Comparison For Histological Changes Of Cerebral Arteries Resulted From Hematodes And Non-hematodes Stimulus In Subarachnoid Space

Objective: The aim of this study was to investigate the pathogenesy of delayed cerebral vasospasm (DCVS) following subarachnoid hemorrhage(SAH) in rabbits through comparing of histological change of basilar artery which induced by injecting hematodes and non-hematodes stimulus into subarachnoid space.Methods: 40 New Zealand white rabbits were randomly assigned to 5 groups (n=8).①Shamed-operation Group (A):Shamed-injection was only done for animals in the Shamed-operation Group.②SAH Group (B): SAH was simulated by the two-hemorrhage rabbit model through injecting autologous arterial blood 0.8ml/kg into the cisterna magna for those in this group. At 48 hours after the first injection, the second one was completed in the same as the first.③Inactivation Bacterial Group (C): 0.25ml/kg suspension of inactivation bacteria (3×1010 per/ml) was injected in cisterna magna. 48 hours later the second injection was accomplished .④Kaoline Group (D): the operation was done as it described in Group C except for 15% Kaoline suspension replacing inactivation bacteria.⑤Anticoagulated Blood Group (E): the operation was completed as it described in Group B, but for autologous arterial blood was taken by heparinized autologous arterial blood. All rabbits were killed by infusing 4% Polyoxymethylene at day 7 after the first operation. After all samples had been fixated with fixing solution for 24 hours, part brainstem with basilar artery (BA) and its main branches were carefully removed so as not to stretch and injure the BA. The 5μm slices which were obtained at middle of the BA should be prepared for hematoxylin and eosin (H.E) staining to observe the structure of vessel, consecutive slices for immunohistochemical staining would be performed to evaluate expression of TNF-αin the BA wall . D/T ( diameter/thickness ) and CSA( cross section area )value were calculated by measuring inner diameter(hereinafter as"D") of lumens and thickness (hereinafter as"T") of vessel wall. Immunohistochemical expressing of TNF-αwas represented by average optical density (hereinafter as"OD") value of the staining positive cells. All data, including D/T, CSA and OD value, would be processed by SPSS12.0 Statistics Package. The three data sets (D/T,CSA and OD) were described with mean±standard deviation ( x±s). Multiple comparisons of the data were analyzed by multi-sample One-Way ANOVA and SNK-q's method. It was taken as the significant differences when P<0.05.Results: In the Group A, the structure around the BA was normal. There was butterfly-like blood clots adhered to the BA in the Group B, and a few of pink-hematodes cerebrospinal fluid (CSF) could be seen in basal cistern. There was a plenty of white particle of Kaoline on the vessels of the BA completely in the Group D. In the Group E, the ventral surface of brainstem became slight red and a several of small blood clots were diffused around BA. There were less blood clots than it in the Group B significantly. In the Group C, it seemed that the structure of the BA was normal under naked eye. The result of microscope structure: In the Group A, the intima of BA was smooth and integrity and no reductus can be found in it.Endothelial cell was normal. No thickening, apomorphosis and necrosis of smooth muscle in medium were found, which was composed of 2~3 layers of eumorphism smooth muscle cell. The structure of adventitia was complete. There was no inflammatory cells around the BA. In the Group E, adventitia was thickening slightly. There was no hypertrophia, hyperplasy, apomorphosis and necrosis in smooth muscle cell of muscular layer. Significant reductus could not be found in intima. Vasospasm and inflammatory reaction were found in the others. Compared it with the Group A, CSA became narrow and wall of BA were thickness significantly. Typical histological appearances of DCVS can be seen: smooth muscle cell was hypertrophy, distortion, vacuolar degeneration and part necrosis, endochylema could be stained significantly, caryon was pycnotic, adventitia was thicken significantly. In the Group B, a plenty of dated RBC(red blood cell) and inflammatory cell infiltrating around adventitia of the BA. In the Group D, a plenty of white particle of Kaoline and inflammatory cell infiltrating around adventitia of the BA. In the Group C, a plenty of inflammatory cell infiltrating around membrana adventitia of the BA. The result of immunohistochemical staining about TNF-αshowed that: less expression could be seen on intima and adventitia in the Group A and E. In the other groups, some significant differences could be seen: lots of brown-yellow particles aggregated inside of epithelium of the adventitia and endothelial cell of the intima. D/T, CSA and OD in the Group A were 29.66±5.17, 38.61±12.45(U) and 0.1374±0.0151 respectively. In the Group E, those were 29.94±1.91, 36.09±9.53(U) and 0.1431±0.0249 respectively. There was no statistically significant difference between groups A and E, The basilar artery tissues was normal. In the Group B, those were 11.28±2.97, 15.72±7.04(U) and 0.2989±0.0406. In the Group C, those were 16.48±5.12, 26.98±7.03(U) and 0.1805±0.0294. In the Group D, those were 11.83±4.26, 17.34±4.20(U) and 0.2999±0.0389. Among these three groups, it has vasospasm to a different extent. The analysis result (D/T, CSA and OD)of statistics showed that: there was no significant difference between Group A and E, and less vasospasm could be found in these two groups. Other groups had vasospasm to a different extent. Compared with Group A respectively, there was a statistically significant difference with Group B,C and D.Conclusions: Different non-hematodes stimulus in subarachnoid space could led to analogical pathological change as DCVS following SAH, the severer vasospasm of BA was, the more expression of TNF-αwas. The result of this study showed that: inflammatory reaction may be one of the most important reasons of DCVS. In addition, the result of the Group E, which has no blood clots around the BA, no significate vasospasm can be found. All those showed that: excessive inflammatory reaction around blood vessel played an important role in the pathogenesy of DCVS following SAH.