Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resinembedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.Rabies is an acute viral infection of the central nervous system for which there is no effective antiviral therapy in humans (14). Despite its lethality, only relatively mild histopathological lesions are typically found under natural conditions, leading to the concept that neuronal dysfunction, rather than structural neuronal injury or cell death, is responsible for the clinical features and fatal outcome (8, 13). However, mechanisms of neuronal dysfunction in rabies virus infection are still not understood. Studies have investigated ion channel dysfunction (12), abnormalities in neurotransmitters (2, 3, 5), and electroencephalographic changes (9, 10) in rabies virus infection, but no consistent abnormality has been identified. Both nitric oxide neurotoxicity and excitotoxicity have been evaluated with experimental animal models of rabies, but no important role has yet been established for these mechanisms (13,27). Li and colleagues (19) have suggested that the degeneration of neuronal processes and disruption of synaptic structures may for...

Cultures derived from the cerebral cortices and hippocampi of 17-day-old mouse fetuses infected with the CVS strain of rabies virus showed loss of trypan blue exclusion, morphological apoptotic features, and activated caspase 3 expression, indicating apoptosis. The NMDA (N-methyl-D-aspartate acid) antagonists ketamine (125 M) and MK-801 (60 M) were found to have no significant neuroprotective effect on CVS-infected neurons, while the caspase inhibitor Ac-Asp-Glu-Val aspartic acid aldehyde (25 M) exerted a marked neuroprotective effect. Glutamate-stimulated increases in levels of intracellular calcium were reduced in CVS-infected hippocampal neurons. Ketamine (120 mg/kg of body weight/day intraperitoneally) given to CVS-infected adult mice produced no beneficial effects. We have found no supportive evidence that excitotoxicity plays an important role in rabies virus infection.

Minocycline is a tetracycline derivative with antiapoptotic and anti-inflammatory properties, and the drug has been shown to have beneficial effects in a variety of models of neurological disorders. The potentially neuroprotective role of minocycline was assessed in experimental in vitro and in vivo models of rabies virus infection. In this study, 5 nM minocycline did not improve the viability of embryonic mouse cortical and hippocampal neurons infected in vitro with the attenuated SAD-D29 strain of rabies virus, based on assessments using trypan blue exclusion. Two-day-old ICR mice were inoculated in the right hind limb thigh muscle with SAD-D29, and they received daily subcutaneous injections of either 50 mg/kg minocycline or vehicle (phosphate-buffered saline). Infected minocycline-treated mice experienced an earlier onset of neurologic signs and greater mortality (83% versus 50%) than those receiving vehicle (log rank test, P ‫؍‬ 0.002 and P ‫؍‬ 0.003, respectively). Immunohistochemical analysis of rabies virus antigen distribution was performed at early time points and in moribund mice. There were greater numbers of infected neurons in the regional brain areas of minocycline-treated mice than in vehicle-treated mice, which was significant in the CA1 region of the hippocampus. There was less apoptosis (P ‫؍‬ 0.01) and caspase 3 immunostaining (P ‫؍‬ 0.0008) in the midbrains of mice treated with minocycline than in mice treated with vehicle, consistent with a neuroprotective role of neuronal apoptosis that may have had a mild effect of inhibiting viral spread. Reduced infiltration of CD3 ؉ T cells was observed in the pons/medulla of moribund mice that received minocycline therapy (P ‫؍‬ 0.008), suggesting that the anti-inflammatory actions of minocycline may intensify the neurologic disease. These findings indicate that minocycline has important detrimental effects in the therapy of experimental rabies. Empirical therapy with minocycline should therefore be approached with caution in cases of human rabies and possibly other viral encephalitides until more experimental data become available.New therapeutic agents are needed for the therapy of rabies encephalitis in humans. Despite initial enthusiasm about therapy with the anesthetic agent ketamine (6), recent experimental (19) and clinical (4) evidence have raised questions about the therapeutic usefulness of this agent in human rabies. A combination of therapeutic agents is a reasonable approach for consideration in the treatment of rabies as it is used for other viral infections and diseases (6). Minocycline is a tetracycline derivative with antiapoptotic and anti-inflammatory properties, and the drug has been shown to have beneficial effects in a variety of neurological disorders (21). The efficacy of minocycline therapy has also been reported for a variety of experimental models of viral infections in animals, including neuroadapted Sindbis virus infection of mice (2), reovirus infection of neonatal mice (14), and simian immunodeficiency virus infection of...