| Objective:To establish the rat model of caudate nucleus intracerebral hemorrhage(ICH), and given different doses of four single sialic acid ganglioside hexose intervention, by observing the impact of GM1 for nerve function score, brain water content, pathological change, degree of inflammation, degree of brain tissue around hematoma cells apoptosis, poly adenosine diphosphate ribose transferase 1(PARP) and apoptosis inducing factor(AIF) expression in secondary brain injury caused by cerebral hemorrhage, To study the reaction of cell apoptosis and its protection after GM1 on the secondary brain injury after intracerebral hemorrhage.Methods: 1. The method of autologous blood injection to establish the rat caudate nucleus hemorrhage model.2. The adult 80 healthy male SD rats are randomly divided into four groups:Sham group(5), cerebral hemorrhage group(ICH group)(25), small doses(15 mg/kg) GM1 treatment group [GM1(S) group](25), high-dose(30 mg/kg) GM1 treatment group [GM1(L) group](25).Then according to the time points, cerebral hemorrhage group and GM1 therapy group was divided into 6 h, 1d,2 d, 3d, 5d five subgroups after operation, There are 5 rats in each group.Sham group rats only pin injection without autologous blood, only after 6h time points. 3. The dosing method: GM1 method : GM1 treatment group rats with cerebral hemorrhage 5 min and 60 min after the surgery, according to the weight respectively give low-dose(15 mg/kg) or high dose(30mg/kg) GM1 by intraperitoneal injection at a time, Sham group and ICH group rats give the same amount of saline solution at the same time by intraperitoneal injection.4.To evaluate building situation after 6 hours and to evaluate nerve function score at each time point.5.The dry-wet weight method is used to measure the postoperative hematoma surrounding brain tissue water content in different time points.6. HE staining observation of brain tissue around hematoma inflammatory reaction and pathological changes in different time points.7. TUNEL staining to observe apoptosis brain tissue around hematoma in different time points. 8. Immunohistochemical method to observe the brain tissue around hematoma expression of PARP 1 and AIF protein in different time points Results: 1. The behavior score: Sham group rats had no obvious postoperative neurological dysfunction, ICH group, GM1 group compared with the Sham group showed different degrees of neurological dysfunction, the difference was statistically significant P < 0.05;ICH group and GM1 treatment group at 2d compared with 3d time point had no statistical difference P > 0.05,but at 6 h, 1d, 5d time points after surgery compared with 2d time points had statistic significance P < 0.05;In cerebral hemorrhage at 6h time point GM1(S) group has no significant change compared with ICH group, there was no statistically significant difference P > 0.05, but at 1d, 2d, 3d and 5d time point after cerebral hemorrhage were all higher than ICH group, which had statistically difference P < 0.05, GM1(L) treatment group nerve function score at each time point were significantly higher than ICH group, which had statistically difference P < 0.05, GM1(L) treatment group nerve function score at each time point were significantly higher than GM1(S) treatment group, which had statistically difference P < 0.05.2. Brain tissue water content: ICH group and GM1 trestment group after each time point compared with the Sham group were statistically significant P < 0.05;ICH group and GM1 treatment group at 6 h, 1d, 5d time points after surgery compared with 2d time points had statistic significance P<0.05, 2d compared with 3d time point had no statistical difference P > 0.05;The tissue water content of GM1(S) treatment group lower than ICH group within 1d, 2 d, 3 d and 5d time point, the difference has statistical significance P < 0.05, but the tissue water content compared with ICH group has no statistical difference at 6h time point P > 0.05,This brain water content at each time point GM1(L) group were relatively lower than ICH group, there are statistically significant P < 0.05,The brain water content of GM1(L) treatment group was lower than GM1(S) treatment at each time point,the difference is statistically significant P < 0.05,3.pathology change and the degree of the inflammatory response: observing the brain tissue beside the pinhole of Sham group after HE staining under the light microscopy,we can see that the brain structure was clear and complete, neurons in neat rows,the morphological structure in good condition, no obvious cell swelling, the nucleus is complete, no deformity, the interstitial no inflammatory cell infiltration and edema;ICH group and GM1 treatment group were visible different degrees of brain hyperemia and edema at each time point after cerebral hemorrhage, some nerve cells was swelling, cells stroma broadening, the vacuoles degeneration of neurons,the structure of neurons was damage, even degeneration necrosis, the nucleus was degenerate,dissolved and solid shrinkage,the density of chromatin increase, eosin staining deep, the gap around the cells widened, glial cell proliferation can be seen in the stroma, a large number of inflammatory cells infiltration, the pathological changes and inflammatory responsein reached a high degree at 2-3d and then as time prolonged decline;GM1 treatment group compared with ICH group show the degree of inflammation and pathological changes lighten, the GM1(L) treatment group is more obvious on the improvement of the inflammatory reaction and pathological change.4. The comparison of the degree of brain tissue cells apoptosis around hematoma :observing the brain tissue beside the pinhole of Sham group after TUNEL staining,we can see that there are no obvious cell apoptosis;ICH group and GM1 treatment group around the hematoma after cerebral hemorrhage postoperative can be visible different degrees of apoptosis cells at different time, compared with the Sham group was statistically significant P < 0.05;the degree of the apoptosis of ICH group and GM1 treatment group at 6 h, 1d, 3d and 5d after surgery compared with 2d time point had statistically significant P < 0.05;GM1(S) treatment group compared with ICH group at 6h time points was no significant statistical difference P > 0.05, but at 1d, 2 d, 3d, 5d time point all reduced, the difference is statistically significant P < 0.05, GM1(L) treatment group at each time point compared with ICH group were significantly less apoptosis, the difference was statistically significant P < 0.05,GM1(L) treatment group at each time point compared with GM1(S) treatment group were significantly less apoptosis, the difference was statistically significant P < 0.05.5. PARP 1 protein changes: Sham group of brain tissue around hemorrhage did not see obvious PARP-1 positive expression, ICH group and GM1 treatment group compared with the Sham group PARP-1 protein levels were significantly increased at all time points,P < 0.05;at 6h,At 1d,3d and 5d the ICH group and GM1 group is statistically significant difference compared with 2d after surgery P<0.05;There was no significant statistical difference between PARP-1 protein content of GM1(S) group and ICH group at 6h time points P > 0.05, but at 1d, 2 d, 3 d, 5 d time point were all reduced, the difference is statistically significant P < 0.05, PARP-1 protein levels of GM1(L) treatment group were significantly reduced compared with ICH group at various time points,the difference was statistically significant P < 0.05,PARP-1 protein levels of GM1(L) treatment group were significantly reduced compared with GM1(S) treatment group at various time points,the difference was statistically significant P < 0.05.6.AIF protein changes: Sham group of brain tissue around hemorrhage did not see obvious AIF positive expression, ICH group and GM1 treatment group compared with the Sham group, AIF protein levels were significantly increased at all time points, there are statistically significant,P < 0.05;At 6h,1d,3d and 5d the ICH group and GM1 group is statistically significant difference compared with 2d after surgery P<0.05;there are no significant statistical difference between GM1(S) treatment group and ICH group in AIF protein content at 6h time points P > 0.05, but at 1d, 2 d, 3 d, 5 d time point were all reduced, there are statistically significant difference,P < 0.05, AIF protein levels of GM1(L) treatment group were significantly reduced compared with ICH group at various time points,the difference was statistically significant P < 0.05,AIF protein levels of GM1(L) treatment group were significantly reduced compared with GM1(S) treatment group at various time points,the difference was statistically significant P < 0.05.Conclusion: 1. using autologous blood injection method building the rats cerebral hemorrhage model is similar to the physiological pathology change after human cerebral hemorrhage,the operation is relatively simple,which is an ideal animal model of cerebral hemorrhage.2. PARP-1 expression surrounding the cerebral hemorrhage hematoma issues increased, can promote the expression of AIF, thereby promoting apoptosis, increase nerve dysfunction and brain edema, aggravating pathological change and degree of inflammation, cause secondary brain damage.3, GM1 treatment after cerebral hemorrhage can cut the expression of PARP-1, reduce AIF expression, so that reduce the level of apoptosis, relieve nerve dysfunction and brain edema, relieve the pathological change and inflammation, protective to secondary brain injury. |
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