Effects of combined prenatal stress and toluene exposure on apoptotic neurodegeneration in cerebellum and hippocampus of rats.

Pregnant Wistar rats were exposed to 1500 ppm toluene 6 hr/day from gestational day 7-20 or to chronical mild stress from gestational day 9-20 as single exposure or in combination. Behavioural, immunohistopathological, molecular biological, and neurochemical methods were applied to investigate the offspring for developmental neurotoxicity and level of apoptosis in the brain. The number of apoptotic cells in cerebellum postnatal day 22, 24, and 27 and in hippocampus (postnatal day 22, 24, and 27) were counted after visualization by the TUNEL staining or measured by DNA-laddering technique. Caspase-3 activity was determined in cerebellum (postnatal day 6, 22, 24, and 27) and in hippocampus (postnatal day 6 and 22). TUNEL staining and DNA-laddering technique showed a marked decrease in number of apoptotic cells from postnatal day 22 to 27 in both cerebellum and hippocampus. Apparently, a peak in the number of TUNEL positive cells was identified in cerebellum at postnatal day 22. There was no statistically significant influence of exposure except that DNA-laddering in cerebellum at postnatal day 27 was increased by toluene exposure. Caspase-3 activity decreased in cerebellum and hippocampus with age. At postnatal day 6 stress and toluene, when singly exposed, increased activity in cerebellum whereas co-exposure to stress and toluene did not. Stress increased caspase-3 activity in hippocampus postnatal day 22. There was overall consistency between the results obtained by the three supplementary methods regarding the influence of exposure and age on apoptotic activity in cerebellum and hippocampus. New methods to quantitate the relative level of apoptosis measured as DNA-laddering and the caspase-3 activity in tissue are presented.