大鼠创伤性脑损伤炎性细胞因子的变化及意义

发布时间：2018-06-04 06:27

本文选题：脑损伤 + 炎性细胞因子　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:复制大鼠Marmarou’s闭合性脑损伤模型,研究大鼠创伤性脑损伤后血清及脑组织中炎性细胞因子的表达变化,探寻脑损伤生物标志物,为创伤性脑损伤临床诊断、治疗及预后评估提供理论基础。方法:选雄性SD大鼠随机分为手术对照组及损伤后1h、4h、8h、12h、24h、48h、72h组,复制Marmarou’s分级脑损伤模型。分别进行神经行为学评价、HE染色、焦油紫染色和酯化银染色进行病理学观察,测定脑组织及血清中MDA含量及MPO、SOD活性,检测血脑屏障通透性的改变;采用QAR-CYT-3芯片和ELISA技术检测炎性细胞因子在闭合性脑损伤中的变化。结果:1临床表现:轻度损伤组大鼠于打击后出现短暂呼吸暂停、心跳加速,但迅速恢复;中度损伤组大鼠95只,其中6只(6.31%)于打击后死亡,11只(11.58%)于打击后出现抽搐并伴有呼吸暂停,持续约30s,无惊厥;重度损伤组大鼠125只,其中41只(32.8%)于打击后全身抽搐、心跳停止死亡,68只(80.95%)出现抽搐并伴有呼吸暂停,持续约5min,28只(33.33%)出现惊厥,持续7-8s。2神经行为学评价:损伤后各组大鼠平衡能力、行走、逃避和四肢功能均较损伤前有所减弱,mNSS评分结果显示,重度损伤组评分最高,中度损伤组次之,轻度损伤组最低,随时间延长,神经行为学功能逐渐恢复,评分下降,至48h,轻度损伤组基本恢复正常。3病理学改变:轻度损伤组大鼠大体病理学无明显改变,脑干偶见少量蛛网膜下腔出血;中度损伤组蛛网膜下腔出血明显,主要分布在脑底及脑干,无挫伤,切面偶见双侧脑室少量出血;重度损伤组可见大量蛛网膜下腔出血,主要分布在脑底、小脑及脑干,切面可见胼胝体点状出血及双侧脑室出血,顶叶偶见轻微挫伤。HE染色可见手术对照组大鼠海马区神经元排列整齐,胞核清晰呈淡紫色,包浆呈淡粉色。轻度损伤组大鼠在损伤后1~4h时,CA1区锥形细胞肿胀变圆,排列疏松,包浆淡染;损伤后8h,海马ca1区出现散在分布的红色神经元;损伤后12h,坏死神经元细胞增多,胞核固缩细胞固缩成三角形,呈强嗜酸性。中、重度损伤组病理学改变较轻度损伤组明显,坏死神经元数量增多。焦油紫染色可见坏死神经元呈深紫色,损伤后各组坏死神经元分布趋势同he染色。在酯化银染色中,损伤神经元呈黑色,神经纤维呈波浪状改变。4生化指标改变:(1)tbi大鼠脑组织mpo活性变化:轻、中、重度损伤组大鼠脑组织mpo活性分别于损伤后12h、8h和4h开始升高,至24h达高峰,然后逐渐下降,损伤程度越重,升高幅度越明显,与对照组相比有显著性差异(p0.05或p0.01)。(2)tbi大鼠脑组织及血清sod活性变化:损伤后各组大鼠脑组织sod活性于8h开始降低,损伤后12h达低谷,然后逐渐恢复,损伤程度越重,降幅越明显。损伤后8~48h,损伤各组大鼠脑组织sod活性均低于对照组,有显著性差异(p0.05或p0.01)。损伤后72h,损伤各组脑组织sod活性恢复正常,与对照组相比无显著性差异(p0.05)。损伤后各组大鼠血清sod活性逐步降低,至12h达低谷,然后逐渐恢复,变化大体同脑组织sod活性改变一致。(3)tbi大鼠脑组织及血清中mda含量的变化:损伤后各组大鼠脑组织mda含量逐渐上升,至12h达高峰,然后逐渐下降。与手术对照组相比,损伤后8h,轻度损伤组脑组织mda含量升高;损伤后1~12h,中度损伤组脑组织mda含量逐步上升;损伤后1~72h,重度损伤组脑组织mda含量均升高,与手术对照组相比差别均有有显著性差异(p0.05或p0.01)。损伤后各组大鼠血清mda含量逐渐上升,至8~12h达高峰,然后逐渐下降。损伤后12~24h,轻度损伤组血清mda含量升高;损伤后12~48h,中度损伤组血清mda含量升高;损伤后1~72h,重度损伤组血清mda含量升高,与手术对照组相比,差别均有显著性差异(p0.05或p0.01)5血脑屏障的改变:脑皮质eb含量检测结果显示,大鼠血脑屏障通透性在损伤后迅速增加,至4h达高峰,然后逐渐下降。损伤后4~48h,轻度损伤组大鼠血脑屏障通透性均高于手术对照组,差别有显著性差异(p0.01)。中、重度损伤组大鼠血脑屏障通透性均高于手术对照组和轻度损伤组(p0.05或p0.01)。重度损伤组大鼠各组血脑屏障通透性均高于中度损伤组,差别有显著性差异(p0.05或p0.01)。6细胞因子的检测:(1)il-10:脑损伤组大鼠血清il-10含量呈先上升后下降而后逐渐恢复趋势。损伤后8h,中度损伤组大鼠血清il-10含量升高,与手术对照组相比有显著性差异(p0.05);损伤后24h,轻、中度损伤组血清il-10含量下降,与手术对照组相比有显著性差异(p0.05);至72h,轻、中度损伤组血清il-10含量逐渐恢复正常。损伤组大鼠海马组织il-10表达先升高后下降。损伤后24h,轻度损伤组il-10含量升高,与手术对照组相比有显著性差异(p0.05),而后逐渐下降。损伤后8~12h,中度损伤组il-10含量升高,与手术对照组、轻度损伤组相比均有显著性差异(p0.05),而后逐渐下降。损伤后4h,重度损伤组il-10含量升高,与手术对照组相比有显著性差异(p0.05),然后逐渐下降,于12h到达最低点,与手术对照组相比有显著性差异(p0.05或p0.01),损伤后24~48h,il-10含量逐渐恢复正常。(2)fractalkine:脑损伤后,轻度损伤组大鼠血清fractalkine含量逐渐下降,至24h达到低谷,与手术对照组相比有显著性差异(p0.05),然后逐渐升高;中度损伤组大鼠血清fractalkine含量逐渐升高,至24h达到高峰,与手术对照组、轻度损伤组相比有显著性差异(p0.05)。大鼠海马组织fractalkine的表达呈先升高后下降趋势。损伤后12~24h,轻度及中度损伤组fractalkine含量升高,与手术对照组相比有显著性差异(p0.05或p0.01)。损伤后12h,重度损伤组fractalkine含量升高,与手术对照组、轻度损伤组相比均有显著性差异(p0.01);损伤后24h,fractalkine含量下降,与中度损伤组相比有显著性差异(p0.05)。(3)prolactinr(prlr):脑损伤后,轻度损伤组血清中prlr含量呈先升高后下降、随后又升高趋势;中度损伤组血清中prlr含量先下降,而后逐渐恢复正常。损伤后8h,轻度损伤组血清中prlr含量升高,与手术对照组相比有显著性差异(p0.05);中度损伤组prlr含量下降,与手术对照组及轻度损伤组相比有显著性差异(p0.05)。损伤后24h,轻度损伤组prlr含量下降,与手术对照组及中度损伤组相比有显著性差异(P0.05)。损伤后72h,轻度损伤组PRLR含量升高,与手术对照组相比有显著性差异(P0.05)。各损伤组海马组织中PRLR含量升高,4h达高峰,随后逐渐下降,恢复至对照组水平。损伤后4~12h,轻度损伤组PRLR含量升高,与手术对照组相比,均有显著性差异(P0.05)。损伤后4h~8h,中度损伤组PRLR含量升高,与手术对照组相比有显著性差异(P0.05),损伤后12~24h,PRLR含量逐渐下降,与手术对照组相比无显著性差异(P0.05),损伤后48~72h,PRLR含量进一步下降,与手术对照组相比有显著性差异(P0.05或P0.01)。损伤后4~8h,重度损伤组PRLR含量升高,与手术对照组相比有显著性差异(P0.05),然后逐渐降低,至72h,PRLR含量降低,与手术对照组、轻度损伤组、中度损伤组相比均有显著性差异(P0.05)。结论:1成功复制大鼠分级TBI动物模型,损伤越重,病理学改变、急性炎症反应、氧化应激反应及血脑屏障损害越严重。2脑损伤可引起血清及海马组织中炎性细胞因子表达变化。血清和海马中检测到的炎性细胞因子表达存在差异,提示脑损伤后引发的免疫应答存在延迟反应。3在血清和海马中检测到的差异表达炎性细胞因子可能是介导继发性脑损伤的关键靶点,对其深入研究探索,有助于了解脑损伤的分子生物学机制,为脑损伤的诊断和治疗提供理论依据。
[Abstract]:Objective: to reproduce the model of Marmarou 's closed brain injury in rats, to study the expression of inflammatory cytokines in the serum and brain tissue of the rat after traumatic brain injury, to explore the biomarkers of brain injury, and to provide a theoretical basis for the clinical diagnosis, treatment and prognosis evaluation of traumatic brain injury. Methods: the male SD rats were randomly divided into surgical control. Group and after injury 1H, 4h, 8h, 12h, 24h, 48h, 72h, replicate Marmarou 's classification brain damage model. Neurobehavioral Evaluation, HE staining, tar purple staining and esterified silver staining were performed to observe the pathological changes of MDA content in brain tissue and serum, MPO, activity and detection of blood brain barrier permeability. The changes of inflammatory cytokines in closed brain injury were detected by A. Results: 1 clinical manifestations: mild injury group had transient apnea, rapid heartbeat, but rapid recovery; 95 rats in moderate injury group, of which 6 (6.31%) died after the attack, 11 (11.58%) had convulsions and apnea after the attack. There was no convulsion in 30s, and 125 rats in severe injury group, of which 41 (32.8%) were convulsive after the attack, the heart beat was stopped, 68 (80.95%) had convulsions with apnea, and continued about 5min, 28 (33.33%) had convulsions and continued 7-8s.2 Neurobehavioral Evaluation: the balance ability, walking, escape and limb function of all rats after injury were compared. The mNSS score showed that the score of the severe injury group was the highest, the moderate injury group was the highest, the mild injury group was the lowest, the neurobehavioral function gradually resumed with the time, the score decreased, to 48h, and the mild injury group basically recovered the normal.3 pathological changes: the mild injury group had no obvious changes in general pathology, brain stem pairs. A small amount of subarachnoid hemorrhage was seen. The subarachnoid hemorrhage in the moderate injury group was obvious, mainly in the brain and brain stem, without contusion, and a small amount of hemorrhage in the bilateral ventricle, and a large number of subarachnoid hemorrhage in the severe injury group, mainly in the brain fundus, cerebellum and brain stem, and the punctate hemorrhage of the corpus callosum and bilateral ventricle hemorrhage were seen in the tangent surface. Top of the subarachnoid hemorrhage and the top of the ventricle were seen in the cutting surface. .HE staining of slight contusion showed that the neurons in the hippocampus of the operation control group were arranged neatly, the nucleus of the cell was clear in the light purple, and the pulp was pale pink. In the mild injury group, the conical cells in the CA1 area were swollen and round when the injury was 1~4h, and the cells were loosely arranged and the pulp was pale dye; after the injury, the red neurons scattered in the hippocampus CA1 area appeared in the red neurons; injury was damaged in the hippocampus CA1 area. After 12h, the necrosis neuron cells increased and the nucleus retraction cells of the nucleus became triangular and strongly eosinophilic. In the severe injury group, the pathological changes were more obvious than those in the mild injury group, and the number of necrotic neurons increased. The necrotic neurons were dark purple in the tar purple staining. The injured neurons were black and the nerve fibers changed the biochemical index of.4 in wave form: (1) the activity of MPO in the brain tissue of TBI rats was changed: the activity of MPO in the brain tissue of the rats of the moderate and severe injury groups began to rise after the injury, respectively, 12h, 8h and 4h, and then to the peak of 24h, then gradually decreased, the more significant the damage was, the more obvious the increase was, compared with the control group. There were significant differences (P0.05 or P0.01). (2) the changes in the activity of brain tissue and serum SOD in TBI rats: the activity of SOD in the brain tissue of the rats after injury was reduced after injury, and the 12h reached the low valley after injury, and then gradually recovered, the more serious the damage was, the more obvious the decrease was. The activity of SOD in the brain tissue of rats after injury was lower than that of the control group, which was significantly lower than that of the control group. There was a significant difference. Different (P0.05 or P0.01). After 72h injury, the activity of SOD in the brain tissue of each group recovered to normal, and there was no significant difference compared with the control group (P0.05). The serum SOD activity decreased gradually after the injury, to the valley of 12h, and then gradually recovered. (3) the changes of MDA content in the brain tissue and serum of the rats were changed. After injury, the content of MDA in brain tissue increased gradually after injury to the peak of 12h, and then decreased gradually. Compared with the operation control group, the content of MDA in the brain tissue of the mild injury group increased after the injury, 8h and the mild injury group. After the injury, the content of MDA in the brain tissue of the moderate injury group was gradually increased, and the level of MDA in the brain tissue of the severe injury group increased after 1 ~72h, and the operation was the same as that in the severe injury group. There were significant differences between the groups (P0.05 or P0.01). After the injury, the serum MDA content increased gradually, to the peak of 8~12h, and then decreased gradually. After the injury, the serum MDA content in the mild injury group increased after 12~24h injury, and the serum MDA content in the moderate injury group increased after the injury, and the serum MDA content of the severe injury group was raised after injury. Higher, compared with the operation control group, the difference was significant (P0.05 or P0.01) 5 blood brain barrier change: the cerebral cortex EB content detection results showed that the blood brain barrier permeability of rats increased rapidly after injury, to the peak of 4h, and then gradually decreased. After injury, the permeability of blood brain barrier in the mild injury group was higher than that of the operation control group. The difference was significant (P0.01). In the severe injury group, the permeability of blood brain barrier in the rats was higher than that of the operation control group and the mild injury group (P0.05 or P0.01). The blood brain barrier permeability in the severe injury group was higher than that of the moderate injury group. There was a significant difference (P0.05 or P0.01).6 cytokine detection: (1) the rats of il-10: brain injury group Serum IL-10 content increased first and then decreased and then gradually recovered. After 8h injury, the serum IL-10 content in the moderate injury group increased significantly (P0.05) compared with the operation control group (P0.05). The serum level of 24h, light and moderate injury group decreased after injury (P0.05) compared with the operation group (P0.05); to 72h, light, moderate injury. The content of serum IL-10 in the group of the group gradually recovered to normal. The expression of IL-10 in the hippocampus of the injured group first increased and then decreased. The content of IL-10 in the mild injury group increased after 24h injury, and there was a significant difference compared with the operation control group (P0.05), and then gradually decreased. The content of IL-10 in the moderate injury group was higher than that in the moderate injury group, compared with the operation control group and the mild injury group. There were significant differences (P0.05), and then decreased gradually. The content of IL-10 in 4H and severe injury group increased significantly after injury (P0.05), and then decreased gradually and reached the lowest point at 12h (P0.05 or P0.01) compared with the operation control group (P0.05 or P0.01), and the IL-10 content gradually returned to normal after injury (2) fractalk. After ine: brain injury, the content of serum fractalkine decreased gradually in the mild injury group, and reached the low valley to 24h. Compared with the operation control group, there was a significant difference (P0.05), and then increased gradually. The serum fractalkine content in the moderate injury group increased gradually and reached the peak to 24h (P0). Compared with the operation control group, the mild injury group had a significant difference (P0 .05). The expression of fractalkine in the hippocampus of rats was increased first and then decreased. The content of fractalkine in 12~24h, mild and moderate injury group was significantly higher than that in the control group (P0.05 or P0.01). The content of fractalkine in the severe injury group was higher than that in the severe injury group, and the level of fractalkine in the severe injury group was significantly higher than that in the operation control group and the mild injury group. The sex difference (P0.01); the content of 24h and fractalkine decreased after injury (P0.05). (3) prolactinr (PRLR): after brain injury, the serum level of PRLR in the mild injury group increased first and then decreased, then increased, and the serum PRLR content in the moderate injury group decreased first and then gradually returned to normal. 8h, light after injury, was light. The level of PRLR in the serum of the degree injury group was significantly higher than that in the operation control group (P0.05). The PRLR content in the moderate injury group decreased significantly (P0.05) compared with the operation control group and the mild injury group (P0.05). After the injury, the content of the mild injury group decreased, and there was a significant difference compared with the operation control group and the moderate injury group (P0.05 After the injury, the content of PRLR in the mild injury group increased, and compared with the operation control group (P0.05). The content of PRLR in the hippocampus of the injured group increased, the 4H reached the peak, and then decreased gradually to the control level. After the injury, the PRLR content in the mild injury group increased, and there were significant differences (P0.05) compared with the operation control group (P0.05). The content of PRLR in 4h~8h and moderate injury group was higher than that of the control group (P0.05). The content of 12~24h and PRLR decreased gradually after the injury, and there was no significant difference compared with the operation control group (P0.05). The content of 48~72h and PRLR decreased further after the injury. There was a significant difference (P0.05 or P0.01) compared with the operation control group (P0.05 or P0.01). 4~8h after injury. The content of PRLR in severe injury group was significantly higher than that in the operation control group (P0.05), and then decreased gradually, to 72h, and PRLR content decreased. Compared with the operation control group, the mild injury group and the moderate injury group, there were significant differences (P0.05). Conclusion: 1 the successful reproduced rat classification TBI animal model, the heavier the injury, the pathological changes, acute inflammation. The expression of inflammatory cytokines in serum and hippocampal tissues can be induced by.2 injury, the more severe the oxidative stress reaction and the damage of the blood brain barrier, the difference in the expression of inflammatory cytokines in the serum and hippocampus suggests the difference in the detection of delayed response.3 in the serum and hippocampus after the brain injury. The expression of inflammatory cytokines may be the key target to mediate secondary brain injury. It is helpful to understand the molecular biological mechanism of brain injury and provide a theoretical basis for the diagnosis and treatment of brain injury.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

【参考文献】