Chang The Content Of Serum Interleukin-17 Following Subarachnoid Hemorrhage In Rats And Investigate The Sense Of It

IntroductionSubarachnoid hamehorrhage(SAH) is caused by spontaneous(rather than traumatic) arterial bleed into the subarachnoid space. The incidence of subarachnoid hemorrhage 9-22.5 cases per 100 Cerebral vessels cases in t-he statistics of domestic information. Cerebral vasospasm (CVS) is one of the most important complication of SAH , which is an essential cause of mortality and neurological morbidity in patiens. The process of CVS is divided into an initial acute phase and a subsequent delayed phase. The initial acute phase is temporary or early onset CVS that the bleed in subarachoind space mechanical irritates to Cerebral vessels. The other maybe caused by subsequent Cerebral vasospasm or delayed cerebral vasospasm(DC-VS), which is more persistence time and still unclear mechanism nowadays .But the incident of DCVS is more in the clinical. Nowadays, many scholar consider that factor of immunization is significant role to cause dcvs following SAH. Moreover, many documents report that catabolin of blood clot around cerebral arteries precipitates the generation of the DCVS. The subsequent immunological reaction in cerebral arteries wall precipitates the generation of the DCVS. Accordingly, a new pathway is established to invest-ingate pathogenesy and therapy of the DCVS. Interleukin-17 is one of powerful Pre-inflammatory cell factors, and one of readjust factors of Inflammatory reaction. Interleukin-17 can stimulate many cells to secrete cell factors, and produce synergistic effect with many cell factors to enlarge.Inflammatory reaction. Many writings' investigation manifests that the level of Interleukin-17 of patients steps up in cerebral arterial thrombosis, the cells of which have close correlation with damage degree nervous system. The experimental model of SAH was completed by double injection autogeneic arterial blood into cisterna magna, through which way the content of Interleukin-17 of bloodplasma was detected and the relation of Interle-ukin-17 and cerebral angio spasm was approached and this provided experimental data to degrade cerebral angiospasm following SAH clinically.Materials and Methods一,Materials1,Reagens and Devicessym-enzyme immunoreagent kit of Interleukin-17 of rat(sen xiong technology industry company limited of Shang Hai) chloral hydrate, paraformaldehyde, hematoxylin, eosin, laser Doppler flowmetry (FP3type , madein Sweden PERIMED Company) , paraffin- secting cutter, enzyme-marking meter, thermostatic waterbath box, centrifuger, micropipette, surgical instruments, electronic balance refrigerator, microscope etc.2,Objects40 healthy rats of both sexes weighing 300-350 were used in this study(supplied by the experimental animal center, China Medical University), they were randomly assigned to two groups:①sham SAH group(NS 20 rats):injecting liquor natrii chloridi isotonicus into cisterna magna of rats.②haemato-injecting groups (SAH,20rats): injecting fresh anticoagulationless autoallergic arterial blood into cisterna magna of rats.二,Methods1,Animal ProceduresThe experimental model of SAH was completed by double injection autogeneic arterial blood into cisterna magna. The rats were anesthetized with intraperitoneal 10% chloral hydrate (40mg/100g). Under sterile conditions, the posterior scalp was incised in the midline and the occipital bone was exposded at the junction if the occipital bone and the arch if C1. The occipital muscles were carefully dissected off the occipital bone. The atlanto-occipital membrane was then exposed, covered with sterile gauze, a 0.3ml of blood sample was drawn from femoral artery which had been exposed, after an equivalent volume of cerebrospinal fluid(CSF) was drawn from intracisternal space, the 0.3ml of heparinized autoblood sample was injected slowly into cisterna magna by a 4-gaugeneedle over a period of 2 min. Notable changes in animals' repiration pattern even transient apnea was observed by the end of injection. Animals were immediately placed in a head-down position for 30 min to facilitate the diffusion of the autoblood in the basal cisterns. After no significant blood or cerebrospinal fluid leakage from the insertion site was found, the muscles and the antibiotic was administered by mouth for infection prevention. Forty-eight hours after the initial intracisternal blood injection into the cisterna magna. The sham SAH group underwent the same basic procedure as the SAH-animals, except that physiological saline was injected into the intracisternal space rather than blood. The control group was untreated.2,Measurement of Cerebral Blood Flow(CBF)The SAH-animals were reanesthetized as described above for the SAH procedure at 1 hour to 7 days post-SAH. The posterior scalp was incised in the midline and a hole(D=3mm) was drilled by a dental drill at the point which was 3mm away from the blood flow(CBF) was measured by a detecting head(D=2.5) of laser Doppler flowmetry which was placed vertically on the cerebral dura mater. Experimental data was statistically analyzed.3,Determination of Interleukin-17(1) Extraction of Interleukin-17 samples1ml peripheral bloods was adopted and put into EP test tubes respectively on the 2nd ,3rd,5th,7th day after experimental animals being injected doubly and preserved at -20℃. Samples were dislodged and melt automaticly at room temperature after being fully collected and then centrifuged at 4℃(3000r/min). Discard the supernatant 15 min later.(2) MethodsABC-ELISA method of sandwich of double antibody was adopted. OD value was measured at 492nm with scale-ase meter and handled with SOFTMax software(Immuno-laboratory of China Medical University) and the corresponding content of Interleukin-17 of rats yielded.4,observation of the cindition of animalsSurvival of animals or no, general states of survive animals including self-cleaning, ingestion, hydroposia quantity, hyperspasmia and other phenomena. Observation of base of skull dissection and pathology after animals being executed according to set-time.5,Sacrificing and dissection of Rats and patho- observationAfter the CBF of all rats was measured, the cardiac apex was cannulated retrogradeiy through a thoracotomy and auricle of right atrium was cut simultaneously. The systemic circulation was perfused with 200～300ml of physiologic saline(at 37℃) until the fluid from the auricle of right atrium was clean. The samples were taken from all rats by cutting brain stem at the level of C1 and were subdivided into two segments at the level of middle pons, and a part of the samples which were below the pons were fixed in 4% paraformaldehyde for 24 hour, in order to observe the conditions of blood injection and the anatomic structure of bases of skull. The fixed samples were embedded in liquid paraffin for Hematine-Eosin stain and immunohistochemical stain. The basal artery was observed under 40-time light microscope after the samples embedded in liquid paraffin being sectioned and stained with Hematine-Eosin .6,Statistical Analysis The data which were measured as described above expressed as mean±standard deviation, the data were evaluated by t test and correlation analysis, and the differences were considered significant if P<0.05.Results1,rCBF in haemato-injecting groups obviously decreases on the 2nd,3rd,5th day compared to that in sham SAH group(p<0.05), but there is not statistically significance between the two groups on the 7th day(p>0.05).2,Under light microscope, major inflammatory cell, haemorrhage, endotheliocy te degeneration and necrosis, elasticity membrane wrinkle and breakage, smooth muscle fiber degeneration and necrosis and so on are found in tunica adventitia of basilar artery of rats of SAH Group, hoewer, which changes are not found in NS Group.3,The content of Interleukin-17 in haemato injecting groups obviously inceases on the 2nd day compare to that in sham SAH group (p<0.01), reachesma xlum on the 3rd (p<0.001),comes to get down from then on and the content of Interleukin-17 still remains obvious distinction comparsion to that in sham SAH group until the seventh day(p<0.05).ConclusionCerebrovascular Spasm(CVS) is one of frequent complications after SAH. Interleukin-17, one of important vasoactive substances that cause Cerebrovascular Spasm, participates in patho-physiology procedure of SAH. All above hints that antagon of Interleukin-17 treats SAH and prevents Cerebrovascular Spasm will be one of possible pathways.