摘要
背景:近年来,吉兰-巴雷综合征和眼肌麻痹-共济失调-无反射综合征屡有发现中枢神经系统受累,受累部位包括视神经、脑干和小脑。眼肌麻痹-共济失调-无反射综合征患者的脑干和脊髓小脑束发现MRI异常信号。因此建立空肠弯曲菌感染后脑炎的动物模型并通过影像学、免疫学及病理学等方法手段探讨其形成机制是实验出发点。目的:建立空肠弯曲菌Penner4型感染后中枢神经系统病变的动物模型。设计:随机分组设计、对照动物实验。单位:河北医科大学第二医院影像科、神经内科。材料:实验于2003-08/12在河北医科大学第二医院分子影像实验室完成。选择健康大耳白兔15只,随机分为实验组10只,对照组5只。方法:实验组于第1周将空肠弯曲菌灭活菌液与等体积的完全弗氏佐剂对抽充分乳化后,分别于双腋窝、双腹股沟及背部脊柱旁皮下多点注射免疫,每处1mL,每只总量5mL。以后每2周以单纯空肠弯曲菌灭活菌液腹腔注射加强免疫,每只每次总量5mL,共5次。对照组以相同体积的生理盐水代替空肠弯曲菌菌液,注射方法及时间与实验组完全相同。评价方法:①症状与体征:观察动物精神状态、饮食、尿便及肢体活动状况等。②血清学检查:用酶联免疫吸附法分别检测动物血清中抗空肠弯曲菌抗体、抗IgG型GM1抗体及髓鞘碱性蛋白含量。③影像学观察:使用TOSHIBA1.5T磁共振成像设备分别于每次免疫前随机抽取两组实验动物行MRI检查。扫描序列包括:自旋回波序列T1加权像,扫描参数为500/15ms(TR/TE);快速自旋回波序列T2加权像,4000/108ms(TR/TE);液体衰减反转恢复序列,参数为10000/120ms(TR/TE),翻转角90°,以上扫描层厚4.0mm,层间距0.8mm。④组织学检查:发病动物于初次免疫后4周经心脏灌注致死,立即开颅取视神经、部分白质、海马、脑干、小脑及颈、胸、腰各段脊髓,固定于40g/L甲醛溶液,分别行苏木精-伊红染色、坚固蓝染色及髓鞘碱性蛋白免疫组化染色。免疫后10周于实验组内随机选取5只及对照组5只以同样方法获得组织学标本。主要观察指标:症状与体征;动物血清中抗空肠弯曲菌抗体、抗IgG型GM1抗体及髓鞘碱性蛋白含量;影像学观察;组织学观察。结果:纳入动物15只,14只进入结果分析,实验组中1只动物于免疫后4周时死亡。①实验组1只动物于免疫后2周出现精神症状及肢体活动障碍。②实验组动物血清抗空肠弯曲菌-IgG抗体滴度在2～4周达到高峰,自第2周起,实验组动物血清A值显著大于对照组(实验组、对照组分别为1.923±0.403,0.973±0.633,P<0.05)。IgG型GM1(A值)在第8周时明显升高,但与对照组比较差异无显著性(实验组、对照组分别为0.115±0.042,0.097±0.039,P>0.05)。血清髓鞘碱性蛋白含量(A值)均在第8周时显著上升(0.134±0.041)。③影像学检查发现实验组动物在免疫后2～4周出现不同程度的颅脑MRI异常信号。④组织学改变见实验组动物脑干、延髓、颈髓、胸髓及腰髓等部位髓鞘肿胀,未见炎细胞浸润及髓鞘脱失。对照组上述部位均未见明显改变。结论:空肠弯曲菌Penner4型可诱发中枢神经系统病变。
BACKGROUND：It has been found that central nervous system is involved in Guillain-Barre syndrome and Miller-Fisher syndrome, and the involved sites include optic nerve, brain stem and cerebellum. Abnormal signal of MRI can be observed in the brainstem and spinocerebellar tract of patients with Miller-Fisher syndrome. To establish an animal model of encephalitis after infection of Campylobacter jejuni, and investigate the mechanism of formation by means of imaging, immunology and pathology.
OBJECTIVE： To construct an animal model of lesion of central nervous system after infection of Campylobacterjejuni Penner 4.
DESIGN： A randomized grouping designed, controlled animal experiment.
SETTING： Department of Imaging and Department of Neurology, Second Hospital of Hebei Medical University.
MATERIALS： The experiment was carried out in the Laboratory of Molecular Imaging, the Second Hospital of Hebei Medical University between August and December 2003. Fifteen healthy flap-eared rabbits were randomly divided into experimental group （n=10） and control group （n=5）.
METHODS： In the experimental group, Campylobacter jejuni inactivated bacteria liquor was completely emulsified with complete Freund adjuvant （CFA） of the same volume in week 1, and then the rabbits were immunized with subcutaneous injection at multiple points of bilateral axilla, bilateral groins and side of back spine, 1 mL for each site, and 5 mL for each rabbit; The rabbits were further immunized with intraperitoneal injection of simple Campylobacterjejuni inactivated bacteria liquor in the following every two weeks, 5 mL for each time in each rabbit for 5 times. In the control group, the Campylobacter jejuni inactivated bacteria liquor was replaced by saline of the same volume, the injected method and time were all the same as those in the experimental group.
Evaluative methods： ① Symptoms and physical signs： their mental status, conditions of diet, urine and excrement, and activities of limbs were observed; ② Serological examination： the contents of anti-Campylobacterjejuni antibody, anti-IgG GM1 antibody and myelin basic protein （MBP） were detected with enzyme-linked immunoadsorbent assay （ELISA）; ③ MRI examination was applied to the randomly selected rabbits before every immunization with Toshiba 1.5 T MRI instrument. The scanning sequence included spin-echo T1-weighted image with the scanning parameter of 500/15 ms （TR/TE）; rapid spin-echo T2-weighted image, 4 000/108 ms （TR/TE）; fluid attented inversion recovery （Flair） sequence, the parameter was 10 000/120 ms （TR/TE）, inversion angle was 90°. The thickness of Scanning layer was 4.0 mm, and the layer space was 0.8 mm. ④ Histological examination： At 4 weeks after the first immunization, the attacked animals were induced to death by cardiac perfusion, and the skull was opened immediately to remove optic nerve, part white matter, hippocampus, brainstem, cerebellum and spinal cords of neck, chest and waist, which were fixed with formaldehyde solution （40 g/L）, and hematoxylin-eosin （HE） staining, fast blue staining and MBP immunohistochemical staining were performed respectively. At 10 weeks after immunization, 5 randomly selected rabbits in the experimental group and the 5 rabbits in the control group were treated with the same methods to obtain the histological samples.
MAIN OUTCOME MEASURES： The symptoms and physical signs, contents of anti-Campylobacterjejuni antibody, anti-IgG GM, antibody and MBP, imaging observation and histological examination were mainly observed.
RESULTS： Fifteen animals were enrolled, 14 were involved in the analysis of results, 1 rabbit in the experimental group died at 4 weeks after immunizatian. ① Mental symptoms and disorder of limb＇s activity occurred in 1 rabbit in the experimental group at 2 weeks after immunization. ② In the experimental group, titre of anti-Campylobacterjejuni-IgG antibody in serum reach the peak at 2-4 weeks. From week 2, the serum A value was significantly higher in the experimental group than in the control group （1.923±0.403, 0.973±0.633, P 〈 0.05）. The lgG type GM1 （A value） was obviously elevated at week 8, but insignificantly different from that in the control group （0.115±0.042, 0.097±0.039, P 〉 0.05）. The MBP content （A value） in serum was significantly elevated at the 8th week （0.134±0.041）. ③ The imaging examination showed that abnormal MRI signal of different degree occurred at 2-4 weeks after immunization in the experimental group. ④ The histological changes showed that there was swelling of myelin sheath at the sites of brainstem, medulla oblongata, cervical spinal cord, thoracic spinal cord and lumbar spinal cord in the experimental group, no inflammatory cell infiltration and deletion of myelin sheath were observed. No obvious changes at the above site were observed in the cantrol group.
CONCLUSION： Campylobacterjejuni Penner 4 can induce lesion of cen- tral nervous system.
出处
《中国临床康复》
CSCD
北大核心
2006年第16期174-177,F0003,共5页
Chinese Journal of Clinical Rehabilitation
基金
河北省科技厅资助项目(01276180D)~~
