The Role And Mechanism Of NGF On The Angiogenesis After The Permanent Focal Cerebral Ischemia In Rats

Objective To observe the role and mechanism of NGF on the angiogenesis in the MatrigelMethod 24 SD male rats are randomly divided into 4 groups: IgG group, NGF group, anti-NGF group, TAE226 group. The HE staining is used to evaluate the change of histology structure. The expressions and distributions of CD31 and a-SMA in Matrigel are detected with immunohistochemistry and the expression of p-FAK is investigated with Westem Blot.Results1. The new blood vessel distribute around the Matrigel in NGF group, the cell infiltrations are found in the group of NGF. The histology score of NGF group is 2 times compared with IgG group and 3 times compared with and anti-NGF group TAE226 group.2. The expression of CD31 are largely increased in the Matrigel of NGF group, fBut there are few expression of CD31 in the Matrigel after neutralizing the endogenous NGF and TAE226 treatment. There are significant statistic differences when positive cell numbers in NGF group are compared with other groups (P＜0.05). The expressions ofα-SMA are few in the group of IgG, TAE226 group and anti-NGF group, but the expression is obviously increased after NGF treatment.3. The P-FAK expression is substantially decreased in TAE226 group because of blocking FAK phosporalation, but the protein car been detected after NGF treatment. The level of FAK is decreased after neutralize the endogenous NGF compared with IgG group.Result:NGF can increase the angiogenesis in Matrigel. And the mechanism of NGF on the angiogenesis is related with P-FAK dependen integrin-FAK signal pathway. Objective To observe the role of NGF and Integrin-FAK signal pathway on angiogenesis, and to evaluate how NGF impacts the pathway, and reveal the effect of NGF on angiogenesis and its probably mechanism after cerebral ischemia.Method 282 healthy male SD rats with 10-12 weeks old, weight 250±20 gram, were randomly divided into 6 groups: normal group,sham group,cerebral ischemia group,TAE226 intervention group,NGF therapy group,NGF therapy + TAE226 intervention group, based on different time. After injecting into lateral cerebral ventricle, NGF dense levels were measured by enzyme-linked immunosorbent assay (ELISA) in the peripheral ischemic zone. The expression of CD31 andα-SMA was tested by immunohistochemistry in order to view the number of regenerated vessels around the peripheral ischemic part. The variable expression of integrinαvβ₃ and TrkA mRNA was detected by RT-PCR around the peripheral ischemic zone of parietal cortex. P-FAK protein levels were investigated by westem blot.Results1. Endogenous NGF was found at 3 hours after cerebral ischemia. The levels inceased to about 8～9 ng/g at the first day after ischemia. Significant increase of NGF levels were found around the peripheral ischemic zone at 3 hour in the NGF group, got peak after 1 day, significant decreased after 3 days. Statistically significant differences (P＜0.01 or P＜0.05) were found between the NGF and the cerebral ischemia group.2. The number of regenerated vessels in the NGF group was markedly higher than those in cerebral ischemia group (P＜0.01), the cerebral ischemia group was higher than the TAE226 group and NGF + TAE226 group (P＜0.05), NGF + TAE226 group was higher than the TAE226 group, but lower than the cerebral ischemia group.3. The expression of CD31 in cerebral ischemia group was found after cerebral ischemia 1 day, arrived at the peak after cerebral ischemia 7 days, decreased after cerebral ischemia 28 days. The expression of CD31 was decreased in TAE226 group, which was found statistically significant differences (P＜0.01) compared with cerebral ischemia group after cerebral ischemia 7 days and 14 days. The expression of CD31 in NGF group was found obviously to increase after cerebral ischemia 1 day, to arrive at the highest point after cerebral ischemia 7 days, to the expression continued with high level until the post cerebral ischemia 28th day, which was found statistically significant differences (P＜0.05) compared with cerebral ischemia group on the 7th day, 14th day, and 28th day. The expression of CD31 in NGF + TAE226 group was more than the TAE226 group, but fewer than the cerebral ischemia group. A few expression ofα-SMA in parietal cortex was found in normal and sham group. The expression ofα-SMA in cerebral ischemia group was found after 1 day of ischemia, to arrive at the peak point after 14 days, to decrease 28 days later. The expression ofα-SMA decreased in TAE226 group, which was found statistically significant differences (P＜0.01 or P＜0.05) compared with cerebral ischemia group after the 7, 14, and 28 day ischemia. The expression ofα-SMA in NGF group was showed obviously more than the ischemia group on the 1st day, to arrive at the point on the 14th and 28th day, which was found statistically significant differences (P＜0.01) compared with cerebral ischemia group. The expression ofα-SMA in NGF+TAE226 group was more than the TAE226 group, but less than the cerebral ischemia group, which was similar to CD31 expression.4. The expression of TrkA mRNA in the parietal cortex in cerebral ischemia group was found to increase obviously after 3 days of ischemia, to hold the peak from the 7th day to the 14th day, to decrease 28th days later. The expression of CD31 decreased in TAE226 group. The expression of TrkA mRNA in NGF group was found to increase obviously and show statistically significant differences (P＜0.01) with cerebral ischemia group on the 3rd day. 5. The expression of Integrinαvβ₃mRNA in cerebral ischemia group was found to show after 1 day of ischemia, to increase obviously after 7 days ischemia, to arrive at the peak point on the 14th day. The expression of Integrinαvβ₃mRNA in NGF group was found to increase obviously and show statistically significant differences (P＜0.05 or P＜0.01) with cerebral ischemia group after 3 days. The expression of Integrinαvβ₃mRNA in TAE226 group and NGF + TAE226 group were similar to cerebral ischemia group and NGF group, which showed no statistically significant differences (P＞0.05).6. The expression of P-FAK in cerebral ischemia group was found to increase before the post-ischemia 14 days, to decrease dramatically after 14 days. The expression of P-FAK in NGF group was found to increase obviously (P＜0.05 or P＜0.01) with cerebral ischemia group on the 3rd,7th,14th and 28th day. The expression of P-FAK in TAE226 group and NGF + TAE226 group were low, and showed statistically significant differences (P＜0.05 or P＜0.01) with cerebral ischemia group on the 7th and 14th day.ConclusionNGF could promote angiogenesis after ischemia stroke in rats, which due to the close relationship between the angiogenesis of NGF and Integrin-FAK signal pathway. And NGF could activate Integrin-FAK signal pathway through binding to its high affinity receptor TrkA. Objective: To explore the relation of NGF on the angiogenesis and neurogenesis. The neurogenesis in SVZ and SGZ are investigated with Brdu labelling through suppression of the phosphoralation of FAK after cerebral ischemia 14 days.Methods: Male SD rats are randomly divided into 4 groups: sham operation group, cerebral ischemia group, NGF group, NGF+TAE226 group. BrdU are injected subcutaneously 3 days before the animals are sacrificed. Immunoflurorescence histochemistry are employed to detect the cell proliferation and differentiation in SVZ and SGZ.Results:1 .The numbers of Brdu positive cells are most in NGF group than the other three groups (P＜0.01), the Brdu positive cell numbers are more in the group of NGF+TAE226 group than the group of only cerebral ischemia group (P＜0.05)2 .The expression of DCX are increased in SVZ and SGZ of the NGF group, particularly in the SVZ. There are little DCX expression in the only cerebral ischemia group and sham operation group. The numbers of Brdu differentiation into immature neuron are less in the group of NGF+TAE226 than the group of NGF.3 .The expressions of NG2 are low in the group of only cerebral ischemia group and sham operation group, NG2 are increased in the group of NGF group and NGF+TAE226 group, but there are no statistically difference (ρ＞0.05)4. The expression of GFAP are robustly increased in the group of NGF, the double-labeling cell numbers of Brdu and GFAP are more in the group of NGF group than NGF+TAE226 group.Conclusion: The increased neurogenesis by NGF after cerebral ischemia is maybe correlated with the increased angiogenesis caused by NGF.