Neuroanatomy and Sampling of Central Projections for the Visual System in Mammals Used in Toxicity Testing.

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.

Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Robust amyloid clearance in a mouse model of Alzheimer's disease provides novel insights into the mechanism of amyloid-beta immunotherapy.

Many new therapeutics for Alzheimer's disease delay the accumulation of amyloid-ß (Aß) in transgenic mice, but evidence for clearance of preexisting plaques is often lacking. Here, we demonstrate that anti-Aß immunotherapy combined with suppression of Aß synthesis allows significant removal of antecedent deposits. We treated amyloid-bearing tet-off APP (amyloid precursor protein) mice with doxycycline to suppress transgenic Aß production before initiating a 12 week course of passive immunization. Animals remained on doxycycline for 3 months afterward to assess whether improvements attained during combined treatment could be maintained by monotherapy. This strategy reduced amyloid load by 52% and Aß42 content by 28% relative to pretreatment levels, with preferential clearance of small deposits and diffuse Aß surrounding fibrillar cores. We demonstrate that peripherally administered anti-Aß antibody crossed the blood-brain barrier, bound to plaques, and was still be found associated with a subset of amyloid deposits many months after the final injection. Antibody accessed the brain independent of plasma Aß levels, where it enhanced microglial internalization of aggregated Aß. Our data support a mechanism by which passive immunization acts centrally to stimulate microglial phagocytosis of aggregated Aß, but is opposed by the continued aggregation of newly secreted Aß. By arresting the production of Aß, combination therapy allows microglial clearance to work from a static amyloid burden toward a significant reduction in plaque load. Our findings suggest that combining two therapeutic approaches currently in clinical trials may improve neuropathological outcome over either alone.

Recommended neuroanatomical sampling practices for comprehensive brain evaluation in nonclinical safety studies.

Adequate tissue sampling is known to reduce the likelihood that the toxicity of novel biomolecules, chemicals, and drugs might go undetected. Each organ, and often specific structurally and functionally distinct regions within it, must be assessed to detect potential site-specific toxicity. Adequate sampling of the brain requires particular consideration because of the many major substructures and more than 600 subpopulations of generally irreplaceable cells with unique functions and vulnerabilities. All known neurotoxicants affect specific subpopulations (usually neurons) rather than damaging a certain percentage of cells throughout the brain; thus, all populations should be independently assessed for lesions. Historically, the affected neural cell subpopulation has not been predictable, but it is now clear that sampling selected populations (e.g., cerebral cortex, hippocampus, cerebellar folia) cannot forecast the health of other populations. This article reviews the neuroanatomical domains affected by several model neurotoxicants to illustrate the need for more comprehensive neurohistological evaluation during nonclinical development of novel compounds. The article also describes an easily executed, cost-effective method that uses a set number of evenly spaced coronal (cross) sections to accomplish this comprehensive brain assessment during nonclinical safety studies performed in rodents, dogs, and nonhuman primates.

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.

Gene expression studies reveal that DNA damage, vascular perturbation, and inflammation contribute to the pathogenesis of carbonyl sulfide neurotoxicity.

Carbonyl sulfide (COS) is an odorless gas that produces highly reproducible lesions in the central nervous system. In the present study, the time course for the development of the neurotoxicological lesions was defined and the gene expression changes occurring in the posterior colliculus upon exposure to COS were characterized. Fischer 344 rats were exposed to 0 or 500 ppm COS for one, two, three, four, five, eight, or ten days, six hours per day. On days 1 and 2, no morphological changes were detected; on day 3, 10/10 (100%) rats had necrosis in the posterior colliculi; and on day 4 and later, necrosis was observed in numerous areas of the brain. Important gene expression changes occurring in the posterior colliculi after one or two days of COS exposure that were predictive of the subsequent morphological findings included up-regulation of genes associated with DNA damage and G1/S checkpoint regulation (KLF4, BTG2, GADD45g), apoptosis (TGM2, GADD45g, RIPK3), and vascular mediators (ADAMTS, CTGF, CYR61, VEGFC). Proinflammatory mediators (CCL2, CEBPD) were up-regulated prior to increases in expression of the astrocytic marker GFAP and macrophage marker CSF2rb1. These gene expression findings were predictive of later CNS lesions caused by COS exposure and serve as a model for future investigations into the mechanisms of disease in the central nervous system.

Brain of the African elephant (Loxodonta africana): neuroanatomy from magnetic resonance images.

We acquired magnetic resonance images of the brain of an adult African elephant, Loxodonta africana, in the axial and parasagittal planes and produced anatomically labeled images. We quantified the volume of the whole brain (3,886.7 cm3) and of the neocortical and cerebellar gray and white matter. The white matter-to-gray matter ratio in the elephant neocortex and cerebellum is in keeping with that expected for a brain of this size. The ratio of neocortical gray matter volume to corpus callosum cross-sectional area is similar in the elephant and human brains (108 and 93.7, respectively), emphasizing the difference between terrestrial mammals and cetaceans, which have a very small corpus callosum relative to the volume of neocortical gray matter (ratio of 181-287 in our sample). Finally, the elephant has an unusually large and convoluted hippocampus compared to primates and especially to cetaceans. This may be related to the extremely long social and chemical memory of elephants.

Distribution and Severity of Neuropathology in ß-Mannosidase-Deficient Mice is Strain Dependent.

Neurological dysfunction is common in humans and animals with lysosomal storage diseases. ß-Mannosidosis, an autosomal recessive inherited disorder of glycoprotein catabolism caused by deficiency of the lysosomal enzyme ß-mannosidase, is characterized by intracellular accumulation of small oligosaccharides in selected cell types. In ruminants, clinical manifestation is severe, and neuropathology includes extensive intracellular vacuolation and dysmyelination. In human cases of ß-mannosidosis, the clinical symptoms, including intellectual disability, are variable and can be relatively mild. A ß-mannosidosis knockout mouse was previously characterized and showed normal growth, appearance, and lifespan. Neuropathology between 1 and 9 months of age included selective, variable neuronal vacuolation with no hypomyelination. This study characterized distribution of brain pathology in older mutant mice, investigating the effects of two strain backgrounds. Morphological analysis indicated a severe consistent pattern of neuronal vacuolation and disintegrative degeneration in all five 129X1/SvJ mice. However, the mice with a mixed genetic background showed substantial variability in the severity of pathology. In the severely affected animals, neuronal vacuolation was prominent in specific layers of piriform area, retrosplenial area, anterior cingulate area, selected regions of isocortex, and in hippocampus CA3. Silver degeneration reaction product was prominent in regions including specific cortical layers and cerebellar molecular layer. The very consistent pattern of neuropathology suggests metabolic differences among neuronal populations that are not yet understood and will serve as a basis for future comparison with human neuropathological analysis. The variation in severity of pathology in different mouse strains implicates genetic modifiers in the variable phenotypic expression in humans.

Rat injury model under controlled field-relevant primary blast conditions: acute response to a wide range of peak overpressures.

We evaluated the acute (up to 24 h) pathophysiological response to primary blast using a rat model and helium driven shock tube. The shock tube generates animal loadings with controlled pure primary blast parameters over a wide range and field-relevant conditions. We studied the biomechanical loading with a set of pressure gauges mounted on the surface of the nose, in the cranial space, and in the thoracic cavity of cadaver rats. Anesthetized rats were exposed to a single blast at precisely controlled five peak overpressures over a wide range (130, 190, 230, 250, and 290 kPa). We observed 0% mortality rates in 130 and 230 kPa groups, and 30%, 24%, and 100% mortality rates in 190, 250, and 290 kPa groups, respectively. The body weight loss was statistically significant in 190 and 250 kPa groups 24 h after exposure. The data analysis showed the magnitude of peak-to-peak amplitude of intracranial pressure (ICP) fluctuations correlates well with mortality rates. The ICP oscillations recorded for 190, 250, and 290 kPa are characterized by higher frequency (10-20 kHz) than in other two groups (7-8 kHz). We noted acute bradycardia and lung hemorrhage in all groups of rats subjected to the blast. We established the onset of both corresponds to 110 kPa peak overpressure. The immunostaining against immunoglobulin G (IgG) of brain sections of rats sacrificed 24-h post-exposure indicated the diffuse blood-brain barrier breakdown in the brain parenchyma. At high blast intensities (peak overpressure of 190 kPa or more), the IgG uptake by neurons was evident, but there was no evidence of neurodegeneration after 24 h post-exposure, as indicated by cupric silver staining. We observed that the acute response as well as mortality is a non-linear function over the peak overpressure and impulse ranges explored in this work.

Autoradiographic evidence for the transmeningeal diffusion of muscimol into the neocortex in rats.

Electrophysiological and behavioral studies have demonstrated that muscimol administered through the cranial meninges can prevent focal neocortical seizures. It was proposed that transmeningeal muscimol delivery can be used for the treatment of intractable focal neocortical epilepsy. However, it has not been proved that muscimol administered via the transmeningeal route can penetrate into the neocortex. The purpose of the present study was to solve this problem by using combined autoradiography-histology methods. Four rats were implanted with epidural cups over the parietal cortices. A 50 µL mixture of [³H] muscimol and unlabeled muscimol with a final concentration of 1.0mM was delivered through each cup on the dura mater. After a 1-hour exposure, the muscimol solution was removed and replaced with formalin to trap the transmeningeally diffused molecules. Then the whole brain was fixed transcardially, sectioned, with the sections subjected to autoradiography and thionine counterstaining. Results showed that (1) [³H] muscimol diffused through the meninges into the cortical tissue underlying the epidural cup in all rats. (2) [³H] muscimol-related autoradiography grains were distributed in all six neocortical layers. (3) [³H] muscimol-related autoradiography grains were localized to the cortical area underneath the epidural delivery site and were absent in the cerebral cortical white matter and other brain structures. This study provided evidence that muscimol can be delivered via the transmeningeal route into the neocortical tissue in a spatially controlled manner. The finding further supports the rationale of using transmeningeal muscimol for the treatment of intractable focal neocortical epilepsy.

Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury.

Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration ∼ 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rat's head. The insult produced ∼ 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.

Striatal pathology underlies prion infection-mediated hyperactivity in mice.

Although prion diseases are most commonly modeled using the laboratory mouse, the diversity of prion strains, behavioral testing and neuropathological assessments hamper our collective understanding of mouse models of prion disease. Here we compared several commonly used murine strains of prions in C57BL/6J female mice in a detailed home cage behavior detection system and a systematic study of pathological markers and neurotransmitter systems. We observed that mice inoculated with RML or 139A prions develop a severe hyperactivity phenotype in the home cage. A detailed assessment of pathology markers, such as microglial marker IBA1, astroglial marker GFAP and degeneration staining indicate early striatal pathology in mice inoculated with RML or 139A but not in those inoculated with 22L prions. An assessment of neuromodulatory systems including serotonin, dopamine, noradrenalin and acetylcholine showed surprisingly little decline in neuronal cell bodies or their innervations of regions controlling locomotor behavior, except for a small decrease in dopaminergic innervations of the dorsal striatum. These results implicate the dorsal striatum in mediating the major behavioral phenotype of 139A and RML prions. Further, they suggest that measurements of activity may be a sensitive manner in which to diagnose murine prion disease. With respect to neuropathology, our results indicate that pathological stains as opposed to neurotransmitter markers are much more informative and sensitive as markers of prion disease in mouse models.

Quiescent adult neural stem cells are exceptionally sensitive to cosmic radiation.

Generation of new neurons in the adult brain, a process that is likely to be essential for learning, memory, and mood regulation, is impaired by radiation. Therefore, radiation exposure might have not only such previously expected consequences as increased probability of developing cancer, but might also impair cognitive function and emotional stability. Radiation exposure is encountered in settings ranging from cancer therapy to space travel; evaluating the neurogenic risks of radiation requires identifying the at-risk populations of stem and progenitor cells in the adult brain. Here we have used a novel reporter mouse line to find that early neural progenitors are selectively affected by conditions simulating the space radiation environment. This is reflected both in a decrease in the number of these progenitors in the neurogenic regions and in an increase in the number of dying cells in these regions. Unexpectedly, we found that quiescent neural stem cells, rather than their rapidly dividing progeny, are most sensitive to radiation. Since these stem cells are responsible for adult neurogenesis, their death would have a profound impact on the production of new neurons in the irradiated adult brain. Our finding raises an important concern about cognitive and emotional risks associated with radiation exposure.

Somatosensory nuclei of the manatee brainstem and thalamus.

Florida manatees have an extensive, well-developed system of vibrissae distributed over their entire bodies and especially concentrated on the face. Although behavioral and anatomical assessments support the manatee's reliance on somatosensation, a systematic analysis of the manatee thalamus and brainstem areas dedicated to tactile input has never been completed. Using histochemical and histological techniques (including stains for myelin, Nissl, cytochrome oxidase, and acetylcholinesterase), we characterized the relative size, extent, and specializations of somatosensory regions of the brainstem and thalamus. The principal somatosensory regions of the brainstem (trigeminal, cuneate, gracile, and Bischoff's nucleus) and the thalamus (ventroposterior nucleus) were disproportionately large relative to nuclei dedicated to other sensory modalities, providing neuroanatomical evidence that supports the manatee's reliance on somatosensation. In fact, areas of the thalamus related to somatosensation (the ventroposterior and posterior nuclei) and audition (the medial geniculate nucleus) appeared to displace the lateral geniculate nucleus dedicated to the subordinate visual modality. Furthermore, it is noteworthy that, although the manatee cortex contains Rindenkerne (barrel-like cortical nuclei located in layer VI), no corresponding cell clusters were located in the brainstem ("barrelettes") or thalamus ("barreloids").

Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase (SSADH) deficiency, a rare genetic defect of GABA degradation recently modelled in mice (SSADH(-/-) mice), manifests early absence seizures that evolve into generalized convulsive seizures and lethal status epilepticus in gene-ablated mice. Disrupted GABA homeostasis, in conjunction with the epileptic phenotype and increased gamma-hydroxybutyric acid (GHB), suggested that expression profiling with the U74Av2 Affymetrix system would reveal dysregulation of receptor genes associated with GABAergic and glutamatergic neurotransmission. Unexpectedly, we found significant downregulation for genes associated with myelin biogenesis and compaction, predominantly in hippocampus and cortex. These results were confirmed by: (1) myelin basic protein (MBP) immunohistochemistry; (2) western blotting of myelin-associated glycoprotein (MAG) and MBP; (3) qRT-PCR analyses of myelin-associated oligodendrocytic basic protein (MOBP), MAG, MBP and proteolipid protein (PLP) in hippocampus, cortex and spinal cord; (4) quantitation of ethanolamine and choline plasmalogens, all core myelin components; (5) evaluation of myelin content in brain sections employing toluidine blue staining; and (6) ultrastructural evaluation of myelin sheath thickness via electron microscopy. We speculate that increased GABA/GHB, acting through GABAergic systems, results in decreased levels of the neurosteroids progesterone and allopregnanolone [Gupta et al (2003) Ann Neurol 54(Supplement 6): S81-S90] and phosphorylation of mitogen-activated protein (MAP) kinase, with resulting myelin protein abnormalities primarily in the cortex of SSADH(-/-) mice.