engleski

Expression and activation of ERK and JNK in the optic nerves of rats exposed to global cerebral ischemia

Effects of the global cerebral ischemia on the ERK and JNK expression and activation signals in the optic nerves of rats exposed to global cerebral ischemia were studied. Animals were exposed to global cerebral ischemia (20 min duration) and after different reperfusion periods (5 and 10 min ; 1 ; 6 and 12 h after ischemia), ERK and JNK expression and activation signals were determined. Transient global cerebral ischemia was induced by the four-vessel occlusion method described by Pulsinelli and Buchan (1988). The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and their vertebral arteries were cauterized bilaterally with a bipolar coagulator (Geiger cautery Unit Model 100). Threads were then placed around the common carotid arteries, but carotid blood flow was not interrupted. The rats recovered for 24 hrs after the surgery, were placed on TSE operating table with temperature control (Model No 908100-OPT-R/MH). Their common carotid arteries were exposed by pulling the threads under light anesthesia with ether. After recovery from anesthesia, the carotid arteries were occluded with aneurysm clips during a period of 20 min. Rats that had undergone vertebral artery cauterization and carotid artery occlusion and that had lost their righting reflexes during the period of ischemia were assigned to the ischemic group. Body temperature was continuously measured and maintained at 37°C by external heating. Total protein (35 ug) (determined by Bio Rad protein assay dye reagent) was loaded for each sample onto a 12% polyacrylamide gel usually runat 100 V (Bio Rad model 1000/500), Bio rad Laboratories). Transfer onto nitrocellulose (0.2 µ ; ; ; ; m ; Bio Rad Laboratories) was conducted at 250 mA for 90 min. Membranes were probed with anti-ERK/JNK and anti-phospho ERK/JNK antibody (diluted 1:5000 in blocking buffer) overnight at 4 C. A α -rabbit horseradish peroxidase-conjugated secondary antibody (diluted 1:4000 in 5% low fat milk in TBS+T) was utilized to allow detection of the appropriate bands using enhanced chemiluminescence reagent and film Three animals in each experimental group were used to analyse the levels of proteins by Western blot. Three samples derived from three animals in each experimental group were run on the same gel. The expression levels of JNK in the optic nerves increased with the time with a striking increase after 12 h while expression of ERK remained constant. The levels of phosphorylated ERK and JNK increased with time during the first hour of reperfusion. After 6 h, the amount of phosphorylated ERK and JNK had returned to control levels. The levels of both kinases appeared to return to peak levels after 12 h of reperfusion in the second wave of kinase activation. Our results indicated that expression and activation of ERK and JNK were implicated in the early and the later periods of reperfusion injury in optic nerves of global cerebral ischemia exposed rats.

ERK; JNK; optic nerve; global cerebral ischemia; rat

nije evidentirano