Expression Of Oxygen Free Radical And Intercellular Adhesion Molecule-1 Following Subarachnoid Hemorrhage In Rats

IntroductionCerebral Vasospasm (CVS) was one of the most important complication of aneurismal Subarachnoid Hemorrhage (SAH) , which was an essential cause of mortality and neurological morbidity in patients. The research on the reason and pathogenesis of CVS following SAH continued decades, but there never was systematic theory or hypothesis to elucidate hitherto. The process of CVS was divided into an initial acute phase and a subsequent delayed phase. Nowadays most scholars accept the view that CVS was induced by the association with multiple factor and link. Analyzing literature, the author found that there were two paths to induce CVS following SAH: ①the mechanism of microvascular dysfunction and autoregulatory failure;②the mechanism of immune - inflammatory reaction. At present, many institutions and scholars in the world carried out penetrating study on the reasons which had been found of CVS following SAH and the evidence were sufficient. They identified clearly the existence of the two mechanisms of CVS, but they studied the pathogenesis by one single mechanism and lost with one - sidedness. Moreover, they explained the degree of effect of each factor on the phase of CVS and the relation of them in the two mechanisms ambiguously. In view of this, the author proposed a new hypothesis as follows after referring to pertinent literature: ①During the development of CVS following SAH, both the two mechanisms may be to coexist ( Fig. 1 );②The two mechanisms may be to change as follows on the phase of CVS following SAH ( Fig. 2 ).In order to authenticate this hypothesis and provide objective evidence, we selected oxygen free radical (OFR) and intercellular adhesion molecule - 1(ICAM - 1 ) as indexes because they were essential factors in the two paths, and detected the expression and dynamic variance of the indexes in the animal model.Materials and Methods1. Materials1. 1 Reagents and DevicesRabbit anti ICAM -1 and rabbit IgG SABC kit (supplied by the Boster Biology Company) ,SOD^MDA and Coomassie brilliant blue albumen kit (supplied by the Nanjing Jiancheng Biotechnology Institute) , chloral hydrate, paraformal-dehyde, PBS, DAB developer, hematine, eosin, laser Doppler flowmetry (PF3type,made in Sweden PERIMED Company) , paraffin section cutter, spec-tro photometer, thermostatic waterbath cabinet, centrifuger, micropipettor, microimage analytical system ( MetaMorph/C - 5050/BX41 UIC/OLYMPUS, US/ JP) ,surgical instruments, electronic balance etc.1.2 Objects45 Wistar rats of both sexes weighing 300 - 350g were used in this study (supplied by the experimental animal centre, China Medical University) , they were randomly assigned to control group ^ sham SAH group ^ SAH group (include post - SAH lh^6h^l2h^ld^3d^5dN7d) ,5 rats each group.2. Methods2. 1 Animal ProceduresThe experimental model of SAH was completed by double injection autoge-neic arterial blood into cisterna magna. The rats were anesthetized with intraper-itoneal 10%chloral hydrate (40mg/100g). Under sterile conditions, the posterior scalp was incised in the midline and the occipital bone was exposed at the junction of the occipital bone and the arch of Cl. 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 cere-brospinal fluid (CSF) was drawn from intracisternal space, the 0. 3ml of hepa-rinized autoblood sample was injected slowly into cisterna magna by a 4 - gauge needle over a period of 1 min. Notable changes in animals' respiration 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 incisions were closed with suture and sterilized. All the rats were fed cleanly and antibiotic was administered by mouth for infection prevention. Forty - eight hours afer the initial intracisternal blood injection, the rats in SAH group were reanesthe-tized and 0. 3ml of autoblood was reinjected 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.2.2 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 ( control group and sham group at a fixation time). The posterior scalp was incised in the midline and a hole ( D = 3 mm) was drilled by a dental drill at the point which was 3 mm away from the midline and the bregma respectively on the left of the skull. The rats cerebral blood flow(CBF) was measured by a detecting head ( D =2. 5mm) of laser Doppler flowmetry which was placed vertically on the cerebral dura mater.2. 3 Sacrificing of Rats and Preparation of SamplesAfter the CBF of all rats was measured, the cardiac apex was cannulated retrogradely through a thoracotomy and the auricle of right atrium was cut simultaneously. The systemic circulation was perfused with 200 - 300ml of physiologic saline (at 37 °C ) 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 Cl 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 (at 4^ )for 24 h. The fixed samples were embedded in liquid paraffin for He-matine - Eosin stain and immunohistochemical stain.The basal artery (BA) N Willis arterial circle and bole of cerebral arterieswere isolated from the remaining parts of samples and were homogenized in ice -cold physiological saline with a homogenizer (at 15000r. p. m for 2 min at + 4ct! ). The 1% homogenates were used to measure the level of MDA and SOD.2.4 Measurement of IndexesThe paraffin wax samples were sectioned, and stained with Hematine - Eo-sin and anti ICAM - 1 immunohistochemically. The intensity of ICAM - 1 immu-noreactivity in the vessel wall of basal artery was detected by microimage analytical system by means of gray scale. Protein level in homogenates was measured by the method of Coomassie brilliant blue. The activity of hepatocuprein ( SOD) in homogenates was measured by the method of xanthine oxidase at 550 nm in a spectrophotometer, results were reported as U/mgprot. The content of malondi-aldehyde(MDA) in homogenates was measured by the method of thiobarbituric acid at 532 nm in a spectrophotometer, results were reported as nmol/ mgprot.3. Statistical AnalysisThe data which were measured as described above were 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.ResultsStatistical analysis of the CBF revealed that there was significant difference in post - SAH lh ~5d groups compared with control group and sham SAH group ( P <0. 05) . The variance of the CBF in SAH group is diphasic. Hematine — E-osin stain revealed twice marked narrowing in the lumens of basilar arteries on 1 hour group and 3 days following SAH. There were significant pathological changes in the vessel wall of basilar arteries after 1 day post -SAH. The expression of ICAM - 1 immunoreactivity in the vessel wall gradually increased beginning at 1 hour after SAH and the most obviously on 3 days after SAH. The activity of SOD in homogenates of SAH group decreased the lowest at 1 hour after SAH compared with control group (P <0. 01) , from then increased but still lower than control group (P <0. 05) and persisting for 5 days;on the contrary, the content of MDA in homogenates of SAH group increased the highest at 1 hour af-ter SAH compared with control group(P <0.01) , from then decreased but still higher than control group (P <0. 05) and persisting for 5 days( Table 4 ~ 14, Figure 3 -19).DiscussionThe expound of oxygen free radical (OFR) and intercellular adhesion molecule - 1 (ICAM - 1) have been penetrating in references, but the relation of them was explained ambiguously. By the author' s analysis, the relation of them would be intimate: the blood enter the subarachnoid space after SAH, superox-ide free radicals are produced during the process of blood degradation and injure the blood vessel endothelium, and the signal transduction mechanism is activated by the oxidation - reduction reaction, then expression of ICAM - 1 is enhanced;the enhancement of the expression of ICAM - 1 precipitate leukocyte to aggregate and adhere the endothelial cell, and respiratory burst is activated by leukocyte, then superoxide free radicals is released and injure the blood vessel endothelium, so the infernal circle emerges. The research of the predecessor found that the phase of vasospasm in rats is diphasic. In this study, the expression of OFR achieved peak while the expression of ICAM - 1 was light on lh post - SAH, it demonstrated that the factor of the mechanism of microvascular dysfunction played main role, but immune - inflammatory reaction had initiated on the phase of acute CVS. On the contrary, the expression of ICAM - 1 was the most obvious and the level of OFR still higher than control group on 3 days post - SAH, it demonstrated that immune - inflammatory reaction come to climax but the function of microvascular had not yet recovered normal on the phase of delayed CVS.Conclusion1. Both ICAM - 1 and OFR participate the development of CVS following SAH.2. ICAM - 1 and OFR play defferent role on defferent phase of CVS re-spectively, OFR participate the development of acute CVS mainly, and ICAM -1 participate the development of delayed CVS mainly.