Antioxidant protective effects of stobadine against ischemia|[sol]|reperfusion (I|[sol]|R) induced alterations of muscular and endothelial functions of rat middle cerebral arteries (MCA)

The reperfusion step during cerebral I/R is known to be responsible for the generation of free radicals which play significant role in the development of alterations of cerebral tissue and vessels. The aim of our study was to investigate the effects of stobadine (STO), a pyridolindole derivative, previously experienced in different models of pathologies for its scavenging effects towards free radicals1. Rats were ischemized for 1 h then allowed to reperfusion for 24 h. STO (1 mg/kg) or vehicle (VEH) was administered (IV) at t=0 and 5 h of reperfusion. Brain infarct volumes were quantified and MCA studied either with patch-clamp technique for KIR2.1 of smooth muscle cells (SMC), either by vasomotricity for endothelium and smooth muscle functions (Halpern myograph). STO reduced brain infarct volumes from 2389 mm3 on VEH-treated rats (n=18) to 1829 mm3 (p<0.005, n=21). KIR2.1, an inward rectifier potassium current of SMC was drastically reduced after I/R2. Tested on SMC of I/R STO-treated rats, the KIR2.1 density was significantly preserved (-1.060.17 pA/pF, n=9, p<0.05) compared to –0.480.07 pA/pF (n=6) in I/R VEH-treated rats and -1.230.11 pA/pF in SHAM VEH-treated rats (n=9). The endothelial-dependent vasorelaxation was induced by ACh (3.10–5 M) on serotonin-preconstricted (10-6 M) MCA. The percentage of diameter increase induced by ACh was 22.201.60% (n=11) on sham-VEH MCA but was significantly reduced to 12.311.42% (n=6) on I/R VEH-treated MCA. After STO treatment, endothelium-dependent vasodilation was significantly preserved on I/R MCA (20.732.63%, p<0.01, n=6). Concerning the smooth muscle dependent relaxation induced by KIR2.1 activation, stobadine pretreatment didn't allow any protection towards the I/R induced reduction of this vasodilation. Application of 15 mM KCl allowed SHAM VEH-treated MCA to relax to a 25.532.25% increase of its basal tone (n=15). Same application on I/R VEH-treated MCA induced only 5.001.35% of increase basal diameter (n=8). After STO treatment, vasodilation reached 11.452.28% (n=11) on SHAM MCA and 6.242.18% (n=8) on I/R MCA. These data revealed the potent protective effects of the antioxidant agent stobadine towards neuronal tissue (i.e. reduction of infarct size) in parallel to endothelial vascular function. Smooth muscle KIR2.1 density is also protected while the relaxation depending on is still altered. This result suggested another step between KIR2.1 activation and the triggering of the smooth muscle dependent relaxation. However stobadine appeared to be a promising tool to reveal new therapeutic strategies against cerebral ischemia.