Role of Oxidative Stress in Rabies Virus Infection of Adult Mouse Dorsal Root Ganglion Neurons

Abstract

Rabies virus infection of dorsal root ganglia (DRG) was studied in vitro with cultured adult mouse DRG neurons. Recent in vivo studies of transgenic mice that express the yellow fluorescent protein indicate that neuronal process degeneration, involving both dendrites and axons, occurs in mice infected with the challenge virus standard (CVS) strain of rabies virus by footpad inoculation. Because of the similarities of the morphological changes in experimental rabies and in diabetic neuropathy and other diseases, we hypothesize that neuronal process degeneration occurs as a result of oxidative stress. DRG neurons were cultured from adult ICR mice. Two days after plating, they were infected with CVS. Immunostaining was evaluated with CVS-and mock-infected cultures for neuron specific ␤-tubulin, rabies virus antigen, and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE) (marker of lipid peroxidation and hence oxidative stress). Neuronal viability (by trypan blue exclusion), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and axonal growth were also assessed with the cultures. CVS infected 33 to 54% of cultured DRG neurons. Levels of neuronal viability and TUNEL staining were similar in CVS-and mock-infected DRG neurons. There were significantly more 4-HNE-labeled puncta at 2 and 3 days postinfection in CVS-infected cultures than in mock-infected cultures, and axonal outgrowth was reduced at these time points in CVS infection. Axonal swellings with 4-HNE-labeled puncta were also associated with aggregations of actively respiring mitochondria. We have found evidence that rabies virus infection in vitro causes axonal injury of DRG neurons through oxidative stress. Oxidative stress may be important in vivo in rabies and may explain previous observations of the degeneration of neuronal processes.Rabies is an acute viral infection of the central nervous system (CNS) that is usually fatal in humans and animals (10). Despite its lethality, under natural conditions only relatively mild histopathological lesions are typically found in association with a paucity of degenerative neuronal changes (9, 21). Li et al. (13) showed severe destruction and disorganization of axons and disruption of synaptic structures in silver-stained hippocampal sections from mice infected intracerebrally with the pathogenic N2C strain of rabies virus. Infection with the challenge virus standard (CVS) strain of rabies virus has recently been evaluated using hindlimb footpad inoculation in adult transgenic mice expressing yellow fluorescent protein (26). In this model, dendritic beading and axonal swellings were prominent, and neuronal process degeneration appeared to account for the severe clinical disease and fatal outcome. The axonal swellings exhibited striking morphological similarities to the neurodegenerative changes that occur in the axons of neurons in diabetic sensory and autonomic neuropathy and in human immunodeficiency virus (HIV) infection (4,12,24,35). Studies of cultured adult dorsal root ganglio...

Role of Nuclear Factor-κB in Oxidative Stress Associated with Rabies Virus Infection of Adult Rat Dorsal Root Ganglion Neurons

“…the cause of this neuronal process degeneration in cultured DRG neurons because these neurons are permissively infected by CVS, which allows the degenerative effects on their axons to be studied in detail. We have previously demonstrated that oxidative stress occurs in CVS-infected cultured adult mouse DRG neurons (14). The infected neurons maintained viability and demonstrated axonal swellings containing adducts of 4-HNE without evidence of neuronal apoptosis as assessed by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining (14).…”

“…Immunostaining for adducts of 4-hydroxy-2-nonenal (4-HNE), which is associated with lipid peroxidation (and, hence, oxidative stress), is found at sites of axonal swellings. CVS infection induces oxidative stress in axons and generates abnormal axon morphology (e.g., swellings), and the degenerative changes closely mimic what is seen in dendrites and axons of CVS-infected mice (14).…”

Amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea

“…As previously reported by Kim [18] , our data demonstrate that EGCG may interrupt the early stages of the viral replication cycle. Elevated ROS production and oxidative stress play a pivotal role in exacerbation of various clinical conditions, such as those associated with malignant diseases, chronic inflammation, angiogenesis, and viral infection [14,23,24] . As intracellular free radicals, ROS may induce transient or permanent cellular alterations [25] .…”

Computational Analysis of the Effects of Antineoplastic Agents on Axonal Transport

“…For double staining with CM-DiI and mitoTracker or erTracker, the cultured cells were incubated for 30 min in L-15 medium containing CM-DiI (2 μM) and mitoTracker (500 nM) or erTracker (1 μM), fixed, and analyzed under laser-scann ing microscopy (LSM510; Carl Zeiss, Jena, Germany). Cell viability was assessed via trypan blue exclusion cell counts (12). The procedures involving the experimental animals complied with the animal care guidelines of the National Institutes of Health and the animal ethics committee of Yokohama City University.…”