Development of a Protocol to Evaluate Neuronal Injury and Loss Following Soman-Induced Seizures Using NeuN and Fluoro-Jade C

摘要

Previously, we examined regional neuronal damage following soman- induced seizures using Fluoro-Jade (FJ), which exhibits a marked affinity for degenerating neurons. However, with FJ alone, the degree of neuronal loss cannot be ascertained. The present study was conducted to develop a protocol to double-label a single paraffin-embedded rat brain section with both Fluoro-Jade C (FJ-C) and Neuronspecific nuclear protein (NeuN) to evaluate neuronal injury. Rats pretreated with HI-6 (125 mg/kg, i.p.) were challenged with soman (1.6 LD50; 180 g/kg, s.c.) and administered atropine methyl nitrate (2.0 mg/kg, i.m.) to minimize peripheral toxic effects. All soman-exposed animals displayed prolonged convulsive behavior consistent with seizures. Brains harvested at 24 hr were paraffin-processed, sectioned, and stained with NeuN monoclonal antibody and FJ-C. The extent of neuronal injury was determined by measuring NeuN and FJ-C labeling using imaging analysis. In the piriform cortex of soman- exposed rats, FJ-C positive cells were detected in areas where NeuN immunofluorescence was lost, and pronounced necrotic lesions with depletion of NeuN staining and remnants of neuronal FJ-C fluorescence were observed. Co- localization of NeuN and FJ-C suggests that neurons are damaged, but surviving. To determine whether or not these neurons are capable of recovering, a timecourse evaluation of NeuN and FJ-C beyond 24 hr after exposure is necessary. Results indicate that double-labeling sections with NeuN and FJ-C is an effective method for assessing neuronal injury and loss.