Pathologic characterization of canine multiple system degeneration in the Ibizan hound.

Canine multiple system degeneration (CMSD) is a progressive hereditary neurodegenerative disorder commonly characterized by neuronal degeneration and loss in the cerebellum, olivary nuclei, substantia nigra, and caudate nuclei. In this article, we describe 3 cases of CMSD in Ibizan hounds. All patients exhibited marked cerebellar ataxia and had cerebellar atrophy on magnetic resonance imaging. At necropsy, all cases showed varying degrees of cerebellar atrophy, and 2 cases had gross cavitation of the caudate nuclei. Histologic findings included severe degeneration and loss of all layers of the cerebellum and neuronal loss and degeneration within the olivary nuclei, substantia nigra, and caudate nuclei. Pedigree analysis indicated an autosomal recessive mode of inheritance, but the causative gene in this breed is yet to be identified. CMSD resembles human multiple system atrophy and warrants further investigation.

Comparative Pathology of the Peripheral Nervous System.

The peripheral nervous system (PNS) relays messages between the central nervous system (brain and spinal cord) and the body. Despite this critical role and widespread distribution, the PNS is often overlooked when investigating disease in diagnostic and experimental pathology. This review highlights key features of neuroanatomy and physiology of the somatic and autonomic PNS, and appropriate PNS sampling and processing techniques. The review considers major classes of PNS lesions including neuronopathy, axonopathy, and myelinopathy, and major categories of PNS disease including toxic, metabolic, and paraneoplastic neuropathies; infectious and inflammatory diseases; and neoplasms. This review describes a broad range of common PNS lesions and their diagnostic criteria and provides many useful references for pathologists who perform PNS evaluations as a regular or occasional task in their comparative pathology practice.

Common Structural Lesions of the Peripheral Nervous System.

This review illustrates common lesions of peripheral nerve myelinated fibers that occur in toxic neuropathy. These distinctive structural changes help to define the site of toxicant activity and thus predict the course of neurotoxic disease and recovery. Neuronopathy is the condition where the primary injury is directed to the neuronal cell body giving rise to a peripheral nerve axon. Axonopathy occurs when the axon is the primary target, and myelinopathy develops where the Schwann cell and/or myelin sheath is the primary target; these conditions can be discriminated early during the course of nerve fiber degeneration, but reciprocal influences between axon and myelin result in degeneration of both structures late in the disease.

Nerve Fiber Regeneration in Toxic Peripheral Neuropathy.

There is a striking difference in the potential for regeneration of injured axons in the central and peripheral nervous systems, which is important in neurotoxicologic studies. In contrast to the former, there is a ready mechanism for replacement of peripheral nerve axons that have degenerated following exposure to toxins, where long-distance axon regeneration and substantial functional recovery can occur. This relates at least in part to the nature of the glial and other supporting cells of the peripheral nerve. To provide background for these events, data on regeneration following traumatic injury to peripheral nerve are reviewed. This is followed by descriptions of nerve fiber regeneration after experimental exposure to 3 peripheral nerve axonopathic toxins, organophosphate tri-ortho-tolyl phosphate, the industrial chemical carbon disulfide, and the antituberculosis drug isoniazid.

Toxicologic Pathology of the Peripheral Nervous System (PNS): Overview, Challenges, and Current Practices.

Peripheral nervous system (PNS) toxicity is a frequent adverse effect encountered in patients treated with certain therapeutics (e.g., antiretroviral drugs, cancer chemotherapeutics), in occupational workers exposed to industrial chemicals (e.g., solvents), or during accidental exposures to household chemicals and/or environmental agents (e.g., pesticides). However, the literature and expertise needed for the effective design, conduct, analysis, and reporting of safety studies to identify and define PNS toxicity are hard to find. This half-day course familiarized participants with basic PNS biology; causes and mechanisms of PNS pathology; classic methods and current best practice recommendations for PNS sampling, preparation, and evaluation; and examples of commonly observed lesions and artifacts. Three concluding case presentations synthesized information from the prior technical lectures by presenting real-world examples of lesions caused by drugs and chemicals to demonstrate how PNS toxicity may be addressed in evaluating product safety during nonclinical studies. Topics emphasized comparative and correlative data among animal species used in toxicity studies and clinical evaluation in humans in order to facilitate the translation of animal data into human risk assessment with respect to PNS toxicologic pathology.

STP Position Paper: Recommended Best Practices for Sampling, Processing, and Analysis of the Peripheral Nervous System (Nerves and Somatic and Autonomic Ganglia) during Nonclinical Toxicity Studies.

Peripheral nervous system (PNS) toxicity is surveyed inconsistently in nonclinical general toxicity studies. These Society of Toxicologic Pathology "best practice" recommendations are designed to ensure consistent, efficient, and effective sampling, processing, and evaluation of PNS tissues for four different situations encountered during nonclinical general toxicity (screening) and dedicated neurotoxicity studies. For toxicity studies where neurotoxicity is unknown or not anticipated (situation 1), PNS evaluation may be limited to one sensorimotor spinal nerve. If somatic PNS neurotoxicity is suspected (situation 2), analysis minimally should include three spinal nerves, multiple dorsal root ganglia, and a trigeminal ganglion. If autonomic PNS neuropathy is suspected (situation 3), parasympathetic and sympathetic ganglia should be assessed. For dedicated neurotoxicity studies where a neurotoxic effect is expected (situation 4), PNS sampling follows the strategy for situations 2 and/or 3, as dictated by functional or other compound/target-specific data. For all situations, bilateral sampling with unilateral processing is acceptable. For situations 1-3, PNS is processed conventionally (immersion in buffered formalin, paraffin embedding, and hematoxylin and eosin staining). For situation 4 (and situations 2 and 3 if resources and timing permit), perfusion fixation with methanol-free fixative is recommended. Where PNS neurotoxicity is suspected or likely, at least one (situations 2 and 3) or two (situation 4) nerve cross sections should be postfixed with glutaraldehyde and osmium before hard plastic resin embedding; soft plastic embedding is not a suitable substitute for hard plastic. Special methods may be used if warranted to further characterize PNS findings. Initial PNS analysis should be informed, not masked ("blinded"). Institutions may adapt these recommendations to fit their specific programmatic requirements but may need to explain in project documentation the rationale for their chosen PNS sampling, processing, and evaluation strategy.

Exploratory Studies With BT-11: A Proposed Orally Active Therapeutic for Crohn's Disease.

Lanthionine synthetase cyclase-like receptor 2 (LANCL2) is a novel therapeutic target for Crohn's disease (CD). BT-11 is a small molecule that binds LANCL2, is orally active, and has demonstrated therapeutic efficacy in 3 validated mouse models of colitis at doses as low as 8 mg/kg/d. Exploratory experiments evaluated BT-11 in male Harlan Sprague Dawley rats with a single oral dose of 500 mg/kg and 80 mg/kg/d for 14 days (n = 10 rats dosed/group). Treated and control rats were observed for behavioral detriments, and blood and tissues were collected for clinical pathology and histopathological examination. A functional observational battery demonstrated no differences between treated and control groups over multiple times of observation for quantal, categorical, and continuous end points, including posture, in cage activity, approach, response to touch, weight, grip strength, body temperature, and time on a rotarod. Histopathological examination of the brain, kidney, liver, adrenal gland, testes, stomach, small and large intestines, duodenum, pancreas, heart, lungs, spleen, thymus, and rib found no significant differences between the groups. Plasma enzymes associated with liver function were transiently elevated 2 to 4 days after the 500 mg/kg single dose but returned to normal values by 8 days and were not observed at any time in rats given 80 mg/kg/d for 14 days. One hour after oral administration of a single dose of 80 mg/kg, BT-11 had a maximal concentration of 21 ng/mL; the half-life was 3 hours. These experimental results demonstrated that BT-11 is well tolerated in rats, and, with further testing, may hold promise as an orally active therapeutic for CD.

Preparation and analysis of the peripheral nervous system.

This article is from a presentation at the 2010 STP/IFSTP Symposium on Neuropathology. The organization and basic structure of the peripheral nervous system is reviewed. Examples of toxicant-induced peripheral nerve injury such as neuronopathy, axonopathy, and myelinapathy are discussed, as are contemporary methods for examination of these tissues.

Modern pathology methods for neural investigations.

This session at the 2010 joint symposium of the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP) explored modern neuropathology methods for assessing the neurotoxicologic potential of xenobiotics. Conventional techniques to optimally prepare and evaluate the central and peripheral neural tissues while minimizing artifact were reviewed, and optimal schemes were set forth for evaluation of the nervous system during both routine (i.e., general toxicity) studies and enhanced (i.e., specialized neurotoxicity) studies. Stereology was introduced as the most appropriate means of examining the possible impact of toxicants on neural cell numbers. A focused discussion on brain sampling took place among a panel of expert neuroscientists (anatomists and pathologists) and the audience regarding the proper balance between sufficient sampling and cost- and time-effectiveness of the analysis. No consensus was reached on section orientation (coronal sections of both sides vs. a parasagittal longitudinal section with several unilateral hemisections from the contralateral side), but most panelists favored sampling at least 8 sections (or approximately double to triple the current complement) in routine toxicity studies.

Effect of stress at dosing on organophosphate and heavy metal toxicity.

This paper reviews recent studies assessing the effect of well-defined, severe, transient stress at dosing on two classical models of toxicity. These are the acute (anticholinesterase) toxicity seen following exposure to the organophosphate insecticide chlorpyrifos, and the nephrotoxicity elicited by the heavy metal depleted uranium, in rats. Stress was induced by periods of restraint and forced swimming in days to weeks preceding toxicant exposure. Forced swimming was far more stressful, as measured by marked, if transient, elevation of plasma corticosterone. This form of stress was administered immediately prior to administration of chlorpyrifos or depleted uranium. Chlorpyrifos (single 60 mg/kg subcutaneously) elicited marked inhibition of brain acetylcholinesterase 4-day post-dosing. Depleted uranium (single intramuscular doses of 0.1, 0.3 or 1.0 mg/kg uranium) elicited dose-dependent increase in kidney concentration of the metal, with associated injury to proximal tubular epithelium and increases in serum blood urea nitrogen and creatinine during the 30-day post-dosing period. Stress at dosing had no effect on these toxicologic endpoints.

The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation.

A common histological artifact of brain is the presence of contracted, intensely stained neurons. Such cells are termed dark neurons, and can be produced by post-mortem manipulation or trauma in brain tissue. In a number of recently published experimental neurotoxicology reports cells of this nature are interpreted as representing dying or degenerating neurons, thus assigning inappropriate neurotoxic potential to the compounds administered. The latter are the commonly used drugs and pesticides DEET (N,N-diethyl m-toluamide), malathion, permethrin and pyridostigmine bromide. In this paper I review the nature and genesis of the dark neuron, critically discuss the neuropathologic validity of indicated studies and make a plea for increased awareness of this common artifact.

Optic nerve astrocytoma in a dog.

Intracranial astrocytomas are relatively uncommon in dogs and optic nerve astrocytomas even more so. This neoplasm should be considered as differential in canine patients with vision loss, retinal detachment, ocular mass, and histopathologic findings of infiltrative fusiform to polygonal glial cells possibly associated with glomeruloid vascular proliferation.

Gamma-Diketone central neuropathy: quantitative analyses of cytoskeletal components in myelinated axons of the rat rubrospinal tract.

Loss of axon caliber is a primary component of gamma-diketone neuropathy [LoPachin RM, DeCaprio AP. gamma-Diketone central neuropathy: axon atrophy and the role of cytoskeletal protein adduction. Toxicol Appl Pharmacol 2004;199:20-34]. It is possible that this effect is mediated by changes in the density of cytoskeletal components and corresponding spatial relationships. To examine this possibility, morphometric methods were used to quantify the effects of 2,5-hexanedione (HD) intoxication on neurofilament-microtubule densities and nearest neighbor distances in myelinated rubrospinal axons. Rats were exposed to HD at one of two daily dose-rates (175 or 400 mg/kg per day, gavage) until a moderate level of neurotoxicity was achieved (99 or 21 days of intoxication, respectively) as determined by gait analysis and measurements of hindlimb grip strength. Results indicate that, regardless of dose-rate, HD intoxication did not cause changes in axonal neurofilament (NF) density, but did significantly increase microtubule (MT) density. No consistent alterations in interneurofilament or NF-MT distances were detected by ultrastructural morphometric analyses. These data suggest that the axon atrophy induced by HD was not mediated by major disruptions of stationary cytoskeletal organization. Recent biochemical studies of spinal cord from HD intoxicated rats showed that, although the NF protein content in the stationary cytoskeleton (polymer fraction) was not affected, the mobile subunit pool was depleted substantially [LoPachin RM, He D, Reid ML, Opanashuk LA. 2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol 2004;198:61-73]. The stability of the polymer fraction during HD intoxication is consistent with the absence of significant ultrastructural modifications noted in the present study. Together, these findings implicate loss of mobile NF proteins as the primary mechanism of axon atrophy.

Chlorpyrifos alters functional integrity and structure of an in vitro BBB model: co-cultures of bovine endothelial cells and neonatal rat astrocytes.

The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.

The effect of stress on the temporal and regional distribution of uranium in rat brain after acute uranyl acetate exposure.

Long-term exposure to depleted uranium (DU) has been shown to increase brain uranium and alter hippocampal function; however, little is known about the short-term kinetics of DU in the brain. To address this issue, temporal and regional distribution of brain uranium was investigated in male Sprague-Dawley rats treated with a single intraperitoneal injection of 1 mg uranium/kg as uranyl acetate. Due to the inherent stress of combat and the potential for stress to alter blood-brain barrier permeability, the impact of forced swim stress on brain uranium distribution was also examined in this model. Uranium in serum, hippocampus, striatum, cerebellum, and frontal cortex was quantified by inductively coupled plasma-mass spectrometry (ICP-MS) at 8 h, 24 h, 7 d, and 30 d after exposure. Uranium entered the brain rapidly and was initially concentrated in hippocampus and striatum. While multiple phases of uranium clearance were observed, overall clearance was relatively slow and the uranium content of hippocampus, cerebellum, and cortex remained elevated for more than 7 d after a single exposure. Prior exposure to stress significantly reduced hippocampal and cerebellar uranium 24 h post-exposure and tended to reduce uranium in all brain regions 7 d after exposure. The application of stress appeared to increase brain uranium clearance, as initial tissue levels were similar in stressed and unstressed rats.

Chronic inflammatory demyelinating polyradiculoneuropathy with cholesterol deposits in a dog.

Chronic inflammatory demyelinating polyradiculoneuropathy occurred in an 11-year-old Labrador Retriever dog. Spinal cord compression resulted from massive radiculitis with prominent cholesterol granulomas. Cholesterol deposition and associated granuloma formation is unique in chronic inflammatory demyelinating polyradiculoneuropathy, in both its human and canine expressions.

Paradoxical effects of all-trans-retinoic acid on lupus-like disease in the MRL/lpr mouse model.

Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.

Morphological effects of neuropathy-inducing organophosphorus compounds in primary dorsal root ganglia cell cultures.

Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds. This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then exposed to 1 microM of the OP compounds phenyl saligenin phosphate (PSP) or mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle. The medium containing these toxicants was removed after 12 h, and cultures maintained for 4-7 days post-exposure. Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure. Ultrastructural alterations of neurons treated with PSP and mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria. Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes. This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds.

Animal models of peripheral neuropathy due to environmental toxicants.

Despite the progress in our understanding of pathogeneses and the identification of etiologies of peripheral neuropathy, idiopathic neuropathy remains common. Typically, attention to peripheral neuropathies resulting from exposure to environmental agents is limited relative to more commonly diagnosed causes of peripheral neuropathy (diabetes and chemotherapeutic agents). Given that there are more than 80,000 chemicals in commerce registered with the Environmental Protection Agency and that at least 1000 chemicals are known to have neurotoxic potential, very few chemicals have been established to affect the peripheral nervous system (mainly after occupational exposures). A wide spectrum of exposures, including pesticides, metals, solvents, nutritional sources, and pharmaceutical agents, has been related, both historically and recently, to environmental toxicant-induced peripheral neuropathy. A review of the literature shows that the toxicity and pathogeneses of chemicals adversely affecting the peripheral nervous system have been studied using animal models. This article includes an overview of five prototypical environmental agents known to cause peripheral neuropathy--namely, organophosphates, carbon disulfide, pyridoxine (Vitamin B6), acrylamide, and hexacarbons (mainly n-hexane, 2,5-hexanedione, methyl n-butyl ketone). Also included is a brief introduction to the structural components of the peripheral nervous system and pointers on common methodologies for histopathologic evaluation of the peripheral nerves.

A neonatal gnotobiotic pig model of human enterovirus 71 infection and associated immune responses.

Vaccine development and pathogenesis studies for human enterovirus 71 are limited by a lack of suitable animal models. Here, we report the development of a novel neonatal gnotobiotic pig model using the non-pig-adapted neurovirulent human enterovirus 71 strain BJ110, which has a C4 genotype. Porcine small intestinal epithelial cells, peripheral blood mononuclear cells and neural cells were infected in vitro. Oral and combined oral-nasal infection of 5-day-old neonatal gnotobiotic pigs with 5×10(8) fluorescence forming units (FFU) resulted in shedding up to 18 days post-infection, with viral titers in rectal swab samples peaking at 2.22×10(8) viral RNA copies/mL. Viral capsid proteins were detected in enterocytes within the small intestines on post-infection days (PIDs) 7 and 14. Additionally, viral RNA was detected in intestinal and extra-intestinal tissues, including the central nervous system, the lung and cardiac muscle. The infected neonatal gnotobiotic pigs developed fever, forelimb weakness, rapid breathing and some hand, foot and mouth disease symptoms. Flow cytometry analysis revealed increased frequencies of both CD4(+) and CD8(+) IFN-γ-producing T cells in the brain and the blood on PID 14, but reduced frequencies were observed in the lung. Furthermore, high titers of serum virus-neutralizing antibodies were generated in both orally and combined oral-nasally infected pigs on PIDs 7, 14, 21 and 28. Together, these results demonstrate that neonatal gnotobiotic pigs represent a novel animal model for evaluating vaccines for human enterovirus 71 and for understanding the pathogenesis of this virus and the associated immune responses.