Essential References for Structural Analysis of the Peripheral Nervous System for Pathologists and Toxicologists.

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.

The Science and Art of Nerve Fiber Teasing for Myelinated Nerves: Methodology and Interpretation.

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.

Case Report: Canine Strain- and Study Condition-Dependent Formation of Renaut Bodies in Sciatic Nerves of Beagle Dogs.

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.

Atlas of Normal Microanatomy, Procedural and Processing Artifacts, Common Background Findings, and Neurotoxic Lesions in the Peripheral Nervous System of Laboratory Animals.

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.

Experience with examination of the spinal cord and peripheral nervous system (PNS) in mice: A brief overview.

The representative areas for examination of the mouse peripheral nervous system are the spinal cord, containing central components of the peripheral nervous system that needs to be examined at least at cervical and lumbar level, the sciatic and the tibial nerve. Skeletal muscle samples should include the soleus muscle and the quadriceps femoris or long digital extensor, as well as the medial gastrocnemius. Examination can be extended to the thoracic spinal cord, lumbar dorsal root ganglia and spinal nerve roots, as well as the plantar nerve, and other areas of interest. Perfusion fixation is considered optimal for the nervous system; however, immersion fixation allows producing microscopic sections of excellent quality as well. Paraffin-embedded, hematoxylin and eosin-stained sections can be made from all areas, save for small nerves such as the tibial or plantar nerve, which are examined with advantage in hard plastic sections. It is possible to produce hard plastic sections also of the vertebral column, including the spinal cord, dorsal root ganglia and nerve roots. For special investigations, mice can be fixed in toto, decalcified, embedded and sectioned to reveal the areas of interest. In the mouse peripheral nerves, myelination progresses until the adult age. In aging peripheral nerves there is axonal atrophy, degeneration, nerve fiber loss, increase of collagen and sporadic demyelination, especially radiculoneuropathy. The dorsal root ganglia of untreated control animals show frequent cytoplasmic vacuolation. Axonal degeneration is distally, primary demyelination proximally accentuated. Mouse is not very sensitive to peripheral neurotoxicity: to induce toxic peripheral neuropathy mostly parenteral administration and/or newborn animals are used. Naturally occurring infection affecting the spinal cord and peripheral nerves is Theiler's encephalomyelitis virus inducing acute poliomyelitis or chronic demyelination. Any experimental results are to be assessed taking into account spontaneous, age-related, background changes.

Useful toxicologic neuropathology references for pathologists and toxicologists.

Investigations in toxicologic neuropathology are complex undertakings because of the intricate spatial and temporal diversity in the anatomic, functional, and molecular organization of the central and peripheral nervous systems. This compilation of toxicologic neuropathology resources has been designed to consolidate a broad range of useful neurobiology, neuropathology, and neurotoxicology resources in a single reference. This collection will increase familiarity with the basic knowledge, skills, and tools required for the proficient practice of toxicologic neuropathology and should help to improve the analysis and interpretation of pathology data sets from neural tissues in toxicology studies.

Cerebral vacuolation induced in rats by the administration of LUP-3FDC, an anti-tuberculosis cocktail.

A study was conducted to determine the subacute oral toxicity of LUP-3FDC (a cocktail composed of rifampicin, isoniazid and pyrazinamide) and LUP-Q1 (gatifloxicin sesquihydrate) as well as the potential effects of their combination when administered as repeated sublethal oral (gavage) doses for a period of 90 days in seven (7) groups of Wistar rats. Three (3) additional groups were allowed to live for 28 days after the end of treatment to evaluate the potential reversibility of any toxic effects observed. Mortality was observed at all dose levels. General body weakness and hind limb paralysis (attributable to peripheral neuropathy) were observed in animals administered 1400mg/kg/day LUP-3FDC, 800mg/kg/day LUP 3-FDC+300mg/kg/day LUP Q1 and 1400mg/kg/day LUP-3FDC+300mg/kg/day LUP-Q1. The administration of LUP-3FDC at doses of 1100 or 1400mg/kg/day or a combination of 1400mg/kg/day LUP-3FDC and 300mg/kg/day LUP-Q1 induced an increased incidence of vacuolation in the brain compared to control animals. This effect, which was observed predominantly in the cerebellar roof nuclei, was attributed to the isoniazid component of the compound. Vacuoles were located primarily in the myelinated areas close to cerebellar roof nuclei, but were also seen in the olfactory tubercle, thalamus, cerebral cortex, septum and basal ganglia. Females were more susceptible to this change; vacuoles were still evident in males and females 28 days after the cessation of compound administration. The rat cerebellum is prone to develop vacuolation with age; isoniazid may "accelerate" or "enhance" this tendency in young rats.