| Insulin-like growth factor 1 (IGF-1)is a kind of polypeptide and involved in many physiological functions, such as cell proliferation,differentiation and maturation of organic tissues. It is named as IGF because its structure has high homogeneity with insulin prosome. Almost all the tissue can excrete IGF-1 and express IGF-1 receptor. In the physiological condition there are extensive IGF-1 immunoreactivities and IGF-1 mRNA expression in brain. It was found that IGF-1 and IGF-1R extensively existed in central nervous system, especially there were higher expression in hypophysis, then in olfactory bulb and superior brain stem, and followed by cerebrum, striatum, hippocampus and cerebral cortex in order. The normal brain in mature can express lower IGF-1 protein and both neurons and neuroglial cells can express IGF-1 protein. It has been demonstrated that IGF-1 is a kind of neurotrophic factor and has various functions. It can not only promote the differentiation and proliferation of neurons and neuroglial cells in brain, but also accelerate the growth and development of brain. It also protects cerebrum from ischemia reperfusion injury. It is believed that IGF-1 play an important role in the pathophysiological process of cerebral infarction. After cerebral ischemia, the IGF-1 system was activated, its expression and distribution in brain were obviously changed, and its protective actions were activated at same time. In clinical reports, it has been reported that IGF expression is correlated with the attack of ischemic stroke.IGF-1 dripped via nasal cavity or intravenous injection could decrease the expression of caspase-1 in brain after hypoxic ischemic injury, and could protect the brain from damage. The mechanism of protecting brain from damage of IGF-1 is not very clear. IGF-1 could adjust forkhead like1 through the phosphatidylinositol-3-kinase/serine/threonine kinase pathway, inhibit caspase-3 activity, restrain apoptosis of neurons and neuroglial cells or change the expression of Bcl protien or increase the level ofγ-aminobutyric acid, restrain aminobutyric acid and reduce metabolic rate of neuron in ischemic infarcs area and protect the brain.Magnetic therapy has a long history and is used in the treatment of various diseases. It possesses certain function of protecting brain ischemia reperfusion injury through holding up the pathophysiological development of brain ischemia injury, but the mechanism are not very clear. In animal experiment, magnetic field exposure can improve rat hemodynamic property, increase antioxidase activity, improve organic anti-oxidation capability, prevente free radicals, adjust the intra-cellular calcium content, inhibit the cell apoptosis and reduce neural damage. In clinical research, Magnetic therapy may reduce disability resulting from stroke, and promote neurological and functional improvement. Magnetic therapy can be recognized as a safe method and worth making popular in clinical practice. The experiment is aimed to study the effect of pulsed magnetic field in inducing IGF-1 expressing and the influence on cerebral infarct volume, pathological changes in focal cerebral ischemica reperfusion rat, for providing new theoretic foundation for magnetic therapy.Buyanghuanwu decoction(BYHWD) is the classical prescriptions for hemiplagia differentiated as qi deficiency and blood stasis created by Wang Qingren,the famous doctor in Qing dynasty in "Yi LingGai Chuo". It has the ability of invigorating qi and activating blood circulation. In all the ingredients of BYHWD, the ingredients invigorating qi act as host, and the ingredients activating blood circulation act as supplement. Many scientists have studied the mechanism of BYHWD and its decomposed formulas on cerebral ischemia. They have demonstrated that BYHWD can improve hemodynamic property, increase antioxidase activity, resist free radicals, decrease the intra-cellular calcium overload, etc. inhibit the cell apoptosis and reduce neural damage. There were no reports about the relationship between BYHWD and IGF-1. In this experiment, cerebral ischemia reperfusion model will be prepared for the observation of the effect of BYHWD on the expresion of IGF-1 in brain tissue, and of the influence of BYHWD on neurologic deficit, cerebral infarct area, pathological changes of brain tissue, to provide new breadboard foundation for BYHWD.Objective:To observe the effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rat, providing new breadboard foundation for magnetic therapy and BYHWD.Design: Randomized controlled animal experiment.Methods: 1. Forty SD rats were randomized into 5 groups, namely, sham-operation group, 2h group, 1d group, 3d group and 7d group with 8 in each group. MCAO method was employed to establish focal cerebral ischemia reperfusion model in 2h group, 1d group, 3d group and 7d group, they were occluded for 2h at the right middle cerebral artery, and then they were reperfused, but 8 rats was only tied up without occlusion in sham-operation group. The rats in the experimental group were assessed according to Zeal Longa standard after 2h reperfusion. Without neurologic deficit was marked as 0 point, flexion of left anterior claws when hanging tail as 1 point, circling to left while walking as 2 points, difficulty in walking and falling down to left as 3 points, unconscious mind as 4 points. The rats assessed 1 to 4 point were successful MCAO rats. 32Rats were successfully MCAO. After that, brain tissues were harvested at Oh (sham-operation group), 2h, 1d, 3d and 7d after cerebral ischemic reperfusion injury, 40 rats at each time point. Main outcome measure: IGF-1 expression in the brain tissue was observed by immunohistochemical method.2. Eighty SD rats were randomized into 5 groups, namely, sham-operation group, model group, pulsed magnetic field group, BYHWD group and pulsed magnetic field combined BYHWD group (combination group) with 16 in each group. Also MCAO method was employed to establish cerebral ischemia reperfusion model in model group, pulsed magnetic field group, BYHWD group and combination group. Rats was only tied up without occlusion in sham-operation group. BYHWD was applied by gastric perfusion at the dosages of 13.3g/kg.d for 7d in BYHWD group and combination group. Rats in pulsed magnetic field group and combination group were exposed to 0 to 0.01T intensity pulsed magnetic field of 50 Hz for 20 minutes per day for 7d. Rats in sham-operation group and model group only eat food and drink freely. The rats in the experimental group were assessed according to Zeal Longa standard after 2h ischemia and 1, 3, 7 d reperfusion respectively. Main outcome measure:①Neurologic deficit test.②Infarct area was detected by TTC staining.③Pathological changes were observed microscopically in HE stained sections.④IGF-1 expression in the brain tissue was observed by immunohistochemical method. Statistical management was carried out with SPSS13.0 software. Data were presented as Mean±SD, Repeated Measures and one-way ANOVE were used.Results: 120 rats were involved in the final analysis without deletion. 1.Expression of IGF-1 in cerebral ischemica reperfusion ratsOne-way ANOVE were used. There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of 2h group were more than sham-operation group (P=0.000) . The IGF-1 positive cells in 1d group were more than sham-operation group and 2h group (P=0.000) . The IGF-1 positive cells of 3d group were significantly more than all other group (P=0.000) . There were no significant difference between 7d group and 1d group (P=0.712) , but the IGF-1 positive cells of 7d group were more than sham-operation group and 2h group (P = 0.000) .2. The effect of pulsed magnetic field and BYHWD in inducing IGF-1 expressing and the influence on neurologic deficit, cerebral infarct area, pathological changes in focal cerebral ischemica reperfusion rats2.1 The neurologic deficitThe scores of neurologic deficit of sham-operation group were 0 point all the time. In othe four groups, repeated measures were used. There were significant difference in the scores of neurologic deficit at 2h, 1d, 3d and 7d (F=106.922, P=0.000) . The scores of neurologic deficit of model group, pulsed magnetic field group and BYHWD group were keeping decreasing all the time. There were significant difference in model group, pulsed magnetic field group , BYHWD group and combination group (F=5.479, P=0.032) .At 2h and 1d there were no significant difference in all the groups(F=0.760, P=0.521; F=1.538, P=0.214). After 3 to 7day reperfusion the scores of neurologic deficit of pulsed magnetic field group and BYHWD group were lower than that of model group ((F=12.612, P=0.000; F=4.609, P=0.002).2.2 Cerebral infarct areaOne-way ANOVE were used. There were no infarcts in sham-operation group. The infarct area of pulsed magnetic field group and BYHWD group were significant less than that of model group (P = 0.000) . There were no significant difference between pulsed magnetic field group and BYHWD group (P=0.552). The infarct area of combination group were significant less than that of pulsed magnetic field group and BYHWD group (P=0.009, P=0.035) .2.3 Pathological changesIn the sham-operation group, the morphology of neurons were ovale or multi-angles in shapes, Nissl bodies were stained as dark blue. So there were no obviously changes in sham-operation group. In model group, many neurons disappeared in the center of the infarcts, the nuclei of the remaining neurons were pyknotic and the neural fibers became vacuolar. There were no Nissl bodies. Many neurons became pyknotic and deep-stained around the infarcs. Neuron defects in the infarcted center of pulsed magnetic field group, BYHWD group and combination group were significantly slighter than that of model group. The morphology of many neurons were also ovale or multi-angles in shapes, Nissl bodies still existed and were stained as blue. A few neurons became pyknotic and deep-stained around the infarcs.2.4 Expression of IGF-1One-way ANOVE were used.There were a few IGF-1 positive cells stained lightly in the sham-operation group. The IGF-1 positive cells of model group were more than sham-operation group (P = 0.007) .The IGF-1 positive cells of pulsed magnetic field group and BYHWD group were more than model group (P = 0.000) . The IGF-1 positive cells in combination group were more than pulsed magnetic field group and BYHWD group (P = 0.000) .Conclusion: The expression of IGF-1 was very low in normal brain tissues. The positive cells of IGF-1 higher expressions maintained from 2h to 7d after cerebral ischemia reperfusion, and reached the peak value at 3d, then decreased at 7d, but maintained a higher level than normal brain tissues. It suggests that cerebral ischemia reperfusion injury can enhanced expression of endogenous IGF-1, but the expression are limited and last for shorter durition. After cerebral ischemia reperfusion the neurological function is damaged, and it has a trend of self-recovery, but self-recovery are very weak. Pulsed magnetic field and BYHWD can promote recovery of neurological function deficit and decrease the infarct area. Pulsed magnetic field and BYHWD can protect the brain tissue after cerebral ischemia reperfusion, and promote IGF-1 expressing. The effect of pulsed magnetic field combined BYHWD on protecting the brain tissue after cerebral ischemia reperfusion, and promoting IGF-1 expressing were more than pulsed magnetic field and BYHWD.
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