The Therapeutic Effect And Mechanism Of Interleukin 10 On Cerebral Vasospasm Caused By Subarachnoid Hemorrhage

Background:Clinically, spontaneous subarachnoid hemorrhage (SAH) is one of common clinical cerebrovascular disease and the common clinical critical illness, 75% to 80% of which is caused by intracranial aneurysm.The high-risk age of SAH is 40 to 60 years old and without the surgical intervention of the ruptured aneurysm can easily rupture again. Cerebral vasospasm (CVS) is one of the most serious complications after subarachnoid hemorrhage and the reason of high mortality and morbidity, the incidence rate of which is as high as 70%. The CVS often occurs a few days to two weeks after subarachnoid hemorrhage and can cause varying degrees of ischemia and a series of delayed ischemic symptoms of nervous system when the cerebrovascular persistent spasm.The CVS maybe the most important reason for the high morbidity and mortality.So the earlier the diagnosis and treatment, the better prognosis of the spontaneous subarachnoid hemorrhageAt present, the exact pathogenesis of cerebral vasospasm has not been entirely clear although there are several hundred research reports each year. So we can not exactly treat the cerebral vasospasm in clinical. According to the extensive researches of CVS after SAH, there are multiple reasons which play an important role in the development of CVS, including the subarachnoid clot irritation, inflammation, endothelial apoptosis, oxidative stress, activation of protein C pathway, inhibition of nitric oxide signaling pathway and the cell membrane dysfunction. More and more evidence indicate that the inflammatory response, especially the interaction of leukocytes and endothelial cells play an important role in the cerebral vasospasm after subarachnoid hemorrhage. There are leukocyte recruitment, infiltration and activation in the cerebral artery, which has been confirmed by lots of animal experiments and clinical studies. McGirtt found that the white blood cell count in patients with greater than 15*109/L, the occurrence of CVS was three times higher, which prove that the leukocyte count can be an independent risk factor for the development of cerebral vasospasm and it can be used for early diagnosis and treatment by monitoring the white blood cell count. The monocyte chemoattractant protein-1 (MCP-1) may promote inflammation by recruiting macrophages and the expression levels of monocyte chemoattractant protein-1 in line with the development of CVS, which suggests the significant process of inflammation in CVS. A study by Bavbek showed that the monoclonal antibody of intercellular adhesion molecule (ICAM-1) could be used to mitigate the cerebral vasospasm after subarachnoid hemorrhage, further confirmed the process of inflammation in the cerebral vasospasm and provided strong evidence for targeted therapy cytokines. At the same time, tumor necrosis factor-a (TNF-a) is one common inflammatory cytokines, play an important role in the inflammatory cascade response. In the animal models of subarachnoid hemorrhage, Vecchione proved that the occurrence of cerebral vasospasm with the increase of TNF-a and inhibit the expression of TNF-a can ease the brain damage caused by blood, reduce the incidence of early cerebral vasospasm, which suggest us that TNF-a can be used as a new therapeutic targets for the prevention and control of cerebral vasospasm. While there are other studies showed that by administration of the auxin which has anti-inflammatory function can reduce oxidative stress after subarachnoid hemorrhage, maintain the balance of oxidant and antioxidant, by inhibiting the release of pro-inflammatory mediators, the auxin can prevent the endogenous antioxidant depletion caused by subarachnoid hemorrhage, which can indicate the significant status of inflammation in cerebral vasospasm after subarachnoid hemorrhage. For instance, in a clinical study of 276 patients with SAH confirmed inflammation was very common after SAH and in those patients with serious bleeding was more common. The degree of inflammatory reaction can indicate the occurrence and prognosis of vasospasm. Lots of scholars try to adopt an anti-inflammatory drug treatment of cerebral vasospasm, which have achieved a certain effect, compared to placebo and low dose of hormones, high doses can alleviate the degree of vasoconstriction and vascular wall reconstruction, also can reduce the mortality and the incidence of delayed cerebral ischemia. Handa found that the cyclosporine as an immunosuppressant could inhibit interleukin-2 (IL-2) transcription, resulting in effecter T cells dysfunction and relieve cerebral vasospasm compared with the control group.However, the hormones and immunosuppressive drugs have huge side effects, limiting their clinical application. In addition to the inflammatory response is a complex process, the involvements of inflammatory cytokines are a complicated network, and therefore it may be limited to just inhibit a single cytokine. According to the results at present, there may be new effective therapeutic strategies to control cerebral vasospasm due to subarachnoid hemorrhage by inhibiting the inflammation.Interleukin-10 (IL-10) has strong immune and inflammatory inhibition, which can be secreted by monocyte, macrophages and T cells. Under normal physiological conditions, IL-10 concentration in vivo is very low, however, the cells can secrete more IL-10 when the body suffering from infectious diseases. IL-10 can inhibit the secretion of IL-6, IL-2 and TNF-a. Due to the important function of immune and inflammatory response in the nerve damage after subarachnoid hemorrhage, so the function of IL-10 in cerebral vasospasm after subarachnoid hemorrhage needs further explore and verification. On the other hand, the expression of heme oxygenase-1 (HO-1) is induced by the blood in the subarachnoid through activating heme oxygenase-1 promoter gene. The reaction can clear the hemorrhage in subarachnoid, while the catabolism of heme and HO-1 over expression may directly or indirectly inhibit inflammation reaction. The products of heme catabolism conclude carbon monoxide (CO), biliverdin and free iron, among which the most important is CO, a vascular relaxing factor involved in regulating the physiological and path physiological processes. The CO also relaxes vascular smooth muscle, inhibit platelet aggregation, the adhesion of nertrophil and involve in the circulatory system, nerve system and endocrine regulation. In the animal model of cardiopulmonary bypass-induced lung inflammation and dysfunction, Goebel found that the lung inflammation was inhibited when the animals were inhalation of 250 ppm CO, suggested us that inhaled CO could increase the expression of IL-10 which inhibited the expression of TNF-a and IL-1.HO-1 can be activated by a variety of factors, such as cytokines(IL-10,IL-13,IL-18),growth factors (TGF -β, Platelet-derived Growth Factor),heme, heavy metals, hypoxia,CO, and oxidant (hydrogen peroxide). But whether the HO-1 can mediate IL-10 in cerebral vasospasm after subarachnoid hemorrhage has not been reported, and there are not similar researches about IL-10 therapeutic effect on CVS after SAHObjective:In this study, the subarachnoid hemorrhage model of the Japanese white rabbits was successfully built and administered by intraperitoneal injection drug intervention (interleukin-10 and zinc protoporphyrin). We used the basilar artery diameter which was the observation index of cerebral vasospasm to study the therapeutic effects of interleukin 10 (IL-10) on cerebral vasospasm following subarachnoid hemorrhage in the Japanese white rabbit on the fifth day after the surgery of infusing autologous artery blood twice into the cisterna magna. We also detected the levels of content of TNF-a and IL-6 in serum by ELISA to observe the degree of inflammation after subarachnoid hemorrhage and the protein heme oxygenase-1 expression levels in the basilar artery through the way of western blot to investigate whether IL-10 could be mediated by heme oxygenase-1 in the treatment of cerebral vasospasm after subarachnoid hemorrhage.Method:Thirty healthy and clean Japanese white rabbits were randomly divided into the Sham operation group (A), S AH group (B)、SAH+IL-10 group (C)、 SAH+Znpp+IL-10 group (D) and SAH+Znpp group (E). All the experimental animals were built under intravenous anesthesia with sodium pentobarbital. The animal model of cerebral vasospasm after subarachnoid hemorrhage was performed by infusing autologous artery blood twice into the cisterna magna. The experimental groups were injected the fresh autologous arterial blood according to their weight 0.8ml/kg while the sham group was same equal amount of physiological saline. The SAH+IL-10 group (C)、SAH+Znpp+IL-10 group (D) and SAH+Znpp group (E) were administered by intraperitoneal injection of IL-10 and zinc protoporphyrin everyday postoperatively. The counts of TNF-α and IL-6 levels in serum were detected by ELISA method five days postoperatively and the diameter and expression of HO-1 protein in basilar artery were observed. The content of serum TNF-α,IL-6,HO-1 and basilar artery diameter were recorded as mean±standard deviation. The changes were compared by the One-way ANOVA using SPSS 13.0 statistical analysis software, P<0.05 considered statistically significant differenceResult:The animal model of cerebral vasospasm after subarachnoid hemorrhage was successfully built.There were not blood clots on the brain surface while there were varying degrees of adhesion and blood clot residues in the cistern magna arachnoid of the brain tissue in the experimental groups. The basilar artery and its branches were wrapped in blood clots and the brain seemed to be swelling in the subarachnoid hemorrhage groups. The basilar artery diameter of SAH groups was significantly shorter than the sham operation group (P<0.05) and the TNF-α and IL-6 levels were higher in SAH group (P<0.05). The diameter of group C and D (733.94±17.28 μm、646.11±9.79 μ m)was increased than group B and E (595.64 ±10.15 μm 532.81 ± 17.09μm), the TNF-α and IL-6 levels in group C (26.27 ±1.64pg/ml、58.15±1.38pg/ml) and D (43.45±1.77pg/ml、77.17±1.09pg/ml) were lower than group B (53.56±1.27pg/ml、115.93±1.47pg/ml) and E (60.56± 1.79pg/ml、136.45±1.73pg/ml).There was not the expression of protein HO-1 in sham operation group and the content of Ho-1 in C group (0.446±0.019) was higher than group B、D、E (0.314±0.014、0.251±0.018、0.160±0.011)Conclusion The cerebral vasospasm after subarachnoid hemorrhage can be alleviated by IL-10. The possible mechanism is that the IL-10 can inhibit the protein HO-1 in the process of blood clot degradation which can trigger the secondary p38 mitogen-activated protein activation system, reduce the system inflammation and inhibit the apoptosis following subarachnoid hemorrhage to play its role in the resistance of cerebral vasospasm.