NADPH-DIAPHORASE HISTOCHEMISTRY AND CYTOCHEMISTRY IN RAT BRAIN UNDER THE EFFECT OF CIRCULATING NEUROTOXINS

CRUZ-HOFLING MA *(1), RAPÔSO C. (1), ZAGO G.M. (1,2), LE SUEUR, L.M. (3), FONTANA K. (1,2), ROCHA T. (1)

(1) Depto de Histologia e Embriologia, IB, (2) Depto de Farmacologia, FCM, UNICAMP, Campinas SP, 3Dept Ciências da Saúde, UNIFESP, Santos SP – Brasil

Neurotoxins may act on specific neural circuits inducing increase or decrease of neurotransmitters release, among which nitric oxide (NO). We recently demonstrate that P nigriventer spider venom (PNV) increases the permeability of blood brain barrier in hippocampus and cerebellum of rats, and activates neuronal pathways in motor- and acute stress related areas, all of which also expressed nNOS. Here, we investigated the NADPH-diaphorase (nicotinamide adenine dinucleotide phosphate) activity as a tool to add new insights on NO involvement in the central action of PNV. Adult rats (200-250 g) received systemic injection of PNV (0.85 mg/ml) or sterile saline and were sacrificed by perfusion after 3 h, 1, 5 and 9 days (n=8). Histochemistry (light microscopy-LM) and cytochemistry (transmission electron microscopy-MET) enzymatic reaction for NADPH-d were done. LM study was performed in 10 µm thick freeze sections, and MET reaction was done on 1.5 mm width tissues blocks, both incubated at 37ºC in a medium containing the reagents. Controls of the reaction were carried out omitting NADPH. The results showed that i.v. injection of PNV increased NADPH-d in cortex, hippocampus, thalamus and hippothalamus. This increase was more remarkable at 3 h and 1 d after envenoming. The cellular distribution of NADPH-d activity was found in neurons, astrocytes, microglia, pericytes and endothelial cells. Subcellular distribution included the cytosol and a discrete reaction attached to membranes of endoplasmic reticulum, mitochondria, Golgi complex and nuclear envelope. Control rats showed very weak presence of NADPH-d activity. We conclude that the spider venom neurotoxins, some of which sodium channels activating- or delaying inactivating-neurotoxins are involved in these effects. The mechanism apparently involves humoral NO signaling through an endothelium-dependent mechanism, since the NADPH-d activity peaked at a time the venom is still systemic.

KEY WORDS: Nitric oxide, venom, NADPH-d

CORRESPONDENCE: hofling@unicamp.br

SUPPORT: FAPESP, CNPq, CAPES, FAEPEX-UNICAMP