Correlative Research Of Secondary Brain Injury In CVS After SAH

PartⅠThe clinical research of the variation of TCD,SOD,MDA,NF-kB,ICAM-1 content of blood in patients with subarachnoid hemorrhage Objective: Dynamic content changes of NF-kB,sICAM-1,sVCAM-1 were observed in patients with ruptured aneurysmal subarachnoid hemorrhage .The relations between their content changes of different degrees of disease and CVS were discussed. Methods: 30 cases of ruptured aneurysms were studied in hospital in 48 after the bleeding. The symptoms were graded according to Hunt and Hess Methods, the degrees of SAH were evaluated according to Fisher CT grade. Positions of aneurysms were identified by DSA and the CVS were confirmed by TCD. VMCA>120cm/S was defined as CVS. Venous blood was analyzed the same hour in 3, 5, 7, 9, 11ds after aneurysms ruptured in SAH group. Anticoagulating plasma and outside monocyte were separated, stored in -80 ℃and determined together. First antibody was the poly-colonal antibody(NF-kB) to rabbit. The activity of leukocyte NF-kB was shown in positive degree scores. Positive cells were seen when brown parcel appeared in the cellular core under microscope. If the core was blank or delicate brown, it was negative. 4 hundred of cells were counted in 4 high microscopic scope, with 100 cells in one scope. The rate of positive cells was recorded. The cells were graded according to the amount of brown parcels.Namely, no parcel in the core, 0 grade; the area was less than 1/2, 1 grade; the parcel area was less than 3/4, 2 grade; the area was more than 3/4, 3 grade. The positive degree were shown as scores, namely, 1×1positive rate + 2×2positive rate + 3×3positive rate = score. ICAM-1/VCAM-1 was analyzed in ELISA methods. The determination of SOD was made by 6-OHDA. The determination of MDA was made by TBA. The control group included 20 healthy people which were examined under the same condition. Results: NF-kB,sICAM-1,sVCAM-1 began to increase 1d after SAH. The expression of NF-kB got to top in 3ds, sustained to 5ds, then decreased and basically back to normal in 9d. The expression of sICAM-1,sVCAM-1 got to top in 5d, began to decrease in 7d, back to normal in 11d. The mean data of NF-kB in 1~3d, 5~7d and sICAM-1,sVCAM-1 in 7~9d were significantly higher than the control group. NF-kB,sICAM-1,sVCAM-1 changed more evidently as the CVS became more severely. MDA,SOD in CSF changed accordingly. CSFMn-SOD content was higher than the control group. There was no difference of CuZnSOD content between the gross and the control group, though the content of CuZnSOD in not severe group wasmuch higher than the more severe group. MDA was much higher and SOD was much lower in more severe CVS group. It showed that pathophysiological changes of cerebral ischemia had correlation with the period and the degree of CVS. Content changes of NF-kB in 1-3d after SAH were significantly earlier than sICAM-1,sVCAM-1,MDA,SOD. Conclusion: Variation of NF-kB,sICAM-1,sVCAM-1,MDA,SOD in blood of patients with SAH had correlation with the progress of CVS . It showed that ICAM-1 and VCAM-1 induced by NF-kB played important roles in the cause and progress of CVS. Part ⅡThe expression of PKC, NF-kB, ICAM-1, VCAM-1 as well as MDA and SOD in the hippocampus of rabbits after SAH. Objective: To discuss the existence of PKC, NF-kB, ICAM-1, VCAM-1 and their significance in the cause and progress in hippocampus injury. Methods: 250 Newzealand pure rabbit of 4~6M were afforded by the animal center of SuZhou University medical college without the limitation of gender with the mean weight of 2.5±0.4kg. Models of SAH and CVS were made via double blood injection into major cistern. Basilar artery spasm was detected by DSA. Basilar artery and hippocampus were separated, preserved under zero and observed collectively. Morphological methods as well asimmuno-histochemistry, hybridization in situ, immunology signal methods were employed to observe the change of basilar artery diameter and that of hippocampal nerve cells. Immuno-histochemistry and Western-blot methods were used to detect the change of PKC,NF-kB,ICAM-1 in protein level. Hybridization in situ was employed to detect the change of PKC,NF-kB,ICAM-1 in molecular level. The determination of MDA was made by TBA. The determination of SOD was made by 6-OHDA. Results: Stenosis of BA were identified by DSA 3d after double blood injection into major cistern and got to the top in 5d which was in accordance with morphological observation and could afford foundation for the observation of patho-physiological change of CVS. The expression of PKC,NF-kB,ICAM-1,VCAM-1 in hippocampal cells increased accompanied with the stenosis of spasmodic artery. The expression of PKC,NF-kB significantly increased 1d after double blood injection, maintained in the high level for 3ds and began to decrease in 5d, back to normal in 9d. The expression of ICAM-1 began to increase in 1d, got to the top in 5d, and decreased in 7d, back to normal in 11d. The expression of PKC,NF-kB,ICAM-1,VCAM-1 in protein and mRNA level had difference in sequence. MDA increased while SOD decreased evidently in SAH brain tissue, which had significant difference with the control group and NS injection group. There was no difference between the control group and NS injection group.Conclusion: Inflammation of spasmodic artery controlled by PKC, NF-kB and induced by ICAM-1, VCAM-1 did exist in the happening and progress of hippocampus. These reactions played important functions in the happening and progress of cerebral injury. PartⅢThe experimental research of inhibitors of PKC and NF-kB in the treatment of cerebral ischemia of rabbits Objectives: We attempted to observe the effect of inhibitors of PKC and NF-kB in CVS by breaking the expression of adhesion molecule and other inflammatory factors in gene level in hippocampus. Methods: The inhibitors of PKC, NF-kB ,monoclonal anti-body and NS were injected through major cistern 3d after the model of CVS were made. The method of injecting NF-kB inhibitor into cistern was 0.25ml bid which was defined according to Scheck research results. Myristoylated PKC Pseudosubstrate Peptide inhibitor was 50ug/kg bid injection into major cistern. The quantity of ICAM-1 monoclonal antibody was defined according to the result of Clark. 2 mm diameter silicon tube was settled after the second injection for the SAH model. The depth into the major cistern was less than 3mm. The tube was fixed by cervical muscle and the end was embedded under the endepidermis. It was pumped to ensure if CSF was seen before every injection. The end was embedded under the endepidermis after injection.Two days later the injection was stopped and the rabbits were killed in 5d. Methods of immuno-histochemistry, hybridization in situ and Western blot were employed to examine the expression and effect of PKC, NF-kB, ICAM-1, VCAM-1 in the hippocamal cells. Results: Intima were plain under light microscope. There were no obvious ripple crimples and the endodermis cells arrayed regularly. The stenosis degree of the vessels decreased obviously and there were no evident thickness of the wall of vessels. The effect of inhibitor treating group was slightly better than monoclonal antibody treating group. There were obvious stenosis of vessels and obvious thickness of the walls of vessels as well as the intima twisted inwards in SAH and NS injecting group. The expression of PKC,NF-kB,ICAM-1 was seen in hippocamal cells. PKC,NF-kB,ICAM-1 had strong immune dye in hippocamal cells in SAH and NS injecting groups. PKC,NF-kB and ICAM-1 had little expression after inhibitor treating. PKC,NF-kB had strong expression and ICAM-1 had little expression after monoclonal antibody treating. The expression of PKC,NF-kB and ICAM-1 in hippocamal cells in mRNA level was in accordance with that in protein level in sequence. PKC,NF-kB and ICAM-1 dye was slight after PKC and NF-kB inhibitor treating. PKC and NF-kB had strong dye signals while ICAM-1 had little expression after monoclonal antibody treating. Strong signals were seen in hippocamal cells in control and NS injecting groups. Western-blotmethod was used to ensure the reliability of indexes for determing. It showed that the expression of NF-kB was significantly decreased after PKC and NF-kB inhibitor treating while the effect of monoclonal treating was slight. MDA and SOD content in PKC,NF-kB treating group changed significantly than NS injecting and ICAM-1 treating groups. There was no significant difference of MDA and SOD content between NS injecting and ICAM-1 treating groups. Conclusion: PKC,NF-kB and ICAM-1 chain reaction did exist in the cause and progress of cerebral injury after CVS. Inhibiting the acting of PKC, NF-kB is very important in preventing and treating brain injury.