Many preclinical investigations limit the evaluation of the peripheral nervous system (PNS) to paraffin-embedded sections/hematoxylin and eosin–stained sections of the sciatic nerve. This limitation ...ignores several key mechanisms of toxicity and anatomic differences that may interfere with an accurate assessment of test article effects on the neurons/neurites peripheral to the brain and spinal cord. Ganglion neurons may be exposed to higher concentrations of the test article as compared to neurons in the brain or spinal cord due to differences in capillary permeability. Many peripheral neuropathies are length-dependent, meaning distal nerves may show morphological changes before they are evident in the mid-sciatic nerve. Paraffin-embedded nerves are not optimal to assess myelin changes, notably those leading to demyelination. Differentiating between axonal or myelin degeneration may not be possible from the examination of paraffin-embedded sections. A sampling strategy more consistent with known mechanisms of toxicity, atraumatic harvest of tissues, optimized fixation, and the use of resin and paraffin-embedded sections will greatly enhance the pathologist’s ability to observe and characterize effects in the PNS.
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Riluzole is the only treatment known to improve survival in patients with Amyotrophic Lateral Sclerosis (ALS). However, oral riluzole efficacy is modest at best, further it is known to have large ...inter-individual variability of serum concentration and clearance, is formulated as an oral drug in a patient population plagued with dysphagia, and has known systemic side-effects like asthenia (limiting patient compliance) and elevated liver enzymes. In this context, we postulated that continuous intrathecal (IT) infusion of low doses of riluzole could provide consistent elevations of the drug spinal cord (SC) concentrations at or above those achieved with oral dosing, without increasing the risk for adverse events associated with systemic drug exposure or off-target side effects in the brain. We developed a formulation of riluzole for IT delivery and conducted our studies in purpose-bred hound dogs. Our non-GLP studies revealed that IT infusion alone was able to increase SC concentrations above those provided by oral administration, without increasing plasma concentrations. We then conducted two GLP studies that combined IT infusion with oral administration at human equivalent dose, to evaluate SC and brain concentrations of riluzole along with assessments of safety and tolerability. In the 6-week study, the highest IT dose (0.2 mg/hr) was well tolerated by the animals and increased SC concentrations above those achieved with oral riluzole alone, without increasing brain concentrations. In the 6-month study, the highest dose tested (0.4 mg/hr) was not tolerated and yielded SC significantly above those achieved in all previous studies. Our data show the feasibility and safety profile of continuous IT riluzole delivery to the spinal cord, without concurrent elevated liver enzymes, and minimal brain concentrations creating another potential therapeutic route of delivery to be used in isolation or in combination with other therapeutics.”
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The neurotoxicity of paraquat dichloride (PQ) was assessed in two inbred strains of 9- or 16-week old male C57BL/6 mice housed in two different laboratories and compared to the effects of ...1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PQ was administered by intraperitoneal injections; either once (20 mg/kg) or twice (10 mg/kg) weekly for 3 weeks, while MPTP-HCl was injected 4 times on a single day (20 mg/kg/dose). Brains were collected 8, 16, 24, 48, 96 or 168 hours after the last PQ treatment, and 48 or 168 hours after MPTP treatment. Dopamine neurons in the substantia nigra pars compacta (SNpc) were identified by antibodies to tyrosine hydroxylase (TH+) and microglia were identified using Iba-1 immunoreactivity. The total number of TH+ neurons and the number of resting and activated microglia in the SNpc at 168 hours after the last dose were estimated using model- or design-based stereology, with investigators blinded to treatment. In a further analysis, a pathologist, also blinded to treatment, evaluated the SNpc and/or striatum for loss of TH+ neurons (SNpc) or terminals (striatum), cell death (as indicated by amino cupric silver uptake, TUNEL and/or caspase 3 staining) and neuroinflammation (as indicated by Iba-1 and/or GFAP staining). PQ, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of TH+ neurons or microglia in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. PQ did not induce neuronal cell loss or degeneration in the SNpc or striatum. Additionally, there was no evidence of apoptosis, microgliosis or astrogliosis. In MPTP-treated mice, the number of TH+ neurons in the SNpc was significantly decreased and the number of activated microglia increased. Histopathological assessment found degenerating neurons/terminals in the SNpc and striatum but no evidence of apoptotic cell death. MPTP activated microglia in the SNpc and increased the number of astrocytes in the SNpc and striatum.
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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.
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Gliosis, including microgliosis and astrocytosis, can be challenging to interpret in nonclinical studies. Incidences of glial foci in brains and spinal cords of control rats and nonhuman primates ...(NHPs) were reviewed in the historical control databases from two contract research organizations, including one specializing in neuropathology. In the brain, minimal to mild (grades 1-2) microgliosis was the most common diagnosis, especially in NHPs, although occasional moderate or marked microgliosis (grades 3 and 4) was encountered in both species. Microgliosis was more common in the cerebral cortex, cerebellum, and medulla oblongata in both species and was frequent in the white matter (brain), thalamus, and basal nuclei of NHPs. Gliosis (“not otherwise specified”) of minimal severity was diagnosed in similar brain sub-sites for both species and was more common in NHPs compared with rats. Astrocytosis was most prominent in the cerebellum (molecular layer) of NHPs but was otherwise uncommon. In the spinal cord, microgliosis was most common in the lateral white matter tracts in rats and NHPs, and in the dorsal white matter tracts in NHPs. These data indicate that low-grade spontaneous glial responses occur with some frequency in control animals of two common nonclinical species.
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•Reversible neuronal atrophy but no neuronal cell death in the sympathetic ganglia.•Stereological analysis showed reversible neuronal atrophy in the superior cervical ganglion.•TrkA ...inhibitor did not induce any detectable dysfunction of the sympathetic and sensory nervous system.
ASP7962 is a small molecule inhibitor for the nerve growth factor (NGF) receptor, tropomyosin-related kinase A (TrkA). NGF contributes to the survival of sensory and sympathetic neurons through TrkA receptor activation. Gross, microscopic, and quantitative effects to the nervous system were evaluated following oral ASP7962 administration to Sprague Dawley rats for 4 weeks and 13 weeks and after a recovery period. Histopathological findings included reversible neuronal atrophy but no neuronal death in the sympathetic ganglia (cervicothoracic ganglion, cranial mesenteric ganglion or superior cranial cervical ganglion). Stereological analysis showed reversible decreased ganglion volume and/or decreased neuron size in the superior (cranial) cervical ganglion in both the 4-week and the 13-week repeated dose studies. There were no test article related changes in the brain, dorsal root ganglia with spinal nerve roots or trigeminal ganglia and no functional deficits. ASP7962 did not cause any detectable dysfunction of the sympathetic and sensory nervous system in either study.
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Assessment of the peripheral nervous system (PNS) tissues during animal toxicity studies generally is included within guiding documents issued by regulatory agencies of individual nations (eg, US ...Environmental Protection Agency, US Food and Drug Administration) and multinational federations (eg, European Medicines Agency) as well as international cooperative efforts (eg, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, Organisation for Economic Co-operation and Development). The present list of major regulatory guiding documents categorizes recommendations from around the world for sampling and processing PNS tissues (nerves and ganglia) for general animal toxicity studies (ie, where neurotoxicity is not expected) and specialized neurotoxicity studies (ie, where neurotoxicity is anticipated or known to occur). In general, regulatory guidelines call for collection of one or more sensorimotor nerves (usually the sciatic trunk and its branches), though details vary among agencies. Regulatory guiding documents represent a “starting point,” after which additional PNS samples and/or special methods may be implemented at the applicant’s discretion. Best practice recommendations for PNS sampling and processing in animal toxicity studies endorsed by multiple global societies of toxicologic pathology encompass and expand on existing regulatory guidelines.
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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.
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A retrospective analysis of microscopic evaluation data from control (device and/or saline-treated) animals in intrathecal studies in monkeys, dogs, sheep, and rats was conducted. The studies were ...performed by multiple testing facilities. All slide preparation and microscopic evaluation were conducted in the laboratory of the author. The data were of observations made at the level of the catheter tip, which typically was located in the intrathecal space near the thoracolumbar region of the spinal canal. The most common microscopic changes in control animals were meningeal infiltrates, catheter track (CT) inflammation, spinal cord compression (at the CT), CT fibrosis, spinal cord gliosis (at the CT), and spinal cord nerve fiber degeneration. Although variable between studies (even within species), in general the average severity of these findings was minimal or less in control animals. CT inflammatory mass/pyogranuloma formation, a known complication following the administration of morphine at higher concentrations/doses, was noted in 3 of 25 control dogs and 2 of 77 control monkeys. These data show that inflammatory mass/pyogranuloma formation may occur in control animals, and this occurrence is most common in dogs as compared to monkeys, sheep, and rats.
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In 2013 and 2015, the Pennsylvania Department of Environmental Protection conducted a survey of lotic habitats within the Susquehanna, Delaware, and Ohio River basins in Pennsylvania, USA, to screen ...for microcystins/nodularins (MCs/NODs) in algae communities and smallmouth bass (Micropterus dolomieu). Periphyton (68 from 41 sites), juvenile whole fish (153 from 19 sites) and adult fish liver (115 from 16 sites) samples were collected and screened using an Adda enzyme-linked immunosorbent assay (ELISA). Samples that were positive for MCs/NODs were further analyzed using LC-MS/MS, including 14 variants of microcystin and NOD-R and the MMPB technique. The ELISA was positive for 47% of the periphyton collections, with NOD-R confirmed (0.7–82.2 ng g−1 d.w.) in 20 samples. NOD-R was confirmed in 10 of 15 positive juvenile whole fish samples (0.8–16.7 ng g−1 w.w.) and in 2 of 8 liver samples (1.7 & 2.8 ng g−1 w.w.). The MMPB method resulted in total MCs/NODs measured in periphyton (2.2–1269 ng g−1 d.w.), juvenile whole fish (5.0–210 ng g−1 d.w.) and adult livers (8.5–29.5 ng g−1 d.w.). This work illustrates that NOD-R is present in freshwater benthic algae in the USA, which has broader implications for monitoring and trophic transfer.
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•Periphyton, smallmouth juvenile bass and adult bass were collected from Pennsylvania, USA.•ELISA, Individual Variant Analysis (LC-MS/MS) & MMPB were used to measure Microcystins/Nodularins.•NOD-R was determined to be present in freshwater periphyton.•NOD-R was also detected in juvenile smallmouth bass and adult livers.
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