Nogo受体拮抗剂与甲基强的松龙联合治疗对脊髓损伤修复的影响
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摘要
第一部分大鼠急性脊髓损伤动物模型的建立及脊髓组织病理的观察
目的：建立大鼠脊髓背侧半横断模型,观察性别差异对脊髓损伤(Spinal cord injury, SCI)后肢自发性功能恢复及脊髓组织病理变化的影响。方法：健康Wistar大鼠40只按性别随机分两组,构建大鼠胸髓(T9)背侧半横断模型,观察后肢自发性功能恢复的规律和特点。术后12h、1d、3d、7d处死动物,取出脊髓组织,用HE染色法观察脊髓的一般组织病理学变化。结果：大鼠胸髓背侧半横断后,损伤脊髓有明显出血、水肿、变性、空泡等,雌、雄大鼠后肢自发性功能恢复BBB评分上无统计学差异,所有动物的后肢运动BBB评分在各个时间的评分都非常接近,在术后4周达到了8分左右,并且在以后的时间里维持在8分的水平不再增加。但术后死亡率、泌尿系统的并发症,雌性大鼠明显少于雄性大鼠,且易于术后护理。结论：结合脊髓的一般组织病理学变化规律,SCI动物模型建立成功,可应用于SCI的实验研究。大鼠术后自发性后肢运动功能恢复与性别无关,因术后雌性大鼠护理上优于雄性大鼠,故建议应用雌性大鼠构建SCI模型。
第二部分Nogo受体(NgR)在大鼠脊髓组织中的表达及意义
目的：探讨SCI后髓鞘相关抑制分子Nogo受体NgR mRNA和蛋白表达的变化规律及意义,并探讨Nogo受体拮抗剂(NEP1-40)应用于SCI动物模型中的疗效。方法：健康Wistar大鼠108只随机分为假手术组、创伤对照组、NEP1-40治疗组,建立大鼠胸髓(T9)背侧半横断模型。NEP1-40的给药方法为术后于硬膜下管给予NEP1-4012.5 ug/(20μl/PBS·d),连续28d,对照组给予等量生理盐水。术后24h、3d、7d、14d、28d、42d处死动物,取出脊髓组织,逆转录-聚合酶链反应(RT-PCR)的方法检测NgRmRNA,免疫荧光及Western Blot的方法检测NgR蛋白表达,观察各组SCI后NgR的变化。结果：假手术组NgR mRNA及蛋白表达在各个时间点无显著变化(P＞0.05)。SCI脊髓组织中NgR mRNA表达量在损伤后24h表达减少,3d后上升,7d后逐渐上升至正常水平(与假手术组比较,P＞0.05)；至14d达到高峰并维持于较高水平,至伤后28d缓慢下降(与假手术组比较,P＜0.05)；伤后42d恢复至伤前水平。SCI脊髓组织中NgR蛋白表达量在损伤后24h表达减少,3d后再次下降,7d后逐渐上升至正常水平(与假手术组比较,P＞0.05)；至第14天达到高峰并维持于较高水平,至伤后28d缓慢下降(与假手术组比较,P＜0.05)；伤后42d恢复至伤前水平。SCI后14d、28d,NEPl-40组NgRmRNA及蛋白较创伤组均明显减少(P＜0.05)。结论：SCI后NgR表达呈现动态变化规律,针对NgR进行干预有望为SCI后的治疗找到新的靶点,NEP1-40在神经再生过程中可能发挥着重要作用。
第三部分甲基强的松龙对大鼠脊髓损伤后细胞凋亡及TNF-α表达的影响
目的：观察大剂量甲基强的松龙(methylprednisolone, MP)治疗对大鼠受损脊髓肿瘤坏死因子-α(TNF-α)、神经细胞凋亡及凋亡基因表达的影响。方法：健康雌性Wistar大鼠124只,随机分为假手术组、创伤对照组、MP治疗组。建立大鼠胸髓(T9)背侧半横断模型。MP的给药方法为术后30 min内经鼠尾静脉给予MP 30 mg／kg；对照组给予等量生理盐水。术后1h、4h、12h、24h、3d、7d处死动物,取出脊髓组织,TUNEL检测细胞凋亡,转录-聚合酶链反应(RT-PCR)的方法检测mRNA,免疫组织化学及Western Blot的方法检测蛋白表达,观察各组SCI后Bcl-2、Bax和TNF-α的变化。结果：采用TUNEL法检测细胞凋亡,结果SCI 24h时,脊髓组织细胞凋亡达高峰,然后逐渐下降,与假手术组比较,差异显著(P＜0.05),给予MP治疗后,凋亡率明显降低,与创伤对照组比较,差异具有显著性(P<0.05)。Bcl-2、Bax mRNA及蛋白结果显示,在SCI后4h、12h、3d、7d时MP组大鼠Bcl-2/Bax比值高于创伤组(P＞0.05),在SCI后24h,MP治疗组大鼠脊髓组织Bcl-2/Bax比值明显高于创伤组(P＜0.01). TNF-αmRNA及蛋白结果显示在SCI第12h时,SCI组大鼠脊髓组织TNF-α表达即达峰值,与假手术组比较,差异具有显著性(P＜0.05)；随着损伤时间的延长,TNF-α表达逐渐降低,至SCI后14d时,趋于正常水平。应用MP治疗后,治疗组大鼠TNF-α表达水平明显降低,与创伤对照组比较,差异具有显著性(P＜0.05)。结论：细胞凋亡是SCI早期主要的生物学事件；细胞凋亡的调控基因家族Bax/Bcl-2通过调节细胞凋亡参与SCI的发生发展；MP可抑制大鼠SCI细胞的凋亡,并可调节TNF-α等炎症相关因子含量,从而实现对SCI的影响。
第四部分Nogo受体拮抗剂与甲基强的松龙联合治疗促进大鼠脊髓损伤后轴突再生和功能恢复
目的：探讨NEP1-40与MP联合治疗对大鼠胸髓背侧半横断伤后脊髓再生修复的影响及可能机制。方法：健康雌性Wistar大鼠108只,随机分为创伤对照组、MP治疗组、NEP1-40治疗组和联合治疗组。建立大鼠胸髓(T9)背侧半横断模型。MP的给药方法为术后30 min内经鼠尾静脉给予MP30mg/kg; NEP1-40的给药方法为术后于硬膜下管给予NEP1-40 12.5 ug/(20μl/PBS·d),连续7d；对照组给予等量生理盐水。在不同时间点,免疫荧光、逆转录-聚合酶链反应(RT-PCR)、Western Blot的方法检测GAP-43、MBP mRNA; GAP-43、MBP、GFAP蛋白的表达,分别采用BBB运动学功能评分、生物素葡聚糖胺(BDA)顺行示踪、透射电镜观察等方法,观察动物的运动功能、脊髓再生的轴突和髓鞘。结果：SCI后14d、28d,联合用药组较其他各组GAP-43、MBP mRNA及蛋白的表达明显增加(P＜0.05),同时星形胶质细胞(GFAP)活化数目和活化持续时间明显减少(P＜0.05)。超微结构显示联合用药组轴突数量多于单独用药组,且髓鞘的完整性强于单独用药组。用药组SCI区及其远端BDA标记的神经纤维数量明显多于创伤对照组,联合治疗组最多(P＜0.05)。BBB行为学评分显示MP组的行为学评分提高在SCI两周内明显,而NEP1-40组行为学评分提高在SCI两周后明显,相对于单独用药干预组,联合用药产生更好的功能恢复与轴突再生(P＜0.05)。结论：本实验证实MP和NEP1-40联合使用后其作用机制是不同的、可以互补的。联合治疗对大鼠SCI后髓鞘再生和功能恢复有明显的促进作用,是SCI一种有效的联合治疗方案。
Part I The animal model establishment and the spinal cord histopathology
observation
Objective:To construct an animal model of spinal cord dorsal hemisection in adult rats, and to observe the differences of spontaneous functional recovery and histopathological changes after spinal cord injury(SCI) under different gender. Methods: 40 Wistar rats were randomized into two groups:male group; female group. Constructing SCI dorsal hemisection model(T9). Observing the hindlimb spontaneous function recovery features after injury.12h、Id、3d、7d postoperation, these rats were all killed, the spinal cord including the iniured site were taken out, and HE stain technique was used to observe the histopathological changes with light microscope in different groups. Results:There was significantly haemorrhage, edema, degeneration, vacuole and so on in spinal cord in rats with T9 dorsal transaction. The spontaneous hindlimb function BBB score have no significant difference between male and female rats. There are very close about hindlimb BBB score in all the time among all animals. Hindlimb BBB score of all animals reached about 8 postoperative 4th week, and at later time maintained at the level of 8. But the postoperative mortality and the urinary system complications in female rats were significantly less than male rats. The postoperative care on female rats is more facilitated than male rats. Conclusion:According to change rule of the common organization pathology change in the spinal cord, the acute spinal cord damage animal model established successfully, may be applied to the experimental study of the SCI. Spontaneous rats hindlimb motor function recovery BBB scores have no relation to animal gender. The postoperative care on female rats is more facilitated than male rats, so it is a good choice that applicate female rats to model.
spinal cord injury in rats
Objective:To investigate the expressions of a receptor of myelin-associated inhibitory molecule Nogo(NgR) at the mRNA and protein levels after SCI in rats, and evaluate the effect of Nogo antagonistic peptide(NEP1-40) on the treatment of SCI of adult rats model. Methods:108 Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a NEP1-40 group. Constructing SCI dorsal hemisection model(T9). NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS (PH7.4),20μl/d for 28 days. the control group was treated with saline injection.24h、3d、7d、14d、28d、42d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. RT-PCR was used to determine the change of NgR mRNA expressions while the immunofluorescence and Western Blot were used to detect the NgR protein in different groups. Results:no significant change of the level of NgR-mRNA and protein was detected at any time points mentioned above in the sham operation group(P>0.05). The level of NgR mRNA started to decrease at the 24th hour after injury and increased again at the 3th day, and increased to the normal level at the 7rd day(P>0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR mRNA kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. The level of NgR protein started to decrease at the 24th hour after injury, decreased again at the 3th day, and increased to the normal level at the 7rd day(P >0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR protein kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. At the 14th and 28th day the expression of NgR mRNA and protein decreased significantly in NEPl-40 group as compared with a trauma control group(P<0.05). Conclusion:NgR expression has dynamic changes after SCI, and NgR could be as one of the targets for neuroprotective therapy after SCI. NEP1-40 may play a pivotal role in regeneration of the nerve.
PartⅢEffect and mechanism of methylpredllisolone on apoptosis and
TNF-αexpression after spinal cord injury in rats
Objective:To investigate the effect of high dose Methylprednisolone(MP) on TNF-αcell apoptosis and correlative genes after acute spinal cord injury(ASCI). Methods:124 healthy Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a MP group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30mg/kg within 30 min after the operation, the control group was treated with saline injection.1h、4h、12h、24h、3d、7d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. The terminal deoxynucleotide transferase-mediated DUTP-biotin nick end labeling(TUNEL) was used to observe neural apoptosis; RT-PCR was used to determine the change of Bcl-2、Bax、TNF-αmRNA expressions while the immunohistochemistry and Western Blot were used to detect the Bcl-2、Bax、TNF-αprotein in different groups.
Results:It was found that the apoptosis cell can be seen clearly 24h after SCI(P<0.05 vs sham group), which can be prevented after treatment with MP(P<0.05 vs trauma control group). The results of image analysis of Bcl-2 and Bax mRNA and protein showed that the index of Bcl-2/Bax of MP group increased at 4h、12h、3d、7d after SCI(P>0.05 vs trauma control), While at 24h after MP treatment, the index of Bcl-2/Bax of MP treated group increased significantly(P<0.05 vs trauma control group). At 12h after SCI, amount of TNF-αmRNA and protein expression was at the peak value (P<0.05 vs sham group). With times go on, the amount of TNF-αexpression were lower gradually, and at 14d after SCI, the amount of TNF-αexpression were access to the normal level. While after MP treatment was applied, TNF-αexpression decreased significantly(P<0.05 vs trauma control group).
Conclusion:Apoptosis is an important event of SCI; Bax/Bcl-2 family take part in the development of SCI through control the process of apoptosis. MP can inhibit the process of apoptosis, regulate the expression of TNF-αafter SCI in rats.
monoclonal antibody promotes axonal regeneration and functional recovery
following spinal cord injury in rat
Objective:The purpose of this study was to investigate the effects of combination therapy with MP and NEP1-40 on morphological and functional recovery in adult rats subjected to thoracic dorsal hemisection SCI, and discusses its potential mechanisms. Methods:108 Female Wistar rats were randomized into four groups:A trauma control group, an MP group, a NEP1-40 group, an combined treatment group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30 mg/kg within 30 min after the operation, NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS(PH7.4),20μl/d for 28 days, the control group was treated with saline injection. At the different time point, immunofluorescence was used to detect the GAP-43、MBP、GFAP protein. RT-PCR was used to determine the change of GAP-43、MBP mRNA expressions while Western Blot was used to detect the GAP-43、MBP protein in different groups. The regenerated axons and the myelin sheaths were determined by BDA anterograrle tracing technique and transmission electron microscopy. Hindlimb functional recovery of rats in each group was evaluated by BBB locomotor functional scale. Results:In comparing with other groups, the results of image analysis of GAP-43、MBP protein of the combined treatment group increased at 14d、28d after SCI(P>0.05), the activated astrocyte number(labed with GFAP) and its activating duration of the combined treatment group decreased obviously after SCI(P<0.05). Electron microscopic analysis showed much more axons and better integrity of the myelin sheaths preservation in rats receiving combination therapy(P<0.05). The regenerated nerve fiber stained by BDA was seen in the defected position in MP group and NEP1-40 group, especially in combined treatment group(P<0.05), but almost nothing in the injured site in trauma control group. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NEP1-40 was not apparent until 2 weeks after SCI. Combined treatment with MP and NEP1-40 had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone(P<0.05). Conclusion: These in vitro data support the hypothesis that MP and NEP1-40 will enhance SCI recovery in a sequentially effective manner and suggest that they may have complementary and synergistic neuroprotective effects. Combination therapy with MP and NEP1-40 promotes significantly remyelination and functional recovery following SCI and is an effective approach to treatment of SCI.
目的：建立大鼠脊髓背侧半横断模型,观察性别差异对脊髓损伤(Spinal cord injury, SCI)后肢自发性功能恢复及脊髓组织病理变化的影响。方法：健康Wistar大鼠40只按性别随机分两组,构建大鼠胸髓(T9)背侧半横断模型,观察后肢自发性功能恢复的规律和特点。术后12h、1d、3d、7d处死动物,取出脊髓组织,用HE染色法观察脊髓的一般组织病理学变化。结果：大鼠胸髓背侧半横断后,损伤脊髓有明显出血、水肿、变性、空泡等,雌、雄大鼠后肢自发性功能恢复BBB评分上无统计学差异,所有动物的后肢运动BBB评分在各个时间的评分都非常接近,在术后4周达到了8分左右,并且在以后的时间里维持在8分的水平不再增加。但术后死亡率、泌尿系统的并发症,雌性大鼠明显少于雄性大鼠,且易于术后护理。结论：结合脊髓的一般组织病理学变化规律,SCI动物模型建立成功,可应用于SCI的实验研究。大鼠术后自发性后肢运动功能恢复与性别无关,因术后雌性大鼠护理上优于雄性大鼠,故建议应用雌性大鼠构建SCI模型。
第二部分Nogo受体(NgR)在大鼠脊髓组织中的表达及意义
目的：探讨SCI后髓鞘相关抑制分子Nogo受体NgR mRNA和蛋白表达的变化规律及意义,并探讨Nogo受体拮抗剂(NEP1-40)应用于SCI动物模型中的疗效。方法：健康Wistar大鼠108只随机分为假手术组、创伤对照组、NEP1-40治疗组,建立大鼠胸髓(T9)背侧半横断模型。NEP1-40的给药方法为术后于硬膜下管给予NEP1-4012.5 ug/(20μl/PBS·d),连续28d,对照组给予等量生理盐水。术后24h、3d、7d、14d、28d、42d处死动物,取出脊髓组织,逆转录-聚合酶链反应(RT-PCR)的方法检测NgRmRNA,免疫荧光及Western Blot的方法检测NgR蛋白表达,观察各组SCI后NgR的变化。结果：假手术组NgR mRNA及蛋白表达在各个时间点无显著变化(P＞0.05)。SCI脊髓组织中NgR mRNA表达量在损伤后24h表达减少,3d后上升,7d后逐渐上升至正常水平(与假手术组比较,P＞0.05)；至14d达到高峰并维持于较高水平,至伤后28d缓慢下降(与假手术组比较,P＜0.05)；伤后42d恢复至伤前水平。SCI脊髓组织中NgR蛋白表达量在损伤后24h表达减少,3d后再次下降,7d后逐渐上升至正常水平(与假手术组比较,P＞0.05)；至第14天达到高峰并维持于较高水平,至伤后28d缓慢下降(与假手术组比较,P＜0.05)；伤后42d恢复至伤前水平。SCI后14d、28d,NEPl-40组NgRmRNA及蛋白较创伤组均明显减少(P＜0.05)。结论：SCI后NgR表达呈现动态变化规律,针对NgR进行干预有望为SCI后的治疗找到新的靶点,NEP1-40在神经再生过程中可能发挥着重要作用。
第三部分甲基强的松龙对大鼠脊髓损伤后细胞凋亡及TNF-α表达的影响
目的：观察大剂量甲基强的松龙(methylprednisolone, MP)治疗对大鼠受损脊髓肿瘤坏死因子-α(TNF-α)、神经细胞凋亡及凋亡基因表达的影响。方法：健康雌性Wistar大鼠124只,随机分为假手术组、创伤对照组、MP治疗组。建立大鼠胸髓(T9)背侧半横断模型。MP的给药方法为术后30 min内经鼠尾静脉给予MP 30 mg／kg；对照组给予等量生理盐水。术后1h、4h、12h、24h、3d、7d处死动物,取出脊髓组织,TUNEL检测细胞凋亡,转录-聚合酶链反应(RT-PCR)的方法检测mRNA,免疫组织化学及Western Blot的方法检测蛋白表达,观察各组SCI后Bcl-2、Bax和TNF-α的变化。结果：采用TUNEL法检测细胞凋亡,结果SCI 24h时,脊髓组织细胞凋亡达高峰,然后逐渐下降,与假手术组比较,差异显著(P＜0.05),给予MP治疗后,凋亡率明显降低,与创伤对照组比较,差异具有显著性(P<0.05)。Bcl-2、Bax mRNA及蛋白结果显示,在SCI后4h、12h、3d、7d时MP组大鼠Bcl-2/Bax比值高于创伤组(P＞0.05),在SCI后24h,MP治疗组大鼠脊髓组织Bcl-2/Bax比值明显高于创伤组(P＜0.01). TNF-αmRNA及蛋白结果显示在SCI第12h时,SCI组大鼠脊髓组织TNF-α表达即达峰值,与假手术组比较,差异具有显著性(P＜0.05)；随着损伤时间的延长,TNF-α表达逐渐降低,至SCI后14d时,趋于正常水平。应用MP治疗后,治疗组大鼠TNF-α表达水平明显降低,与创伤对照组比较,差异具有显著性(P＜0.05)。结论：细胞凋亡是SCI早期主要的生物学事件；细胞凋亡的调控基因家族Bax/Bcl-2通过调节细胞凋亡参与SCI的发生发展；MP可抑制大鼠SCI细胞的凋亡,并可调节TNF-α等炎症相关因子含量,从而实现对SCI的影响。
第四部分Nogo受体拮抗剂与甲基强的松龙联合治疗促进大鼠脊髓损伤后轴突再生和功能恢复
目的：探讨NEP1-40与MP联合治疗对大鼠胸髓背侧半横断伤后脊髓再生修复的影响及可能机制。方法：健康雌性Wistar大鼠108只,随机分为创伤对照组、MP治疗组、NEP1-40治疗组和联合治疗组。建立大鼠胸髓(T9)背侧半横断模型。MP的给药方法为术后30 min内经鼠尾静脉给予MP30mg/kg; NEP1-40的给药方法为术后于硬膜下管给予NEP1-40 12.5 ug/(20μl/PBS·d),连续7d；对照组给予等量生理盐水。在不同时间点,免疫荧光、逆转录-聚合酶链反应(RT-PCR)、Western Blot的方法检测GAP-43、MBP mRNA; GAP-43、MBP、GFAP蛋白的表达,分别采用BBB运动学功能评分、生物素葡聚糖胺(BDA)顺行示踪、透射电镜观察等方法,观察动物的运动功能、脊髓再生的轴突和髓鞘。结果：SCI后14d、28d,联合用药组较其他各组GAP-43、MBP mRNA及蛋白的表达明显增加(P＜0.05),同时星形胶质细胞(GFAP)活化数目和活化持续时间明显减少(P＜0.05)。超微结构显示联合用药组轴突数量多于单独用药组,且髓鞘的完整性强于单独用药组。用药组SCI区及其远端BDA标记的神经纤维数量明显多于创伤对照组,联合治疗组最多(P＜0.05)。BBB行为学评分显示MP组的行为学评分提高在SCI两周内明显,而NEP1-40组行为学评分提高在SCI两周后明显,相对于单独用药干预组,联合用药产生更好的功能恢复与轴突再生(P＜0.05)。结论：本实验证实MP和NEP1-40联合使用后其作用机制是不同的、可以互补的。联合治疗对大鼠SCI后髓鞘再生和功能恢复有明显的促进作用,是SCI一种有效的联合治疗方案。
Part I The animal model establishment and the spinal cord histopathology
observation
Objective:To construct an animal model of spinal cord dorsal hemisection in adult rats, and to observe the differences of spontaneous functional recovery and histopathological changes after spinal cord injury(SCI) under different gender. Methods: 40 Wistar rats were randomized into two groups:male group; female group. Constructing SCI dorsal hemisection model(T9). Observing the hindlimb spontaneous function recovery features after injury.12h、Id、3d、7d postoperation, these rats were all killed, the spinal cord including the iniured site were taken out, and HE stain technique was used to observe the histopathological changes with light microscope in different groups. Results:There was significantly haemorrhage, edema, degeneration, vacuole and so on in spinal cord in rats with T9 dorsal transaction. The spontaneous hindlimb function BBB score have no significant difference between male and female rats. There are very close about hindlimb BBB score in all the time among all animals. Hindlimb BBB score of all animals reached about 8 postoperative 4th week, and at later time maintained at the level of 8. But the postoperative mortality and the urinary system complications in female rats were significantly less than male rats. The postoperative care on female rats is more facilitated than male rats. Conclusion:According to change rule of the common organization pathology change in the spinal cord, the acute spinal cord damage animal model established successfully, may be applied to the experimental study of the SCI. Spontaneous rats hindlimb motor function recovery BBB scores have no relation to animal gender. The postoperative care on female rats is more facilitated than male rats, so it is a good choice that applicate female rats to model.
spinal cord injury in rats
Objective:To investigate the expressions of a receptor of myelin-associated inhibitory molecule Nogo(NgR) at the mRNA and protein levels after SCI in rats, and evaluate the effect of Nogo antagonistic peptide(NEP1-40) on the treatment of SCI of adult rats model. Methods:108 Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a NEP1-40 group. Constructing SCI dorsal hemisection model(T9). NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS (PH7.4),20μl/d for 28 days. the control group was treated with saline injection.24h、3d、7d、14d、28d、42d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. RT-PCR was used to determine the change of NgR mRNA expressions while the immunofluorescence and Western Blot were used to detect the NgR protein in different groups. Results:no significant change of the level of NgR-mRNA and protein was detected at any time points mentioned above in the sham operation group(P>0.05). The level of NgR mRNA started to decrease at the 24th hour after injury and increased again at the 3th day, and increased to the normal level at the 7rd day(P>0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR mRNA kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. The level of NgR protein started to decrease at the 24th hour after injury, decreased again at the 3th day, and increased to the normal level at the 7rd day(P >0.05 vs sham group); Then the expression reached the peak at the 14th day. During the 14th to the 28th day after injury, the expression of NgR protein kept high(P<0.05 vs sham group) and decreased to the normal level(at the 42th day) step by step. At the 14th and 28th day the expression of NgR mRNA and protein decreased significantly in NEPl-40 group as compared with a trauma control group(P<0.05). Conclusion:NgR expression has dynamic changes after SCI, and NgR could be as one of the targets for neuroprotective therapy after SCI. NEP1-40 may play a pivotal role in regeneration of the nerve.
PartⅢEffect and mechanism of methylpredllisolone on apoptosis and
TNF-αexpression after spinal cord injury in rats
Objective:To investigate the effect of high dose Methylprednisolone(MP) on TNF-αcell apoptosis and correlative genes after acute spinal cord injury(ASCI). Methods:124 healthy Female Wistar rats were randomized into three groups:a sham operated group, a trauma control group, a MP group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30mg/kg within 30 min after the operation, the control group was treated with saline injection.1h、4h、12h、24h、3d、7d postoperation, these rats were all killed, the spinal cord including the injured site were taken out. The terminal deoxynucleotide transferase-mediated DUTP-biotin nick end labeling(TUNEL) was used to observe neural apoptosis; RT-PCR was used to determine the change of Bcl-2、Bax、TNF-αmRNA expressions while the immunohistochemistry and Western Blot were used to detect the Bcl-2、Bax、TNF-αprotein in different groups.
Results:It was found that the apoptosis cell can be seen clearly 24h after SCI(P<0.05 vs sham group), which can be prevented after treatment with MP(P<0.05 vs trauma control group). The results of image analysis of Bcl-2 and Bax mRNA and protein showed that the index of Bcl-2/Bax of MP group increased at 4h、12h、3d、7d after SCI(P>0.05 vs trauma control), While at 24h after MP treatment, the index of Bcl-2/Bax of MP treated group increased significantly(P<0.05 vs trauma control group). At 12h after SCI, amount of TNF-αmRNA and protein expression was at the peak value (P<0.05 vs sham group). With times go on, the amount of TNF-αexpression were lower gradually, and at 14d after SCI, the amount of TNF-αexpression were access to the normal level. While after MP treatment was applied, TNF-αexpression decreased significantly(P<0.05 vs trauma control group).
Conclusion:Apoptosis is an important event of SCI; Bax/Bcl-2 family take part in the development of SCI through control the process of apoptosis. MP can inhibit the process of apoptosis, regulate the expression of TNF-αafter SCI in rats.
monoclonal antibody promotes axonal regeneration and functional recovery
following spinal cord injury in rat
Objective:The purpose of this study was to investigate the effects of combination therapy with MP and NEP1-40 on morphological and functional recovery in adult rats subjected to thoracic dorsal hemisection SCI, and discusses its potential mechanisms. Methods:108 Female Wistar rats were randomized into four groups:A trauma control group, an MP group, a NEP1-40 group, an combined treatment group. Constructing SCI dorsal hemisection model (T9). MP was given through vena caudalis at a dose of 30 mg/kg within 30 min after the operation, NEP1-40 was given through sub-meninx fibrosa tube at a dose of 12.5 ug/PBS(PH7.4),20μl/d for 28 days, the control group was treated with saline injection. At the different time point, immunofluorescence was used to detect the GAP-43、MBP、GFAP protein. RT-PCR was used to determine the change of GAP-43、MBP mRNA expressions while Western Blot was used to detect the GAP-43、MBP protein in different groups. The regenerated axons and the myelin sheaths were determined by BDA anterograrle tracing technique and transmission electron microscopy. Hindlimb functional recovery of rats in each group was evaluated by BBB locomotor functional scale. Results:In comparing with other groups, the results of image analysis of GAP-43、MBP protein of the combined treatment group increased at 14d、28d after SCI(P>0.05), the activated astrocyte number(labed with GFAP) and its activating duration of the combined treatment group decreased obviously after SCI(P<0.05). Electron microscopic analysis showed much more axons and better integrity of the myelin sheaths preservation in rats receiving combination therapy(P<0.05). The regenerated nerve fiber stained by BDA was seen in the defected position in MP group and NEP1-40 group, especially in combined treatment group(P<0.05), but almost nothing in the injured site in trauma control group. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NEP1-40 was not apparent until 2 weeks after SCI. Combined treatment with MP and NEP1-40 had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone(P<0.05). Conclusion: These in vitro data support the hypothesis that MP and NEP1-40 will enhance SCI recovery in a sequentially effective manner and suggest that they may have complementary and synergistic neuroprotective effects. Combination therapy with MP and NEP1-40 promotes significantly remyelination and functional recovery following SCI and is an effective approach to treatment of SCI.
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