Real time quantitative gene expression analysis showed that the pro survival Tofacitinib Citrate 540737-29-9 and anti Inhibitors,Modulators,Libraries apoptotic genes have been upregulated. Both caspase 3 and TUNEL assays showed that apoptotic cell death can be reduced upon treatment with nPLA. Methods Purification of nPLA Phospholipase A2 was purified from Naja sputatrix crude venom using Sepha dex G 100 gel filtration followed by reverse phase high performance liquid chromatography. The protein fractions were characterized as described by Armugam et al and quantitated using the Bradford assay. Transient focal cerebral ischemia Male Sprague Dawley rats were obtained from the Laboratory Animal Centre and maintained on an ad libitum intake of standard laboratory chow and drinking water.
All animals were handled according to the guidelines given by the Council for International Organization of Medical Sciences on animal experimentation and the National University of Singapore guidelines for handling labo ratory animals. Animals were Inhibitors,Modulators,Libraries anaesthesized Inhibitors,Modulators,Libraries and left mid dle cerebral artery occlusion was performed as described by Longa et al. The occlusion was con firmed with the real time measurement of cerebral blood flow in the territory of the middle cerebral artery using the Laser Doppler flowmetry and the signals were digitized using a 4 channel Powerlab 4SP and recordings were dis played with Chart 5 software. Reperfusion was initiated by suture withdrawal after 60 min. nPLA was injected intravenously via the femoral vein at various times of post occlusion.
Recombinant tissue plasminogen activator was infused intravenously at a concentration of 10 g g body weight over a period of 60 min, starting 30 min post occlusion. MK801 was administered Inhibitors,Modulators,Libraries intraperitoneally in 3 doses 2. 5 g g body 30 min prior to MCAo followed by 1. 25 g g body weight at 6 and 14 hr post occlusion. Cor responding controls were treated with sterile saline and all animals were sacrificed after a total of 24 hr reperfusion period. Whole brains were rapidly removed and snap frozen in liquid nitrogen. Frozen brains were stored at 80 C until use. Consequently, there were 5 treatment groups Sham operated, Transient MCAo for 60 min and administered with 100 l saline, Transient MCAo for 60 min and administered with nPLA intravenously, Transient MCAo for 60 min and administered with tPA intravenously, Transient MCAo for 60 min and administered with MK801 intraperitoneally.
Quantitation of the ischemic infarct volume Whole rat brains were sliced coronally into 1 mm slices and incubated in 2, 3, 5 triphenyltetrazolium chloride and fixed in 10% buff ered formalin. Inhibitors,Modulators,Libraries selleck chemicals Stained brain slices were scanned and the image was analysed using Scion Image analysis software for measurement of the infarct volume. The infarct size was determined according to Engelhorn et al. His topathological changes in the brains were evaluated from paraffin embedded brain slices.