Antemortem diagnosis and prevention of human rabies

Human rabies still continues to be a significant health problem in India and other developing countries where dogs are the major vectors of transmission. Rabies in humans can present in two clinical forms, i.e., furious and paralytic. While diagnosis of furious rabies can be made based on the typical symptoms and signs, paralytic rabies poses a diagnostic dilemma to the neurologists who may encounter these cases in their practice. Although there are certain clinical features that distinguish this disease from other forms of Guillain-Barre syndromes, confirmation of diagnosis may require laboratory assistance. Conventional techniques such as antigen detection, antibody assays and virus isolation have limited success. The recently introduced molecular techniques show more promise in confirming the cases of paralytic rabies. There has not been much success in the treatment of confirmed rabies cases and recovery from rabies is extremely rare. Therefore, preventive measures of this dreaded disease after an exposure become extremely important. The present article reviews the current status of human rabies with regard to antemortem diagnosis, disease management and post-exposure prophylaxis.     

Immunohistochemical characterization of human masseter muscle spindles

An enzyme- and immunohistochemical study has been performed on human masseter muscle spindles. Antibodies selective for different myosin heavy chain (MHC) isoforms and M-band proteins (M-protein, myomesin, and MM-CK) were used. The expression of these proteins was determined in the different intrafusal fiber types. Nuclear bag₁ and nuclear bag₂ fibers expressed predominantly slow-twitch and slow-tonic MHCs. The bag₂ fibers in addition contained fetal MHC. Nuclear chain fibers coexpressed embryonic, fetal, and fast-twitch MHCs. The bag₂ and chain fibers contained all three M-band proteins, whereas the bag₁ fibers contained only myomesin. In general the MHC expression in the human masseter intrafusal fiber types was similar to that previously reported for limb muscles in man as well as for limb and masseter muscles in other species. However, the number of intrafusal fibers per spindle was unusually high (up to 36). This reinforces the idea that masseter muscle spindles have a strong proprioceptive impact during the control of jaw movements. © 1994 John Wiley & Sons, Inc.     

Immunohistochemical staining of the human forebrain with monoclonal antibody to human choline acetyltransferase

Monoclonal antibody to human choline acetyltransferase (ChAT) was successfully produced from a mouse hybridoma cell line. The antibody was found to be of the IgM molecular species. By using this monoclonal antibody, immunohistochemical staining for ChAT was obtained on human brain sections. Only large sized cells were stained in the putamen and the substantia innominata. The specificity of the staining was comparable to that with polyclonal rabbit antibody to human ChAT produced by standard immunization procedures. No staining was observed when mouse monoclonal antibodies prepared against other human or bacterial antigens, or when normal mouse IgM, was employed.     

Non–rabies Lyssavirus human encephalitis from fruit bats: Australian bat Lyssavirus (pteropid Lyssavirus) infection

A 39-year-old woman died of encephalitis a few weeks after being scratched by fruit bats. Autopsy disclosed meningoencephalomyelitis, and revealed neuronal intracytoplasmic inclusions which had similarities to Negri bodies of rabies. Laboratory investigations detected a Lyssavirus type previously identified only in fruit bats. This appears to be the first human case of encephalitis due to this Lyssavirus type.     

Immunohistochemical localization of carbonic anhydrase isoenzyme C in human brain

Carbonic anhydrase isoenzymes of human brain were examined by the immunoperoxidase method. Only the catalytically highly active isoenzyme C was found in normal cerebral and cerebellar tissues, being located in a limited number of non-neuronal cells interpreted as oligodendrocytes and in myelinated nerve fibres. The enzyme was not evident in the glial cells of astrocytomas.     

Rabies viral antigen in extracranial organs: a post-mortem study

Rabies is a communicable disease and a significant health hazard. Histopathological confirmation of the diagnosis depends on the demonstration of Negri bodies - characteristic intracytoplasmic inclusions. In cases where these are not seen, immunohistochemistry serves as a useful adjunct. After its establishment in the central nervous system, the rabies virus is known to reach peripheral organs by a centrifugal spread. The present study was undertaken with the aim of demonstrating rabies viral antigen (RVAg) in the extracranial organs. Eleven confirmed cases of rabies were analysed and RVAg was found in the adrenal glands, heart, gastrointestinal tract and pancreas, confirming the centrifugal spread of the virus. The detection of RVAg in the extracranial sites may serve as a useful tool in the ante-mortem diagnosis by subjecting the extracranial tissue to biopsy and subsequent immunohistochemistry.     

Immunohistochemical localization of creatine kinase BB-isoenzyme in human brain: Comparison with tubulin and astroprotein

The distribution of creatine kinase BB-isoenzyme in the human central nervous system (CNS) was investigated immunohistochemically and the findings were compared with the distributions of tubulin and astroprotein. Creatine kinase BB-isoenzyme existed both in neurons and astrocytes, and was universally distributed within the CNS. Tubulin was visualized only in the neuronal elements, namely perikarya, dendrites and axons, while astroprotein was exclusively visualized in astrocytes. A combination of immunohistochemistry for structural and soluble proteins may be a useful tool for the investigation of pathophysiological conditions within the CNS.     

Amyotrophic choreo-acanthocytosis: A neuropathological and immunocytochemical study

A 47 year old man, one of a sibship affected by amyotrophic choreo-acanthocytosis was studied neuropathologically after some years of clinical observation. Besides the classic optical findings (neuronal loss, astrocytic gliosis and “status spongiosus” in the basal ganglia, namely in the caudate nucleus) a few MEnk+ and NPY+ neurons were observed immunocytochemically in the striatum. In the spinal cord also, while no neuronal loss was perceivable, both mild demyelination and interfibrillary astrocytic hyperplasia of the long tracts were present. On the other hand, microscopic findings of muscle and peripheral nerve showed no differences from what was previously intra-vitam appreciated in the same patient. The neuropathological and immunocytochemical findings of this case are discussed in relation to the differential diagnosis between amyotrophic choreoacanthocytosis and Huntington's disease. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Lymphocyte subsets in human encephalitic and paralytic rabies

Lymphocyte subsets of 7 patients with encephalitic and paralytic rabies were determined by immunocytochemical techniques using mouse monoclonal antibodies. Almost all patients had diminished mononuclear cells of Leu 7 phenotype (natural killer cells). Cells of Leu 12 marker (B cells) were decreased in 3 paralytic rabies patients compared with those of 4 patients in the encephalitic group.     

Immunohistochemical study of the early human fetal brain

Summary To assess the cytogenesis of the central nervous system we studied the spinal cord and the cerebrum in 11 human embryos and fetuses of gestation age 7–25 weeks immunohistochemically using anti-vimentin, anti-neurofilament protein (NFP), anti-neuron-specific enolase (NSE), anti-glial fibrillary acidic protein (GFAP), anti-S-100 protein, anti-Leu 7 and anti-myelin basic protein (MBP) antibodies. Vimentin was demonstrated in ventricular cells at 7 weeks and older. NFP-68-kDa and-160-kDa components were observed in neuroblastic cells of the neural tube at 7 weeks. NFP (68 and 160 kDa) was mainly located in the marginal zone of the spinal cord and the cerebrum at 8–9 weeks. NSE was not found in the neural tube at 7 weeks, although NSE was demonstrable at 9 weeks both in the spinal cord and in the cerebrum. GFAP-positive cells started to appear at 9 weeks in the spinal cord and at 15 weeks in the cerebrum, respectively. S-100 immunoreactivity was almost coincident with GFAP. S-100, however, was observed in more numerous glioblastic cells. Leu 7 was detected at 7 weeks and located in the neuropil of the central nervous tissue. MBP was not demonstrable in this study. Our study indicates that neuronal differentiation occurs much earlier than glial differentiation in the human brain and that neuronal and glial cell classes do not coexist in the ventricular zone of the early human fetal brain.     

Immunohistochemical localization of γ-enolase in early human embryos

γ-Enolase has been believed to be distributed only in the neurons and it was frequently labelled as neuron-specific enolase. However, recent precise studies have suggested a wider distribution of the protein. It can also be found in neuroendocrine cells, some mesodermal tissues, and some malignant tumors originating from tissues without the antigen in a normal condition. In early rat embryos, before the formation of neural tissues, a sensitive immunoassay system revealed a substantial amount of γ-enolase, though it is not yet clear where the antigen is located. In the present study, tissue distribution of γ-enolase in early human embryos was studied using an immunohistochemical method and it was suggested that the protein is present not only in neural tissue primordium but also in most tissues in the youngest embryo of 6.3 mm crown-rump length. Many of the immunoreactive non-neural tissues, however, lost immunoreactivity with the advancement of the embryonic stage while neural tissues became more intensely stained. In the embryo of 24 mm length, the staining pattern was almost the same as that of reported adult men. In embryonic tissues such as notochord and mesonephros, which disappear in the due course of growth, the antigen was also found. These findings will suggest that the antigen is rather common in undifferentiated tissues but then localizes in neural elements with advancing age. Our findings may be useful in explaining why early rat embryos, before the formation of neural tissues, showed a considerable amount of γ-enolase and why many undifferentiated tumors, originating from tissues which did not have the antigen in normal condition, revealed the antigen.     

Pathology of rabies: A light- and electron-microscopical study with particular reference to the changes in cases with prolonged survival

Summary Light-and electron-microscopical studies were conducted on necropsy material from six cases of rabies encephalitis including three with the unusual feature of surviving for over 14 days as a result of intensive medical care. This included administration of antiviral agents and interferon inducers and prevention of hypoxia by intermittent positive pressure ventilation. In all these cases, typical Negri bodies were demonstrated. Inflammatory reaction was absent or minimal. Unlike the cases with short survival where Negri bodies were infrequently seen and restricted mostly to the hippocampus, in cases with prolonged survival, they were present in large number, widely distributed throughout the grey matter of the brain. The associated inflammatory reaction in these cases, however, did not keep pace with the increase in number of inclusion bodies. Peripheral neuritis was observed in two of these cases, which also showed myelitis involving the cervical region and inflammation of dorsal root ganglia. One of them showed necrosis and severe inflammation of the lower cervical sympathetic ganglion. An electron-microscopical study conducted in four cases showed three forms of the inclusion body in the cytoplasm of neurons.     

Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study

Two types of cannabinoid receptors have been characterized so far, CB1 and CB2. While CB1 receptors are present both in the CNS and in the periphery, CB2 receptors showed an almost exclusive distribution within the immune system. We now report that CB2 receptors are present in a specific microglial cell type of the human cerebellum. Thus, we have performed immunohistochemical analysis of tissue sections of white matter areas of the human cerebellum and detected the presence of CB2 receptors in perivascular microglial cells. These findings match with the well-known immunomodulatory role of CB2 receptors and open new perspectives on the possible role that these receptors may play in pathophysiological events.     

HIV-associated brain pathology: a comparative international study

J. Davies, I. P. Everall, S. Weich, J. Glass, L. R. Sharer, E. S. Cho, J. E. Bell, C. Majtenyi, F. Gray, F. Scaravilli and P. L. Lantos (1998) Neuropathology and Applied Neurobiology 24, 118–124
HIV-associated brain pathology: a comparative international study
Little is known about the frequency and variation of HIV-associated brain pathology in different geographical centres. To assess whether there is an association between the frequency of disease and demographic factors we examined the neuropathological findings in four European and two American cities. The cities included London, Edinburgh, Paris, Budapest, Baltimore and Newark. Information was collected on a total of 1144 cases. HIV encephalitis was the most common observation in all the centres, although its frequency varied between them ( P < 0.01). Furthermore, there were significant differences ( P < 0.001) between the various categories of exposure and the frequency of HIV encephalitis in Edinburgh and other centres. The occurrence of toxoplasmosis, progressive multifocal leukoencephalolpathy (PML) and cryptococcal infection also differed between the various centres ( P < 0.01). None of the findings was attributable to age, sex, or ethnic origin, but the introduction of anti-retroviral treatment, such as Zidovudine, may have been important. Overall, this study highlights geographical variability and the potential importance for group of exposure and anti-retroviral medication as factors affecting the development of various HIV-associated brain lesions.     

Differential localization of GABAA receptor subunits within the substantia nigra of the human brain: an immunohistochemical study

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.     

Expression of peripherin in the brain of macaques (Macaca mulatta and Macaca fascicularis) occurs in astrocytes rather than neurones and is associated with encephalitis

Peripherin is a member of the type III intermediate filament family, expressed in neurones of the peripheral nervous system of many species and in a discrete subpopulation of neurones of the central nervous system (CNS) during early development in rodents. Previous studies on rats have shown that peripherin immunoreactivity increased significantly in cell bodies of spinal motor neurones following axonal injury. Our study examined the expression of peripherin in the cerebrum of normal macaques (Macaca mulatta and Macaca fascicularis) and those with encephalitis of viral (simian immunodeficiency virus and simian virus 40) or autoimmune (experimental allergic encephalomyelitis) aetiology. Immunohistochemistry, immunoelectronmicroscopy, immunofluorescence and confocal microscopy were performed on tissue sections using antibodies against cell-specific markers and peripherin. Peripherin-positive cells were absent in the cerebrum of normal macaques of all ages examined, whereas animals with encephalitis had peripherin-positive cells associated with inflammatory infiltrates. Further evaluation revealed that these peripherin-positive cells were not neurones, but were predominantly astrocytes expressing glial fibrillary acidic protein. Our study suggests that peripherin is not neurone-specific in the CNS of macaques; peripherin is expressed in astrocytes of animals with encephalitis.     

Immunohistochemical localization and dilatatory effect of substance P on human cerebral vessels

Ornithine aminotransferase (Orn-T) activities in Huntington's disease (HD) brains were found to be reduced, when compared to age-matched control brains, by 34-49% in the frontal cortex, parietal cortex, caudate nucleus and putamen. Such changes were not observed in senile dementia of Alzheimer type or schizophrenia. Alterations in choline acetyltransferase activities were consistent with previous findings for these disorders. If Orn-T is involved in the synthesis of neurotransmitter glutamate, the reported losses of Orn-T activity may reflect deterioration of the corticostriatal glutamatergic neurons in HD.     

Hemangioblastomas: Immunohistochemical and ultrastructural study of the stromal cells

An immunohistochemical study of 36 hemangioblastomas (Hmbl) from 32 patients was performed to clarify the cytogenesis of stromal cells (SC). In 19 of 29 Hmbl, SC revealed glial fibrillary acidic protein-immunolabeling with an antigen retrieval by trypsinization, and were also positive for S-100 protein, αB crystallin, neuron-specific enolase and vimentin. Ultrastructurally, SC contained lipid droplets, unevenly distributed intermediate filaments with occasional myelin figures, primitive desmosomal junctions and, although rare, Rosenthal fibers or a cilium. It is concluded that SC associated with Hmbl exhibit markers consistent with an astrocytic origin.     

Glycine receptors in the striatum, globus pallidus, and substantia nigra of the human brain: an immunohistochemical study

Glycine receptors (GlyRs) are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian spinal cord and brain. GlyRs have four functional subunits, alpha1-3 and beta, which likely exist in heteromeric alphabeta combinations. Mutations in GlyR alpha1 and beta subunits are well known for their involvement in hyperekplexia, a paroxysmal motor disorder. In this study we present the first detailed immunohistochemical investigation at the regional, cellular, and subcellular levels of GlyRs in the human basal ganglia. The results show that GlyRs are present at the regional level in low concentrations in the striatum and globus pallidus and are present in the highest concentrations in the substantia nigra. At the cellular level, GlyRs are present only in discrete populations of neurons immunoreactive for choline acetyltransferase (ChAT), parvalbumin, and calretinin in the human striatum, on a subpopulation of parvalbumin- and calretinin-positive neurons in the globus pallidus, and in the substantia nigra GlyRs are present on approximately three-fourths of all pars compacta and one-third of all pars reticulata neurons. They also form a distinct band of immunoreactive neurons in the intermedullary layers of the globus pallidus. At the subcellular level in the substantia nigra pars reticulata (SNr), GlyRs show a localized distribution on the soma and dendrites that partially complements but does not overlap with the distribution of gamma-aminobutyric acid (GABA)A receptors. Our results demonstrate the precise cellular and subcellular localization of GlyRs in the human basal ganglia and suggest that glycinergic receptors may play an important complementary role to other inhibitory receptors in modulating cholinergic, dopaminergic, and GABAergic neuronal pathways in the basal ganglia.