The number and distribution of the retinal ganglion cells in the 2 years old Korean native cattle was determined from whole flat mounted preparation stained with methylene blue and thionin. The total number of retinal ganglion cells was estimated to be 3,085,200 in the bovine retina ranging from 2.214mm** (2) in total area. Visual streak was recognized at the area 2.5mm superior to the optic disc and ganglion cell density drops off rapidly to the direction superior to and inferior to the visual streak. Area centralis (6,800 cells/mm** (2)) was located at the area 10mm temporally from the point of 3mm superior to the optic disc. The number of alpha-type ganglion cells (above 15 micro) was 57,000 in the bovine retina and alpha-type ganglion cells constituted 18.5 % of the total cells. The relative frequency of alpha-type ganglion cells was higher in the peripheral regions than in the visual streak, especially higher in the superior-temporal quadrant than in other region of the bovine retina.

This study determined the presence of nitric oxide synthesis isoforms (nNOS, iNOS, and eNOS) in thoracic spinal cord segments and nodose ganglia of rats with gamma-irradiated livers. Male rats (n = 32) were divided into equal groups A, B, C, and D. In group A, the controls, no radiation was applied, while groups B, C, and D received 10 Gy of ionising gamma radiation. The rats of group B were euthanized at the end of the first day (d1), those of group C on the second day (d2), and those of group D on the third day (d3). The liver, spinal cord segments, and nodose ganglion tissues were dissected and fixed, and the liver sections were examined histopathologically. The other tissues were observed through a light microscope. Regeneration occurred at the end of d3 in hepatocytes which were radiation-damaged at the end of d1 and d2. On d1, some nNOS-positive staining was found in the neuronal cells of laminae I–III of the spinal cord and in neurons of the nodose ganglion, and on d3, some staining was observed in lamina X of the spinal cord, while none of note was in the nodose ganglion. Dense iNOS-positive staining was seen on d1 in the ependymal cells of the spinal cord and in the glial cells of the nodose ganglion, and on d3, there was still considerable iNOS staining in both tissues. There was clear eNOS-positive staining in the capillary endothelial cells of the spinal cord and light diffuse cytoplasmic staining in the neurons of the nodose ganglion on d1, and on d3, intense eNOS-positive staining was visible in several endothelial cells of the spinal cord, while light nuclear staining was recognised in the neurons of the nodose ganglion. The nNOS, iNOS, and eNOS isoforms are activated in the spinal cord and nodose ganglion of rats after ionising radiation insult to the liver.

OBJECTIVE To investigate regional differences of relative metabolite concentrations in the brain of healthy dogs with short echo time, single voxel proton magnetic resonance spectroscopy (1H MRS) at 3.0 T. ANIMALS 10 Beagles. PROCEDURES Short echo time, single voxel 1H MRS was performed at the level of the right and left basal ganglia, right and left thalamus, right and left parietal lobes, occipital lobe, and cerebellum. Data were analyzed with an automated fitting method (linear combination model). Metabolite concentrations relative to water content were obtained, including N-acetyl aspartate, total choline, creatine, myoinositol, the sum of glutamine and glutamate (glutamine-glutamate complex), and glutathione. Metabolite ratios with creatine as the reference metabolite were calculated. Concentration differences between right and left hemispheres and sexes were evaluated with a Wilcoxon signed rank test and among various regions of the brain with an independent t test and 1-way ANOVA. RESULTS No significant differences were detected between sexes and right and left hemispheres. All metabolites, except the glutamine-glutamate complex and glutathione, had regional concentrations that differed significantly. The creatine concentration was highest in the basal ganglia and cerebellum and lowest in the parietal lobes. The N-acetyl aspartate concentration was highest in the parietal lobes and lowest in the cerebellum. Total choline concentration was highest in the basal ganglia and lowest in the occipital lobe. CONCLUSIONS AND CLINICAL RELEVANCE Metabolite concentrations differed among brain parenchymal regions in healthy dogs. This study may provide reference values for clinical and research studies involving 1H MRS performed at 3.0 T.

Pseudorabies virus (PRV) latency was investigated, using polymerase chain reaction (PCR). A PCR protocol was developed that specifically amplified a 217-base pair sequence within the gene encoding the essential glycoprotein gp50 of PRV. Using this PCR procedure, the gp50 sequence was amplified from tissues of pigs infected with various doses of PRV (Becker strain). At postinoculation day 64, viral isolation was performed on nasal swab specimens and homogenates of tonsils and trigeminal nerve ganglia obtained from 11 PRV-convalescent, seropositive pigs. Results were negative in all cases. By use of PCR, 11 of 11 pigs had PRV-positive trigeminal nerve ganglia and brain stem, 10 of 11 pigs had PRV-positive tonsils, and 9 of 11 pigs had PRV-positive olfactory bulbs.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

Akabane virus (AKV) strain OBE-1 was inoculated IV into 17 pregnant sheep. Ten fetuses infected at 29 to 45 days of gestation and examined 29 to 30 days later had AKV antigen in the following groups of cells: neuroglial cells in the brain and spinal cord, ganglion cells in the cranial and abdominal ganglia, layer of ganglion cells in the retina, ganglion cells (Auerbach's plexus) in small intestine, hepatocytes, cells in the arterial wall of mesenteric membrane, and trophoblast cells in the placenta. Prior to detection of circulating virus-neutralizing antibody, immunoglobulin-containing cells were found initially at 59 days of gestation in the peripheral portion of white pulp tissue in the spleen. After that, numbers of immunoglobulin-containing cells gradually increased. These results indicated that AKV may have strong affinity for neuronal and ganglional cells in infected fetuses and immunoglobulin-containing cells might be considered the earliest immunologic response to AKV replication in the fetus.

The objective of this study was to evaluate any otopathologic changes in temporal bone specimens from dogs with deafness related to cochleosaccular (Scheibe) dysplasia (CSD). We used the canine temporal bone collections of the Otopathology Laboratory at the University of Minnesota and of the Massachusetts Eye and Ear Infirmary at Harvard University in Boston. Our morphometric analysis included measuring the areas of the stria vascularis and the spiral ligament and counting the number of spiral ganglion cells. In addition, we noted the presence of the organ of Corti and cochlear hair cells, assessed the location of Reissner's membrane and the saccular membrane, and counted the number of both Type I and Type II vestibular hair cells in the macule of the saccule and vestibular ganglion cells. In the group of specimens from dogs with cochleosaccular dysplasia, we observed generalized degeneration in the cochlea and a significantly decreased number of Type I and Type II vestibular hair cells and vestibular ganglion cells. As hereditary deafness is presently untreatable with known therapeutic methods, dogs with cochleosaccular dysplasia should not be considered for breeding. Future therapeutic approaches, such as stem cell therapies, should be designed to target all the elements of the cochlea in addition to the saccule as it was found that both are affected in dogs with CSD.

Objective—To investigate clinical use of proton magnetic resonance spectroscopy (1H MRS) and to compare metabolic brain bioprofiles of dogs with and without hepatic encephalopathy. Animals—6 dogs with hepatic encephalopathy and 12 control dogs. Procedures—Conventional MRI and single-voxel 1H MRS were performed with a 3-T magnet. Images for routine MRI planes and sequences were obtained. Single-voxel 1H MRS was performed with a point-resolved sequence with a short echo time (35 milliseconds) and voxel of interest placement at the level of the basal ganglia. Metabolites of interest included the glutamine-glutamate complex (sum quantification of glutamate and glutamine), myoinositol, N-acetyl aspartate, total choline, and creatine. Data were analyzed with postprocessing fitting algorithm software, and metabolite concentration relative to water and ratios with creatine as the reference metabolite were calculated. Results—Compared with control dogs, dogs with hepatic encephalopathy had specific changes, which included significantly higher concentration relative to water of the glutamine-glutamate complex and significantly lower concentration of myoinositol. Choline and N-acetyl aspartate concentrations were also slightly lower in dogs with hepatic encephalopathy than in control dogs. No differences in creatine concentration were detected between groups. Conclusions and Clinical Relevance—1H MRS aided in the diagnosis of hepatic encephalopathy in dogs, and findings supported the assumption that ammonia is a neurotoxin that manifests via glutamine-glutamate complex derangements. Use of 1H MRS may provide clinically relevant information in patients with subclinical hepatic encephalopathy, equivocal results of bile acids tests, and equivocal ammonia concentrations or may be helpful in monitoring efficacy of medical management.

The Rice strain of pseudorabies virus (PRV) was intranasally instilled in pigs that were seronegative to PRV. Cells were scraped or brushed from tonsillar surfaces biweekly until pigs were euthanatized at either 10 or 16 weeks after infection. The DNA extracted from tonsillar cells or parenchyma were subjected to polymerase chain reaction analysis, using either a single set of oligonucleotide primers or nested primers from the PRV gII glycoprotein gene. Pigs became seropositive to PRV by 3 weeks after infection. The virus was isolated from the trigeminal ganglia and tonsils of pigs that were euthanatized or died 1 to 2 weeks after infection, but not from pigs that were euthanatized 10 or 16 weeks after infection. The PRV gene products were consistently detected in trigeminal ganglia and tonsils of all pigs at 1, 10, and 16 weeks after infection, and sporadically in the nasal mucosa, lymph nodes, and lungs of pigs that were euthanatized or died during the first 2 weeks after infection. Cells collected biweekly from tonsillar surfaces were mostly nucleated, squamous epithelial cells with fewer lymphocytes and neutrophils. Polymerase chain reaction analysis of DNA extracted from these cells revealed PRV DNA in a large proportion of the samples when sufficient cells were collected to provide 1 microgram of extracted DNA for use in the reaction mixtures. A second group of pigs had PRV strain 4892 intranasally instilled. The virus was isolated from tonsillar swab specimens until 3 weeks after infection. Tonsillar brushing specimens were collected biweekly until 14 weeks after infection. Some brushing specimens contained all nucleated, squamous epithelial cells, whereas other specimens contained a mixture of epithelial cells and up to 15% neutrophils, lymphocytes, and small mononuclear cells. Results of polymerase chain reaction analysis of DNA extracted from tonsillar cells collected 5, 11, and 14 weeks after infection were consistently positive for PRV gene products. Intact cells collected from tonsillar surfaces were placed in polymerase chain reaction mixtures with nested oligonucleotide primers from the PRV gII glycoprotein gene and were subjected to multiple amplification cycles. Afterward, the specificity of the amplified PRV gene products was determined by hybridization procedures, using a virus-specific oligonucleotide probe. Most nucleated, squamous epithelial cells stained positive for PRV DNA, suggesting that these cells were the primary source of PRV gene products in tonsillar brushing specimens.

Rabies virus (RV) is highly neurotropic and migrates to the neuronal soma by retrograde axonal transport from nerve terminals, after which it is taken by anterograde axonal transport to be finally released into the central nervous system (CNS) from which it disseminates, resulting in lethal encephalitis. Dorsal root ganglia (DRG) are crucial in the initial events of the infection by RV since they can act as a gate for the viral entrance into the CNS. In the present study, we examined cell tropism of RV and the roles of neuronal cytoskeletal components in the production of viral nucleoprotein (N protein) using cultured nerve cells and non-neuronal cells from DRG of newborn mice. Our in vitro study demonstrated a low propagation rate of RV in nerve cells, susceptibility of non-neuronal cells to RV, and independence of cytoplasmic synthesis of viral N protein from the neuronal cytoskeleton. The present study also suggests that Schwann cells should be considered as another possible candidate supporting RV propagation.