| Stroke often results from focal cerebral ischemia due to occlusion of a cerebral blood vessel. The severe reduction of blood flow to the affected tissue results in a lack of oxygen and nutrient transportation, which ultimately leads to tissue hypoxia and cell death. To compensate for these detrimental effects, the organism responds by trying to increase oxygen delivery to the affected tissue. One potential mechanism used to increase the oxygenation of hypoxic tissue is the induction of angiogenesis. Newly formed vessels would allow increased blood flow, thus increasing the amount of oxygen delivered to the affected tissue. It is vascular endothelial growth factor ( VEGF) and hypoxia inducible factor - 1 (HIF-1) ,two of biological factor, that we extremely pay attention to.Among the factors capable of modulating angiogenesis characterized to date, vascular endothelial growth factor ( VEGF) is the best candidate for a specific regulator of endothelial cell growth and differentiation. VEGF, also known as vascular permeability factor, is a di-meric glycoprotein that is mitogenic for endothelial cells and enhances vascular permeability.It has been shown that hypoxia is a strong inducer of VEGFmR-NA expression in many cells in vitro, in various organs including the brain in vivo and in various models of ischemia.In vitro studies have identified some mechanisms that are responsible for the increase in biologically active VEGF secreted by cells exposed to hypoxia. One is an increased transcription rate mediated by binding of HIF-1 to a hypoxia - responsive element in the 5'- flanking region of the VEGF gene. HIF-1 is a basic helix - loop - helix het-erodimeric transcription factor activated by reduced oxygen tension. HIF-1 is composed of a hypoxia - regulated a - subunit and a β - sub-unit.However, no data are available regarding the temporal kinetics and tissue distribution of these two factors after cerebral ischemia and little is known about the spatial and temporal correlation cooperativity of them.Materials and MethodsA total of 50 healthy adult male Sprague - Dawley (SD) rats , weighting 260 - 280g , randomly divided into 2 groups: ①immunohis-tochemistry group ( n = 25 ) ② Reverse Transcription - Polymerase Chain Reaction ( RT - PCR ) group ( n = 25 ) . ①- ②groups consisted of 5 groups including sham operated group,4h,8h,24h,72h, respectively ( n = 5).Transient focal cerebral ischemia (2h) was induced using in-traluminal filament occlusion of middle cerebral artery introduced by Zea Longas improved line - lock Technique in arteria.Animals were deeply anesthetized with 10% Chloral Hydrate andperfused by intracadiac puncture using 4% poly - formaldehyde -0.01MPBS(PH 7.4) . Brain were then excised and postfixted . Brain tissues were embedded in paraffin, coronally sectioned (4um).Immunohistochemistry analysis was performed to ensure the selective detection of immunoreactive VEGF and HIF-la with an antibody. Temporal, spatial, and cellular changes of immunohistochemi-cal VEGF and HIF-la expression were compared with different periods of reperfusion from 4 hour to 72 hour after transient MCA occlusion and sham opretion.Total RNA in ischemia region was isolated using TRIzol Reagent according to the manufacturer's instructions. First - strand complementary DNA was synthesized using a reverse transcription ( RT) mixture (20ul).Rat β - actin sense and antisense primers were used as controls for determining the quantity of RNA. PCR products were separated on 2% agarose gels using 7ZF ladder DNA standards as a size reference and were detected on 8 % PAGE ( polyacrylamidedel electrophoresis ) finally.Immunohistochemistry ( SABC ) was used to observe the expression of VEGF and HIF-la protein. Reverse Transcription -Polymer-ase Chain Reaction was used to observe the expression of VEGF - A mRNAs and HIF-1a mRNA. Semi -quantitative method was applied to analysis immunohistochemical cells and mRNA level. Comparisons between immunohistochemistry group and Reverse Transcription...
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