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Veterinary pathology credentials serve as a concise means attesting to educational attainments and experiences indicating a readiness for professional practice. Given the cost, time, and stress associated with obtaining different qualifications, pathologists must consider what credentials enhance their readiness. In this commentary, the authors describe how their various degrees and certifications have facilitated their individual and organizational success. The minimum credentials for proficient veterinary pathology practice are a veterinary medical degree (DVM or equivalent) and advanced pathology training (residency and/or on-the-job "apprenticeship") ideally culminating in board certification in pathology (American College of Veterinary Pathologists [ACVP] diplomate status or equivalent). Graduate degrees (MS, PhD, MPH, etc) and/or other qualifications in allied biomedical fields (eg, board certification in internal medicine, laboratory animal medicine, poultry medicine, preventive medicine, or toxicology) may improve employability by affirming specialty knowledge in another complementary discipline. The authors note that pathology positions may be obtained without a long list of degrees or certifications, and that more credentials may provide occupational flexibility for some employers. However, a good work ethic, experience in the field, ability to adapt to changes, job satisfaction, good attitude, and demonstrated productivity are also important, and indeed, they are often the paramount criteria for career success as a veterinary pathologist., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Dorsal root ganglia (DRG), trigeminal ganglia (TG), other sensory ganglia, and autonomic ganglia may be injured by some test article classes, including anti-neoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, nerve growth factor inhibitors, and aminoglycoside antibiotics. This article reviews ganglion anatomy, cytology, and pathology (emphasizing sensory ganglia) among common nonclinical species used in assessing product safety for such test articles (TAs). Principal histopathologic findings associated with sensory ganglion injury include neuron degeneration, necrosis, and/or loss; increased satellite glial cell and/or Schwann cell numbers; and leukocyte infiltration and/or inflammation. Secondary nerve fiber degeneration and/or glial reactions may occur in nerves, dorsal spinal nerve roots, spinal cord (dorsal and occasionally lateral funiculi), and sometimes the brainstem. Ganglion findings related to TA administration may result from TA exposure and/or trauma related to direct TA delivery into the central nervous system or ganglia. In some cases, TA-related effects may need to be differentiated from a spectrum of artifactual and/or spontaneous background changes., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors express no conflict of interest in producing this technical review except that all are employed by organizations involved in the discovery and development of various biomedical products for treating human diseases (including AAV-based gene therapies, ASOs, other neuroactive biologics, and small molecules).

Certain biopharmaceutical products consistently affect dorsal root ganglia, trigeminal ganglia, and/or autonomic ganglia. Product classes targeting ganglia include antineoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, and anti-nerve growth factor agents. This article outlines "points to consider" for sample collection, processing, evaluation, interpretation, and reporting of ganglion findings; these points are consistent with published best practices for peripheral nervous system evaluation in nonclinical toxicity studies. Ganglion findings often occur as a combination of neuronal injury (e.g., degeneration, necrosis, and/or loss) and/or glial effects (e.g., increased satellite glial cell cellularity) with leukocyte accumulation (e.g., mononuclear cell infiltration or inflammation). Nerve fiber degeneration and/or glial reactions may be seen in nerves, dorsal spinal nerve roots, spinal cord, and occasionally brainstem. Interpretation of test article (TA)-associated effects may be confounded by incidental background changes or experimental procedure-related changes and limited historical control data. Reports should describe findings at these sites, any TA relationship, and the criteria used for assigning severity grades. Contextualizing adversity of ganglia findings can require a weight-of-evidence approach because morphologic changes of variable severity occur in ganglia but often are not accompanied by observable overt in-life functional alterations detectable by conventional behavioral and neurological testing techniques.

Beagle dogs are a key nonrodent species in nonclinical safety evaluation of new biomedical products. The Society of Toxicologic Pathology (STP) has published "best practices" recommendations for nervous system sampling in nonrodents during general toxicity studies ( Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the Beagle dog has not been defined specifically. Here we provide 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the dog brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen test articles with unknown or no anticipated neurotoxic potential; this plan using at least 7 coronal hemisections matches the STP "best practices" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses up to 14 coronal levels to investigate test articles where the brain is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves for dogs during nonclinical studies should follow published STP "best practices" recommendations for sampling the central ( Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral ( Toxicol Pathol 46[4]: 372-402, 2018) nervous systems. This technical guide also demonstrates the locations and approaches to collecting uncommonly sampled peripheral nervous system sites.

Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as day 8 in rats (≥5 × 10 13 vg/kg) and day 14 in monkeys (≥3.3 × 10 13 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species., Competing Interests: K.A.F., R.C.K., C.J.S., H.H.W., V.S.V., S.K.R., and M.P.S. are employees of Pfizer Inc.; I.D.P. is now an employee of Biogen. All authors are/were employees of Pfizer Inc. at the time of their contribution to the manuscript., (© 2022 Pfizer Worldwide Research, Development and Medical.)

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 ( SMN1 ) transgene (AAV-PHP.B-CBh- SMN1 ) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 10 13 , 5 × 10 13 , or 1 × 10 14 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 10 13 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Background: CBA/J mice are standard experimental animals in auditory studies, and age-related changes in auditory pathways are well documented. However, changes in locomotion-related brain regions have not been systematically explored., Results: We showed an increase in immunoreactivity for glial fibrillary acidic protein (GFAP) in the cerebellar molecular layer associated with Purkinje cells in mice at 24 weeks of age but not in the younger mice. Increased GFAP immunoreactivity appeared in the form of clusters and distributed multifocally consistent with hyperplasia of astrocytes that were occasionally associated with Purkinje cell degeneration. Three out of 12 animals at 16 and 24 weeks of age exhibited pre-convulsive clinical signs. Two of these 3 animals also showed increased GFAP immunoreactivity in the cerebellum. Rotarod behavioral assessments indicated decreased performance at 24 weeks of age., Conclusions: These results suggest minimal to mild reactive astrocytosis likely associated with Purkinje cell degeneration in the cerebellum at 24 weeks of age in CBA/J mice. These findings should be taken into consideration prior to using this mouse strain for studying neuroinflammation or aging., (© 2021. The Author(s).)

The use of minipigs as an alternative nonclinical species has increased in the last 20 years. The Society of Toxicologic Pathology (STP) has produced generic "best practice" recommendations for nervous system sampling in nonrodents during general toxicity studies ( Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the minipig has not been attempted. Here, we describe 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the minipig brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen agents with unknown or no anticipated neurotoxic potential; this approach using 7 coronal hemisections accords with the published STP "best practice" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses 14 coronal hemisections and 2 full coronal sections to investigate toxicants where the nervous system is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves from minipigs during nonclinical studies should follow published STP "best practice" recommendations for sampling the central (CNS, Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (PNS, Toxicol Pathol 46[4]: 372-402, 2018) nervous systems.

Visual system toxicity may manifest anywhere in the visual system, from the eye proper to the visual brain. Therefore, effective screening for visual system toxicity must evaluate not only ocular structures (ie, eye and optic nerve) but also multiple key brain regions involved in vision (eg, optic tract, subcortical relay nuclei, and primary and secondary visual cortices). Despite a generally comparable pattern across species, the neuroanatomic organization and function of the visual brain in rodents and rabbits exhibit appreciable differences relative to nonrodents. Currently recognized sampling practices for general toxicity studies in animals, which are based on easily discerned external neuroanatomic landmarks and guided by extant stereotaxic brain atlases, typically will permit histopathologic evaluation of many brain centers involved in visual sensation (eg, optic chiasm, optic tract, dorsal lateral geniculate nucleus, primary and secondary visual cortices) and often some subcortical brain nuclei involved in light-modulated nonvisual activities needed for visual attention and orientation (eg, rostral colliculus in quadrupeds, termed the superior colliculus in bipeds; several cranial nerve nuclei). Pathologic findings induced by toxicants in the visual brain centers are similar to those that are produced in other brain regions.

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.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Although manuscripts for multiple species recommending nervous system sampling for histopathology evaluation in safety assessment have been published in the past 15 years, none have addressed the laboratory rabbit. Here, we describe 2 trimming schemes for evaluating the rabbit brain in nonclinical toxicity studies. In both schemes, the intact brain is cut in the coronal plane to permit bilateral assessment. The first scheme is recommended for general toxicity studies (tier 1) in screening agents where there is no anticipated neurotoxic potential; this 6-section approach is consistent with the Society of Toxicologic Pathology (STP) "best practice" recommendations for brain sampling in nonrodents ( Toxicol Pathol 41: 1028-1048, 2013 1 ). The second trimming scheme is intended for dedicated neurotoxicity studies (tier 2) to characterize known or suspected neurotoxicants where the nervous system is a key target organ. This tier 2 strategy relies on coronal trimming of the whole brain into 3-mm-thick slices and then evaluating 12 sections. Collection of spinal cord, ganglia, and nerve specimens for rabbits during nonclinical studies should follow published STP "best practice" recommendations for sampling the central nervous system 1 and peripheral nervous system ( Toxicol Pathol 46: 372-402, 2018 2 ).

Axonal dystrophy (AD) is a common age-related neurohistological finding in vertebrates that can be congenital or induced by xenobiotics, vitamin E deficiency, or trauma/compression. To understand the incidence and location of AD as a background finding in Beagle dogs used in routine toxicity studies, we examined central nervous system (CNS) and selected peripheral nervous system (PNS) tissues in twenty 18- to 24-month-old and ten 4- to 5-year-old control males and females. Both sexes were equally affected. The cuneate, gracile, and cochlear nuclei and the cerebellar white matter (rostral vermis) were the most common locations for AD. Incidence of AD increased with age in the cuneate nucleus, cerebellar white matter (rostral vermis), trigeminal nuclei/tracts, and lumbar spinal cord. Axonal dystrophy in the CNS was not accompanied by neuronal degeneration/necrosis, nerve fiber degeneration, and/or glial reaction. Axonal dystrophy was not observed in the PNS (sciatic nerve, vagus nerve branches, or gastrointestinal mural autonomic plexuses).

The ability to differentiate among normal structures, procedural and processing artifacts, spontaneous background changes, and test article-related effects in the peripheral nervous system (PNS) is essential for interpreting microscopic features of ganglia and nerves evaluated in animal species commonly used in toxicity studies evaluating regulated products and chemicals. This atlas provides images of findings that may be encountered in ganglia and nerves of animal species commonly used in product discovery and development. Most atlas images are of tissues from control animals that were processed using routine methods (ie, immersion fixation in neutral-buffered 10% formalin, embedding in paraffin, sectioning at 5 µm, and staining with hematoxylin and eosin) since these preparations are traditionally applied to study materials produced during most animal toxicity studies. A few images are of tissues processed using special procedures (ie, immersion or perfusion fixation using methanol-free 4% formaldehyde, postfixation in glutaraldehyde and osmium, embedding in hard plastic resin, sectioning at 1 µm, and staining with toluidine blue), since these preparations promote better stabilization of lipids and thus optimal resolution of myelin sheaths. Together, this compilation provides a useful resource for discriminating among normal structures, procedure- and processing-related artifacts, incidental background changes, and treatment-induced findings that may be seen in PNS tissues of laboratory animals.

Toxicologic neuropathology for the peripheral nervous system (PNS) is a vital but often underappreciated element of basic translational research and safety assessment. Evaluation of the PNS may be complicated by unfamiliarity with normal nerve and ganglion biology, which differs to some degree among species; the presence of confounding artifacts related to suboptimal sampling and processing; and limited experience with differentiating such artifacts from genuine disease manifestations and incidental background changes. This compilation of key PNS neurobiology, neuropathology, and neurotoxicology references is designed to allow pathologists and toxicologists to readily access essential information that is needed to enhance their proficiency in evaluating and interpreting toxic changes in PNS tissues from many species.

Two beagle dog strains were used in a 14-day intrathecal infusion study for a small molecule test article. A moderate number of Renaut bodies (RBs) were observed in the sciatic nerves of control and test article-treated adult animals as early as 1 day after test article infusion (ie, 5 days after catheter implantation in the lumbar cistern). In most cases, the sciatic nerve was affected unilaterally, apparently in association with extended lateral recumbency on one side. The lighter beagle strain (Marshall), and especially the females (which weighed less than age-matched Marshall males), developed more RBs. In contrast, neither females nor males of the larger strain (Harlan) developed any nerve lesions. These data support the hypothesis that RBs develop following mechanical stress to sciatic nerves, suggest that this change may develop fairly quickly following an insult, and demonstrate that different dog strains exhibit strain-specific nerve changes.

Nerve fiber teasing is a sensitive technique utilized in diagnostic neuropathology practice, laboratory research, and animal toxicity studies for characterizing changes in single myelinated nerve fibers over extended distances. In animal toxicity studies, a nerve portion (approximately 10 mm in length) is stained with Sudan black for 24 to 48 hours and then transferred into a drop of viscous medium (eg, glycerin) mounted on an adhesive-coated glass slide, positioning it such that the proximodistal orientation is known. Individual fibers are removed using fine forceps while the sample is viewed under a stereomicroscope. In general, lesions can be identified during teasing, but more detailed characterization and photodocumentation is undertaken once nerve fibers have been dried and coverslipped. Nerve fiber teasing is particularly useful for distinguishing early stages of axonal degeneration (which presents as ovoid fiber fragments in the midinternodal region) from segmental demyelination (which presents as loss of original myelin segments and their replacement by thinner, shorter segments in the absence of axonal damage). The slow, laborious nature of nerve fiber teasing dictates that the technique will be employed on a few samples as an auxiliary method to better define the pathogenesis of nerve lesions first identified by conventional histopathologic assessment.

Peripheral nerves are routinely examined microscopically during the nonclinical safety assessment of therapeutics. In addition to test article-related on- or off-target changes, microscopic changes in peripheral nerves may also be caused by study procedures, such as parenteral test article administration and blood or tissue sampling. We present 2 nonclinical case studies in which nonstandard peripheral nerves had study procedure-related histologic changes. The first case study describes mouse trigeminal nerve changes as a result of blood sampling via retro-orbital sinus puncture. These changes included minimal-to-mild nerve fiber (axonal) degeneration associated with macrophage infiltration. The second case study presents rat brachial plexus changes associated with animal handling and blood sampling. Brachial plexus changes included minimal-to-moderate inflammation, focal hemorrhage, and nerve fiber degeneration. In both cases, the histological changes were morphologically indistinguishable from those that might be due to test article. Therefore, careful consideration of the incidence and severity across groups and a review of study procedures to rule out handling-related nerve damage are essential before identifying a test article-related effect on peripheral nerves. Study design considerations to avoid such procedure-related changes will be discussed, as well as sampling strategies to help distinguish these from test article-related effects.

Neuropathology of the peripheral nervous system (PNS) is an underappreciated area in toxicologic pathology. Toxicity to nerves and ganglia can result from toxic insults following exposure to environmental, occupational, and industrial chemicals; drugs and biologics; cosmetics and food additives; and even physical agents such as noise. The following introduction provides an overview of this special issue of Toxicologic Pathology on toxicologic neuropathology of the PNS and highlights the range of key topics in this field that are reviewed in this compilation.

Chemotherapy-induced peripheral neuropathy (CiPN) is a serious adverse effect in the clinic, but nonclinical assessment methods in animal studies are limited to labor intensive behavioral tests or semi-quantitative microscopic evaluation. Hence, microRNA (miRNA) biomarkers and automated in-life behavioral tracking were assessed for their utility as non-invasive methods. To address the lack of diagnostic biomarkers, we explored miR-124, miR-183 and miR-338 in a CiPN model induced by paclitaxel, a well-known neurotoxic agent. In addition, conventional and Vium's innovative Digital Vivarium technology-based in-life behavioral tests and postmortem microscopic examination of the dorsal root ganglion (DRG) and the sciatic nerve were performed. Terminal blood was collected on days 8 or 16, after 20 mg/kg paclitaxel was administered every other day for total of 4 or 7 doses, respectively, for plasma miRNA quantification by RT-qPCR. DRG and sciatic nerve samples were collected from mice sacrificed on day 16 for miRNA quantification. Among the three miRNAs analyzed, only miR-124 was statistically significantly increased (5 fold and 10 fold on day 8 and day 16, respectively). The increase in circulating miR-124 correlated with cold allodynia and axonal degeneration in both DRG and sciatic nerve. Automated home cage motion analysis revealed for the first time that nighttime motion was significantly decreased (P < 0.05) in paclitaxel-dosed animals. Although both increase in circulating miR-124 and decrease in nighttime motion are compelling, our results provide positive evidence warranting further testing using additional peripheral nerve toxicants and diverse experimental CiPN models., Competing Interests: QP, AS, IP, JF-M, AV, MB, CS, JCC and C-NL are employed by Pfizer. Pfizer paid a scientific service fee to Vium Inc. JM and LS are employed by Vium Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

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.

Microscopic examination of the brain of adult Beagle dogs from four different general toxicity studies revealed the presence of ectopic choroid plexus tissue in six individual dogs (4 females and 2 males) with ages ranging from 12 to 18 months. In each dog, this finding was characterized by a well-circumscribed mass localized to a region above and along the corpus callosum without any apparent compression of adjacent brain tissue. Each mass was composed of columnar ependymal cells forming tubular structures surrounded by variable amounts of fibrovascular connective tissue and had the appearance of small rests of ependymal cells that had been penetrated by the leptomeninges during neural development. There were no associated clinical signs or macroscopic correlates. Based on morphologic appearance, a diagnosis of spontaneous ectopic choroid plexus with secondary sclerosis was made. To the authors' knowledge, ectopic choroid plexus has not been reported in Beagle dogs and is rare in humans and horses.

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.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T 2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Chemotherapy-induced peripheral neuropathy (CiPN) is a frequent adverse effect in patients and a leading safety consideration in oncology drug development. Although behavioral assessment and microscopic examination of the nerves and dorsal root ganglia can be incorporated into toxicity studies to assess CiPN risk, more sensitive and less labor-intensive endpoints are often lacking. In this study, rats and mice administered vincristine (75 μg kg -1  day -1 , i.p., for 10 days in rats and 100 μg kg -1  day -1 , i.p., for 11 days in mice, respectively) were employed as the CiPN models. Behavioral changes were assessed during the dosing phase. At necropsy, the sural or sciatic nerve was harvested from the rats and mice, respectively, and assessed for mechanical and histopathological endpoints. It was found that the maximal load and the load/extension ratio were significantly decreased in the nerves collected from the animals dosed with vincristine compared with the vehicle-treated animals (P < 0.05). Additionally, the gait analysis revealed that the paw print areas were significantly increased in mice (P < 0.01), but not in rats following vincristine administration. Light microscopic histopathology of the nerves and dorsal root ganglia were unaffected by vincristine administration. We concluded that ex vivo mechanical properties of the nerves is a sensitive endpoint, providing a new method to predict CiPN in rodent. Gait analysis may also be a useful tool in these pre-clinical animal models., (Copyright © 2017 John Wiley & Sons, Ltd.)

Small molecule DGAT2 inhibitors have shown promise for the treatment of metabolic diseases in preclinical models. Herein, we report the first toxicological evaluation of imidazopyridine-based DGAT2 inhibitors and show that the arteriopathy associated with imidazopyridine 1 can be mitigated with small structural modifications, and is thus not mechanism related.

A half-day American College of Toxicology continuing education course presented key issues often confronted by translational neuroscientists when predicting human risk from animal-derived toxicologic pathology data. Two talks correlated discrete structures with major functions in brains of rodents and nonrodents. The third lecture provided practical advice to obtain highly homologous rodent brain sections for quantitative morphometry in developmental neurotoxicity testing. The last presentation discussed demographic influences (eg, species, strain, sex, age), physiological attributes (eg, body composition, brain vascularity, pharmacokinetic/pharmacodynamic patterns, etc), and husbandry parameters (eg, group housing) recognized to impact the actions of neuroactive chemicals. Speakers described common cases of real-world challenges to animal data interpretation encountered when designing studies or extrapolating biological responses across species. The efficiency of translational neuroscience efforts will likely be enhanced as new methods (eg, high-resolution non-invasive imaging) improve our capability to cross-connect subtle anatomic and/or biochemical lesions with functional changes over time., (© The Author(s) 2016.)

Two young female Beagle dogs in a laboratory colony with clinical signs of loose stools and fecal blood were confirmed to have histiocytic ulcerative colitis by histologic evaluation. This syndrome is well recognized in other dog breeds such as Boxers and related French Bulldogs, Mastiffs, Alaskan malamutes and Doberman Pinschers. Formalin-fixed paraffin sections of large intestine from one dog demonstrated the presence of Escherichia coli strain LF82 by immunohistochemistry and 16S ribosomal RNA gene sequencing. E coli strain LF82 has been implicated in the pathogenesis of Crohn's disease and similar bacteria have been cultured from cases of histiocytic ulcerative colitis in Boxer dogs. Spontaneous histiocytic ulcerative colitis must be differentiated from test article-related findings in nonclinical toxicity studies in Beagle dogs., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

A half-day Society of Toxicologic Pathology continuing education course on "Fundamentals of Translational Neuroscience in Toxicologic Pathology" presented some current major issues faced when extrapolating animal data regarding potential neurological consequences to assess potential human outcomes. Two talks reviewed functional-structural correlates in rodent and nonrodent mammalian brains needed to predict behavioral consequences of morphologic changes in discrete neural cell populations. The third lecture described practical steps for ensuring that specimens from rodent developmental neurotoxicity tests will be processed correctly to produce highly homologous sections. The fourth talk detailed demographic factors (e.g., species, strain, sex, and age); physiological traits (body composition, brain circulation, pharmacokinetic/pharmacodynamic patterns, etc.); and husbandry influences (e.g., group housing) known to alter the effects of neuroactive agents. The last presentation discussed the appearance, unknown functional effects, and potential relevance to humans of polyethylene glycol (PEG)-associated vacuoles within the choroid plexus epithelium of animals. Speakers provided real-world examples of challenges with data extrapolation among species or with study design considerations that may impact the interpretability of results. Translational neuroscience will be bolstered in the future as less invasive and/or more quantitative techniques are devised for linking overt functional deficits to subtle anatomic and chemical lesions., (© 2014 by The Author(s).)

A workshop on Emerging Respiratory Viral Infections and Spontaneous Diseases in nonhuman primates was sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology, held December 1-5, 2012, in Seattle, Washington. The session had platform presentations from Drs Karen Terio, Thijs Kuiken, Guy Boivin, and Robert Palermo that focused on naturally occurring influenza, human respiratory syncytial virus, and metapneumovirus in wild and zoo-housed great apes; the molecular biology and pathology of these viral respiratory diseases in nonhuman primate (NHP) models; and the therapeutic and vaccine approaches to prevention and control of these emerging respiratory viral infections. These formal presentations were followed by presentations of 14 unique case studies of rare or newly observed spontaneous lesions in NHPs (see online files for access to digital whole-slide images corresponding to each case report at http://scanscope.com/ACVP%20Slide%20Seminars/2012/Primate%20Pathology/view.apml). The session was attended by meeting participants that included students, pathology trainees, and experienced pathologists from academia and industry with an interest in respiratory and spontaneous diseases of NHPs.

The Society of Toxicologic Pathology charged a Nervous System Sampling Working Group with devising recommended practices to routinely screen the central nervous system (CNS) and peripheral nervous system (PNS) in Good Laboratory Practice-type nonclinical general toxicity studies. Brains should be weighed and trimmed similarly for all animals in a study. Certain structures should be sampled regularly: caudate/putamen, cerebellum, cerebral cortex, choroid plexus, eye (with optic nerve), hippocampus, hypothalamus, medulla oblongata, midbrain, nerve, olfactory bulb (rodents only), pons, spinal cord, and thalamus. Brain regions may be sampled bilaterally in rodents using 6 to 7 coronal sections, and unilaterally in nonrodents with 6 to 7 coronal hemisections. Spinal cord and nerves should be examined in transverse and longitudinal (or oblique) orientations. Most Working Group members considered immersion fixation in formalin (for CNS or PNS) or a solution containing acetic acid (for eye), paraffin embedding, and initial evaluation limited to hematoxylin and eosin (H&E)-stained sections to be acceptable for routine microscopic evaluation during general toxicity studies; other neurohistological methods may be undertaken if needed to better characterize H&E findings. Initial microscopic analyses should be qualitative and done with foreknowledge of treatments and doses (i.e., "unblinded"). The pathology report should clearly communicate structures that were assessed and methodological details. Since neuropathologic assessment is only one aspect of general toxicity studies, institutions should retain flexibility in customizing their sampling, processing, analytical, and reporting procedures as long as major neural targets are evaluated systematically.

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

Neoplasms of the nervous system, whether spontaneous or induced, are infrequent in laboratory rodents and very rare in other laboratory animal species. The morphology of neural tumors depends on the intrinsic functions and properties of the cell type, the interactions between the neoplasm and surrounding normal tissue, and regressive changes. The incidence of neural neoplasms varies with sex, location, and age of tumor onset. Although the onset of spontaneous tumor development cannot be established in routine oncogenicity studies, calculations using the time of diagnosis (day of death) have revealed significant differences in tumor biology among different rat strains. In the central nervous system, granular cell tumors (a meningioma variant), followed by glial tumors, are the most common neoplasms in rats, whereas glial cell tumors are observed most frequently in mice. Central nervous system tumors usually affect the brain rather than the spinal cord. Other than adrenal gland pheochromocytomas, the most common neoplasms of the peripheral nervous system are schwannomas. Neural tumors may develop in the central nervous system and peripheral nervous system from other cell lineages (including extraneural elements like adipose tissue and lymphocytes), but such lesions are very rare in laboratory animals.

"Animal Models of Neural Disease" was the focus of General Session 5 at a 2010 scientific symposium that was sponsored jointly by the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). The objective was to consider issues that dictate the choice of animal models for neuropathology-based studies used to investigate neurological diseases and novel therapeutic agents to treat them. In some cases, no animal model exists that recapitulates the attributes of the human disease (e.g., fibromyalgia syndrome). Alternatively, numerous animal models are available for other conditions, so an essential consideration is selecting the most appropriate experimental system (e.g., Alzheimer's disease). New technologies (e.g., genetically engineered rodent models) promise the opportunity to generate suitable animal models for syndromes that currently lack any in vivo animal model, while in vitro models offer the opportunity to evaluate xenobiotic effects in specific neural cell populations. The complex nature of neurological disease requires regular reassessment of available and potential options to ensure that animal-derived data sets support translational medicine efforts to improve public health.

Carcinogenicity of chemicals can currently only be evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR).We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 nongenotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 nongenotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected on both day 1 and day 5. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

Non-genotoxic carcinogenicity of chemicals is currently routinely evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for non-genotoxic carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens (NGTCs) or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR). We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 non-genotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 non-genotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected at both 1 and 5 days. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.

Functional evaluation of the pars intermedia (PI) is required for the early diagnosis of equine pituitary PI dysfunction (PPID), yet most assays target the hypothalamic-pituitary-adrenal axis, which regulates the pars anterior. In contrast, the PI is regulated by dopaminergic tone from hypothalamic neurons. Loss of dopaminergic inhibition is hypothesized to cause the PI hypertrophy and hyperplasia that result in the clinical manifestations of PPID. Domperidone, a dopamine receptor antagonist, should exacerbate the loss of dopaminergic inhibition in horses with PPID and increase the release of endogenous adrenocorticotrophic hormone (eACTH) by PI melanotrophs. To test this, plasma eACTH concentration was determined in horses with or without clinical signs of PPID at 0, 4, and 8 hours after oral administration of 3.3 mg domperidone/kg. Pituitary glands were evaluated postmortem by histologic grading and morphometry. In the 33 horses, median age, plasma ACTH concentration 8 hours after domperidone, and PI area in median sagittal sections were associated with histologic grade as follows: pituitary grade 1 (normal), n = 3, 7.5 years, 20.0 pg/ml, 0.16 cm(2); grade 2 (focal hypertrophy or hyperplasia), n = 9, 14.5 years, 27.1 pg/ml, 0.27 cm(2); grade 3 (diffuse adenomatous hyperplasia), n = 5, 21.0 years, 64.4 pg/ml, 0.48 cm(2); grade 4 (microadenomas), n = 12, 23.3 years, 128.0 pg/ml, 0.87 cm(2); grade 5 (adenoma), n = 4, 24.9 years, 720.5 pg/ml, 2.1 cm(2). Results suggest that horses with pituitary histologic grade > or =3 respond to domperidone with increased plasma ACTH concentration.

In 2004, six puppies and one adult dog from a total of four premises were subjected to necropsy evaluation. For five of the seven dogs, disease caused by canine distemper virus (CDV) infection was suspected based on clinical signs. In all of the dogs, a diagnosis of CDV infection was established by the presence of compatible gross and histologic lesions, immunohistochemical labeling for CDV antigen, and detection of CDV RNA by reverse transcription-PCR. To further characterize the CDV strains detected in the four cases, complete gene sequences were determined for the hemagglutinin (H) and fusion (F) protein genes, while partial gene sequencing was performed for the phosphoprotein gene. A total of 4,508 bases were sequenced for the CDV strains detected from each of the four cases. Two cases were found to have identical sequences except for 2 bases in the intergenic region of the F and H genes. Phylogenetic analysis strongly suggested an evolutionary relationship between sequences detected in these two cases and those of phocine distemper virus 2 and two other strains of CDV not previously detected in the continental United States. Clear phylogenetic relationships were not established for viruses detected in the two additional cases; however, one strain showed similarity to CDV strains detected in a panda from China. Importantly, the three CDV strains detected were demonstrated to be genetically distinct from known vaccine strains and strains previously reported in the continental United States.