies are characterized by exaggerated fatigability and episodic weakness (transient but intermittent after minimal exercise) affecting many body regions (selective cranial nerve and esophageal forms occur with myasthenia gravis) or the entire body. Despite the pronounced functional effects, morphologic evaluation generally shows no lesions of the neuromuscular junction (NMJ) synapses at the light microscopic level. Instead, NMJ lesions may sometimes be observed by transmission electron microscopy, including reductions in the synapse size65,105 or folding65,105 as well as decreased numbers of synaptic vesicles. 133 Junctionopathies do not exhibit lesions in skeletal myofibers; in contrast, primary myopathies do feature variable degeneration and/or necrosis of myofibers. Causes for junctionopathies include autoimmune disease associated with anti-NMJ antibodies (i.e., myasthenia gravis) and systemic exposure to some classes of therapeutic products like neuromuscular blocking agents and some toxins.
Autonomic Ganglia and Nerves
Autonomic syndrome
Autonomic syndrome (alternatively: autonomic neuropathy or dysautonomia) generally results from injury to various autonomic centers (in the hypothalamus); autonomic neurons in the lateral (intermediate) horn gray matter of the thoracic spinal cord; or autonomic ganglia and/or nerves (Table 11). These autonomic elements control cardiac and smooth muscle activities throughout the body. 37 Neurological signs for this system are related to lesions in any or multiple autonomic divisions: parasympathetic, sympathetic, and enteric (a gastrointestinal tract-centered network with parasympathetic-like functions 46 ). Neurological signs are associated with dysfunction of structures subject to involuntary neural control, including the cardiovascular, gastrointestinal, and respiratory systems. Hallmark signs may encompass abnormalities in gastrointestinal motility, cardiovascular rhythm (heart) and tone (blood vessels), excretion (fecal and urinary), exercise and/or heat intolerance, secretion (glands), and pupillary abnormalities (aberrant light response and Horner’s syndrome). The constellation of autonomic signs depends on whether the parasympathetic or sympathetic division is most impacted since the two systems have opposing physiological effects.
Autonomic syndrome.
Many causes have been proposed as explanations for autonomic syndrome, depending on the species. Common nontoxic causes include autoimmune diseases (including paraneoplastic syndromes) and putatively chronic hyperglycemia (through factors that include oxidative stress and advanced glycation end-product formation). 141 Toxicants implicated in dysautonomia include chronic alcoholism, heavy metals, and one or more agents from certain drug classes (e.g., cytotoxic chemotherapeutics). In some conditions, the neurotoxic product responsible for extensive autonomic neural damage has yet to be identified (e.g., equine dysautonomia [“grass sickness”]).
Skeletal Muscle
Myopathic syndrome
Myopathic syndrome is a “neurological” presentation reflecting primary damage to skeletal muscle rather than the somatic nerves that innervate them.37,82 The pattern of in-life clinical signs generally resembles those seen with LMN diseases. Common features include weakness (often generalized); abnormal movements (stiff gait that may become worse with exercise, as with polymyositis); altered muscle mass (increased [hypertrophy] or decreased [atrophy], depending on the cause); myalgia (pain or soreness) with myositis; and sometimes cramping. Pathologic findings are often bilateral but may be asymmetric and/or involve specific muscles or muscle groups. Neurologically, reflex circuits and sensory pathways are generally unaffected.
Myopathies arise from a plethora of causes. Common causes in animals (domestic and laboratory) and humans include acquired and inherited myopathies, inflammation (autoimmune [including drug-induced 71 ] or idiopathic), drug-related toxicity,31,71,75,79 or prolonged inactivity. Different toxicants produce various microscopic lesions in myofibers: atrophy (e.g., botulinum toxin, steroids);2,33,71,73,75 inflammation (e.g., certain immunosuppressants, thiols);31,33 motor endplate (NMJ) degeneration (e.g., tetanospasmin); 75 necrosis (e.g., cholinesterase inhibitors, emetine, lipid-lowering drugs [fibrates, statins], local anesthetics, monensin);31,71,75,79 and vacuolation (e.g., antimalarials, colchicine).31,71 Toxic agents may affect myofibers indiscriminately or impact a single fiber class (i.e., “fast twitch” or “slow twitch”). 75 Accordingly, potential test item-related effects on muscle should always be considered in animals with unexplained signs compatible with either neurological or muscular disease.
Localization for Neurological Syndromes With Multisite Lesions
Complex patterns of neurological signs may be too vague to assign to a single syndrome. This spectrum results from damage spanning multiple CNS regions, thereby resulting in the simultaneous presence of two (or several) syndromes. For example, the presence of seizures and paraplegia indicates damage in both the cerebrum (for seizures) and the spinal cord at some site caudal to the T2 spinal cord segment (for paraplegia).
In most cases, overlapping neurological syndromes can be associated with a single etiology. Causes include antineural autoimmune diseases, disseminated infection (most often bacterial or viral), degenerative disorders (storage diseases due to an inherited or toxicant-induced enzyme deficiency), certain metabolic disorders (though conditions such as hepatic encephalopathy and hypoglycemia typically cause forebrain syndrome), metastatic multifocal neoplasia, or trauma (e.g., injury to multiple sites [spinal fracture and subdural hematoma] or secondary phenomena such as disseminated intravascular coagulation or explosive intervertebral disk prolapse leading to widespread myelomalacia). Some multisite syndromes are idiopathic. Neurotoxic agents that cause complex neurological signatures include some plant toxins that induce neuronal storage diseases (Astragalus spp., Swainsona spp.) and agents that can interact with neurotransmitters in both the CNS and PNS (e.g., strychnine, tetanus toxin). 119 Administration of either 6-aminonicotinamide or selenium to young pigs induces both the cervicothoracic and lumbosacral syndromes as a consequence of bilaterally symmetrical cavitation in the ventral (anterior) gray columns of the spinal cord cervical and lumbar intumescences. 119
Practical Application of Neurological Syndromes to Optimize Neuropathology Evaluations in Animal Toxicity Testing
Regulatory guidance for nervous system sampling in animal toxicity testing notes that representative samples should be evaluated, with variable specificity regarding which CNS and PNS structures should be examined.13,14 In recent years, more specific recommendations to optimize neural sampling have been provided (by expert working groups of pathologists or individual authors) to enhance tissue sampling and processing for many scenarios. For example, detailed but flexible practices have been proposed for general toxicity studies16,19 and dedicated neurotoxicity studies.12,17,19,50,51 Supplemental papers have described sampling approaches for the nervous systems of common vertebrate test species—including rodents,106,107 rabbits, 100 dogs,49,96 minipigs, 99 and nonhuman primates10,98—as well as specific organs like the spinal cord11,19 and autonomic constituents.19,96 If implemented consistently for safety testing, this collective body of expert pathology and regulatory agency guidance will provide a suitable screen for large swaths of the CNS and PNS that have been demonstrated to be affected in various natural diseases of animals and/or serve as targets for neurotoxic agents. That said, routine screening often excludes multiple anatomic areas in which damage consistently induces neurological syndromes that are characterized by specific constellations of in-life neurological signs. Conventional practices in the toxicologic pathology setting will consistently screen the areas responsible for the brain-centered syndromes, with the possible exception of the vestibular syndrome. 16 In contrast, routine sampling in animal toxicity studies effectively screens for only some spinal cord syndromes and is “hit or miss” for the various PNS syndromes. 19
For brain, the usual sampling procedures collect multiple slices of cerebral cortex (prefrontal, parietal [motor and sensory], occipital, and temporal lobes); basal nuclei (caudate and putamen at minimum); subcortical centers (thalamus and hypothalamus); limbic centers (hippocampus and frequently amygdala); cerebellum (lateral hemispheres and vermis); and brainstem (midbrain, pons, and medulla oblongata). Taken together, these specimens reliably permit evaluation of brain regions involved in the cerebral, subcortical, cerebellar, and brainstem syndromes. Depending on the precise location of the brainstem slices, presence of the nuclei for CN VIII may allow analysis for the central variant of the vestibular syndrome. Conserved principles of correlative neuroanatomy may be used when warranted to guide expanded brain sampling and/or the choice of special neurohistological methods if a given pattern of neurological signs suggests that a brain region is affected but no lesions were observed during the microscopic evaluation of the routinely recommended brain sections. 12
For spinal cord, sampling is typically planned by rote according to recognized “best practice” recommendations, 19 supplemented when available by functional and anatomic data collected in prior studies and by the spectrum of in-life neurological signs observed during a given study. At minimum, spinal cord sampling in general toxicity studies is limited to the lumbar division (typically focused on segments L4-L5), thus permitting assessment of the area related to the lumbosacral syndrome. More extensive spinal cord sampling as undertaken by many companies also includes segments from the cervical and thoracic divisions. The location of the cervical segment differs based on institutional preference; a section from C1-C4 offers a suitable screen for the cervical syndrome, while a section from C6-T1 is a reasonable screen for the cervicothoracic syndrome; some institutions but not all collect and process both areas. For routine purposes, the thoracic spinal cord is collected from T6-T8, which is an acceptable survey for the thoracolumbar syndrome. Additional sampling at a specific spinal cord level may be necessary if the in-life neurological signs can localize the site of injury to a defined locus. In general, tail samples need not be collected from animals since findings at this site generally have no translational value in assessing the risk for human patients. That said, tail specimens may be collected from certain species (especially mice and rats, which lack hair on their tails) as caudal nerves may show microscopic lesions of length-dependent neurotoxicity equivalent to those observed in long nerves of the thoracic and pelvic limbs with degenerative (“dying back”) neuropathies. 114
For the PNS syndromes, conventional sampling may or may not adequately assess sites of likely neural damage. Routine sampling practices for general toxicity studies commonly are limited to one or a few lumbar DRG, which are often archived without analysis. 19 Additional ganglia may be collected and evaluated in some situations,12,19 but this expanded collection often includes only a few cervical and thoracic DRG and sometimes one or both trigeminal (CN V) ganglia.63,88,96 Until recently, autonomic ganglia have seldom been harvested systematically, although their locations have been defined in various animal species.19,88,96 For this reason, PNS sampling in general toxicity studies is unlikely to reliably detect neuropathic and autonomic syndromes, so situation-dependent increases to collect more somatic elements, autonomic components, or both will need to be implemented. 19
In like manner, standard approaches to skeletal muscle sampling in clinical practice and animal toxicity studies include only a few sites (classically the biceps femoris, gastrocnemius, vastus lateralis head of the quadriceps, and variably, the diaphragm and tongue). 38 This approach to muscle sampling may not provide an effective screen for discriminating neurogenic causes of muscle atrophy from myopathic syndromes (where myofibers are the primary site of disease). Here, clinical evidence of selective muscle atrophy together with electrophysiologic examination of various muscles can guide sampling. Consideration should also be given to selective fiber type involvement with some conditions,56,57 as with type 2 (fast twitch) fiber loss with endocrine disease and some inherited myopathies. As a rule, in a clinical setting in which polymyopathy is suspected, multiple muscles should be examined.
Real-time adjustments to tissue sampling are often problematic in conventional nonclinical studies where the desired endpoints are specified in advance as set forth in a study protocol and any subsequent amendments. The authors recommend three practices to permit the acquisition of other specimens necessary to investigate unusual constellations of in-life neurological signs and increase the likelihood of identifying lesions when clinical signs suggest a specific site of neural involvement. First, in general, the study protocol should be written so that sampling of additional neural sites (as noted in this section) is permitted at necropsy at the discretion of the pathologist without the need for a same-day protocol amendment. Second, odd collections of in-life clinical signs that suggest CNS or PNS involvement (especially if not attributable to damage to neural regions that are sampled well under current industry best practices) should be evaluated by an experienced neurologist and/or neuropathologist so that further study-specific neural tissue sampling can be designed in advance of the intended necropsy date. In the authors’ experience, the second option offers the most value when routine neuropathology endpoints are used in investigating bizarre patterns of neurological signs. However, implementing the second option effectively necessitates early and vigorous communication among many individuals—the study director, study pathologist, necropsy technicians, and sponsor representative for the core team, often supplemented by subject matter experts in neurology and neuropathology—to achieve success. Finally, in some instances the neurological examination will suggest the existence of a focal lesion or strict involvement of one neural region, in which case it is wise to take additional samples from the involved area beyond those routinely assessed based on current industry best practices. Examples would be an animal with UMN paraplegia of the pelvic limbs (suggesting T3-L3 involvement) with no gross lesion, where spinal cord sampling should be extended beyond the baseline T6-T8 segments examined in nonclinical toxicity studies, 16 or a study in which signs indicate that disease is confined to the cerebellum, in which case multiple blocks (for midline and lateral as well as rostral and caudal regions) would be collected to fully assess this part of the brain. In all these situations, neuropathologists with solid understanding of the anatomic-to-functional correlations in the nervous system, aided by veterinary neurologists when feasible, will be essential contributors to the success of nonclinical neurotoxicity screening for novel biomedical products.
Conclusions
In general, detailed practices for CNS and PNS sampling, processing, and evaluation17,16,19,51 devised by expert working groups to implement generic regulatory guidance provide an efficient and effective neurotoxicity screen in many animal toxicity studies. However, routine protocols for neural tissue collection will not provide specimens from all areas with known structure-function correlations.
The “neurological syndrome” concept provides a useful tool to optimize the likelihood that functional abnormalities detected during life can be explained by identifiable morphological lesions. For this purpose, pathologists with a thorough understanding of structure-function correlations in major CNS and PNS structures will find themselves in an excellent position to expand neural sampling (when warranted) to accomplish this purpose. Importantly, we must remember that a given neurological syndrome may be present even if the pattern of in-life signs does not include the entire spectrum of functional alterations. Therefore, it is generally good to focus on what appears to be the most definitive neurologic sign, as with seizures with forebrain syndrome.
The ability to identify microscopic lesions is a critical aspect of successful neuropathology evaluations during animal toxicity testing. In this regard, neurological syndromes offer a scaffold by which suitably trained and experienced practitioners—neurologists, pathologists, and/or toxicologists, depending on the institutional preference—can integrate functional and structural data when preparing study interpretations. The authors hope that the current review provides a solid foundation to improve study interpretations related to neurological dysfunction observed during animal toxicity studies and thus to rationally support science-based risk assessment decisions.
Footnotes
Acknowledgements
The authors gratefully thank Mr. Tim Vojt (Columbus, Ohio) for crafting the figures.
Author Contributions
The authors are solely responsible for the contents and crafting of this paper. Dr. Bolon is an American College of Veterinary Pathologists (ACVP) board-certified veterinary anatomic pathologist. Dr. Kornegay is an American College of Veterinary Internal Medicine (ACVIM) board-certified veterinary neurologist. Dr. Kornegay also has formal residency training in veterinary pathology and is an honorary diplomate of the ACVP.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
