| Background and ObjectiveIschemia-reperfusion injury(IRI) refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia. It is a common clinical pathophysiological phenomenon. Many of diseases can occur ischemia-reperfusion injury, such as:coronary heart disease, cerebral infarction, artery bypass grafting, percutaneous transluminal coronary angioplasty, thrombolytic therapy and other ischemic. In ischemic cerebrovascular disease, how to decrease reperfusion injury is one of the hotspots.GH (growth hormone, GH) is a protein hormone secreted by the pituitary acidophilic cell, which is made of the 191 amino acid non-glycosylated protein. GH is not only involved in brain growth and development, but also a neuroprotective factor. Study shows that GH plays its biological activity directly or through insulin-like growth factor (IGF). GH may inhibit apoptosis, promote angiogenesis, reduce inflammation and promote neural stem cell proliferation and differentiation in vitro. Nowadays, there are a lack of study on GH in apoptosis, angiogenesis arid the expression of Nestin in cerebral ischemia-reperfusion injury. Object to provide to theory evidence for clinical application of GH in our study. In order to explore the neuroprotective effect of GH, we calculated the number of hippocampal apotosis cells by TUNEL method, observed Nestin-positive cells in hippocampus and in ischemic peripheral zone, counted CD34-positive new vessels by immunohistochemistry, and the TTC staining cerebral ischemia.Materials and methods1 Experimental animal grouping and the administration methods90 healthy adult male SD rats, weighing 250-280g (from the Experimental Animal Center of Zhengzhou University) were randomly divided into three groups, the sham operation group (n=30), the control group (cerebral ischemia-reperfusion+ saline group, n=30) and the treatment group (cerebral ischemia-reperfusion+ recombinant human growth hormone group, n=30). Each group was divided into 7d, 14d,21d subgroups after surgery. The treatment group was given subcutaneous injection of rhGH, while the control group and the sham group was given normal saline by subcutaneous injection.2 Prepariation of the tissue sectionAfter the successful models were decapitated, the rats were perfused in the corresponding time points. The brain tissue was then conventionally fixed, dehydrated, embedded in paraffin, made into 4μm-thick slices, respectively count the number of apoptotic cells, TTC staining, Nestin and CD34 immunohistochemical staining.3 Detection of apoptotic cells, CD34-positive vessels and cerebral infarction volume and expression of Nestin(1) TUNEL method:Calculate the number of apoptotic cells.(2) Nestin-positive cells and CD34-positive new vessels were detected by immunohistochemical staining.(3) TTC staining:TTC assay showed cerebral infarction area, sketching the outline of cerebral infarction by Ulead Photo Express 2.0 image analysis software, cerebral infarction was measured by forensic injury measurement software, the approximate volume of cerebral infarction can be obtained through all cerebral surface areas multiplying thickness of slices.(4) Image Analysis Each rat's cerebral tissue was cut into serial sections. One section was drawed from every four serial sections, which was choosen 5 different regions to observe with high-power microscope (400×). We used image analysis system to quantitatively analyze integral optical density of the immune staining positive area, count apoptotic cells and measure infarct size.4 Statistical MethodsAll the data were expressed as x±S and were analyzed with variance ANOVA and t-test by SPSS 13.0 software. A probability value of a=0.05 was considered statistically significant.Results1 TUNEL method to calculate changes in the number of apoptosisNumbers of apoptosis were counted respectively at 7d,14d,21d in sham operation group, they were 9.34±1.41,6.63±1.08,5.16±0.76, The number is no significant change. The control group, numbers of apoptotic nerve cells at each time points were 70.98±1.99,61.85±1.86,46.23±2.13; The treatment group were 59.92±1.92,48.36±2.30,40.76±1.94. In the corresponding point, the number of apoptotic cells in the treatment group were significantly reduced comparing with control group, the difference of which was statistically significant (P<0.05).2 Nestin expression dynamicsThere were a small amount of expression of Nestin-positive cells in the sham-operated group. Nestin expression of the treatment group were 53.18±1.99, 39.26±1.75,31.01±1.67 at 7d,14d,21d; The control group were 35.12±1.94, 29.76±1.93,27.66±1.58. The treatment group was higher than the control group in the corresponding time points, and the difference was statistically significant (P<0.05).3 CD34 marked changes in angiogenesisThere were only a small amount of new vessels in the sham-operated group. There were12.72±1.05,8.61±1.01,7.47±1.37 in the control group at 7d,14d,21d. In the 7d, there were largest number of new vessels, which were reduced over time. The treatment group were 15.43±0.69,13.52±1.37,11.65±1.08 at each time points, but the numbers of the treatment group were higher than that in the control group at the corresponding time points, and the difference was statistical significance(P<0.05). 4 TTC staining and brain morphological changesNo infarct was stained in the sham-operated group. At different time points, the volume of cerebral infarction in the treatment group was significantly reduced compared with the control group at the corresponding time points.Conclusions1.GH has protected effects on cerebral ischemia-reperfusion, which could reduce infarct size by inhibiting cell apoptosis.2.GH can increase the number of neovessels and the number of Nestin-positive cells.
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