Analysis of combined anti-tuberculous drug induced neurotoxicity in the cerebral cortex of adult Wistar rats by immunohistochemistry

Combined Isoniazid (INH) and Rifampicin administration still remain as two of the main drugs of choice in the treatment regimes of tuberculosis (TB) world wide, particularly in Africa, where tuberculosis incidence rates are among the highest in the world. This study evaluated the effects of chronic administration of combined Isoniazid/Rifampicin on the cerebral cortex of the adult rats by immunohistochemistry. Adult wistar rats (average weight of 220 g) maintained under standard laboratory conditions were randomly grouped into treatment and control (n = 10). The treatment group A received combined INH + Rifampicin (50 mg/kg each, i.p) while the control group B received normal saline (0.5 ml, i.p.) only; daily at 1600 h for 15 days. Animals were sacrificed by cervical dislocation for whole brain weight determination (n = 6) and by whole body perfusion following anaesthesia for neurohistology and immunohistochemistry (n = 4) from each group. Coronal samples of the frontoparietal cortex were taken from each hemisphere and processed routinely for paraffin embedding followed by Nissl staining of 6 μm thick sections. 30 μm thick sections were cryo-sectioned at -23°C, after cryoprotection in 30% sucrose/phosphate buffered saline (PBS) from the remaining samples and reacted for flourescent glial fibrillary acidic protein (GFAP) immunoreactivity and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)method for the detection of apoptosis. The treatment group showed highly reactive intracortical and subpial GFAP positive astrocytes, mostly localized as clusters, compared to the control; GFAP immunostaining intensity was quantified using Adobe Photoshop Element 6.0. Cerebral cortical lamina was well preserved in both groups, while neuronal pyknotism were not observed in the treatment group, compared to the controls. The treatment group showed intense darkly stained discrete cells, positive for the TUNEL signals. These microanatomical and microchemical changes suggest neurotoxicity of these drugs, which though was not overt clinically.   Key words: Tuberculosis, anti-tuberculous drugs, neurotoxicity, quantitative immunocytochemistry, adobe photoshop elements, astrogliosis, apoptosis, cerebral cortex, isoniazid, rifampicin.