周围神经移植联合aFGF治疗急性脊髓损伤的实验研究

目的:探讨周围神经移植联合酸性成纤维细胞生长因子(aFGF)治疗大鼠脊髓损伤(SCI)的可行性及效果.方法:雌性Sprague-Dawley大鼠115只;随机分为4组.A组(自体肋间神经移植组;n=30):在T9水平横行切断脊髓并切除3mm;植入自体肋间神经;B组(自体肋间神经联合aFGF移植组;n=30):同法制备脊髓横断模型;脊髓缺损处植入自体肋间神经和含aFGF的纤维蛋白凝胶;C组(脊髓横断组;n=30):同法制备脊髓横断模     

胶原支架结合脑源性神经营养因子移植促进全横断脊髓损伤大鼠轴突再生及运动功能恢复的研究

目的评价胶原支架结合脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)移植修复大鼠全横断脊髓损伤的效果。方法将32只成年雌性SD大鼠随机分成4组(n=8):A组为假手术组,只暴露T_9、T_(10)段脊髓;B、C、D组切除长4 mm的T_9、T_(10)段脊髓后,C、D组分别于损伤处植入相应长度的线性有序胶原支架(linear ordered collagen scaffolds,LOCS)和结合了胶原结合结构域(collagen binding domain,CBD)-BDNF的LOCS。术前及术后3个月内每周对大鼠进行BBB运动功能评分。术后3个月,实施神经电生理检测各组大鼠运动诱发电位(motor evoked potential,MEP);然后于L_2段脊髓组织注射荧光金(fluorogold,FG)实施逆行示踪,1周后取大鼠大脑及胸、腰段脊髓组织,脱水后观察脊髓组织形态;取包含损伤区的胸、腰段脊髓组织作切片。其中,脊髓冠状切片于激光共聚焦显微镜下进行观察,计算FG阳性细胞积分吸光度(IA)值;胸段脊髓组织水平切片采用免疫荧光染色,观察全横断脊髓损伤造模情况、脊髓损伤区轴突再生情况、D组再生轴突的突触形成情况。结果术后各时间点B、C、D组BBB评分均显著低于A组(P0.05);术后2～12周D组BBB评分均明显高于B、C组(P0.05)。电生理检测示,B组未观测到MEP;C、D组MEP潜伏期显著长于A组,C组显著长于D组,差异均有统计学意义(P0.05)。脊髓组织形态观察示,B组脊髓损伤区域向两端延伸,损伤部位组织破坏严重;C、D组脊髓形态恢复较好,D组更接近正常组织形态。逆行示踪结果显示,各组大鼠损伤区以下的腰段脊髓灰质中均充满了FG阳性细胞;在损伤区以上的胸段脊髓中,A组FG阳性区域IA值显著大于B、C、D组(P0.05),C、D组大于B组(P0.05),C、D组间差异无统计学意义(P0.05)。免疫荧光染色示,自同一脊髓背侧至腹侧选出的组织切片显示了明显异于正常组织的全横断脊髓损伤区域。A组NF阳性轴突数明显多于B、C、D组,C、D组多于B组,D组多于C组,差异均有统计学意义(P0.05)。结论 LOCS结合CBD-BDNF移植可以促进大鼠全横断脊髓损伤后轴突再生以及后肢运动功能恢复。     

利用一级神经元重建完全性脊髓损伤大鼠后肢运动功能的实验研究

目的探讨利用一级神经元重建完全性脊髓损伤大鼠后肢运动功能的效果。方法取40只成年雌性SD大鼠,体质量300~350 g,制备L1椎体水平横断型完全性脊髓损伤模型;模型制备后2周,将大鼠随机分为对照组和实验组(n=20);其中实验组行右后肢一级神经元直接重建手术,对照组除不缝合胫神经远端与股神经近端外,其余处理同实验组。术后观察大鼠一般情况,并于7、30、50、70 d对双侧后肢行BBB评分,观察后肢运动功能恢复情况;于70 d取材行HE染色、神经丝蛋白200(neurofilament 200,NF-200)免疫组织化学染色及辣根过氧化物酶(horseradish peroxidase,HRP)示踪标记染色观察脊髓重建区变化。结果饲养期间6只大鼠死亡。两组右后肢运动功能无明显恢复,各时间点BBB评分均为0分;左后肢运动功能均有不同程度恢复,但各时间点两组BBB评分比较,差异均无统计学意义(P0.05)。实验组HE染色可见脊髓重建部位坐骨神经嵌入脊髓中,坐骨神经束膜显示清楚,连接部位脊髓无明显萎缩;NF-200免疫组织化学染色可见脊髓重建部位周围神经内神经元轴突呈阳性染色,且周围神经与脊髓形成联系。HRP逆行示踪标记染色示,实验组脊髓重建部位头端见HRP显色,对照组无着色。结论一级神经元直接重建手术可使大鼠周围神经与中枢神经通过残留神经元轴突残端再生,重建相应的神经通路,但未见到周围神经支配靶肌肉运动功能的恢复。     

蛛网膜下腔灌注胶质细胞源性神经营养因子对损伤脊髓再生及功能恢复的影响

目的：探讨胶质细胞源性神经营养因子（GDNF）对大鼠脊髓完全性横断后脊髓再生及功能恢复的影响。方法：采用大鼠胸段（T7-T8）脊髓完全横切损伤模型，将SD雌性大鼠随机分为正常组（n=6）、假手术组（n=6）、单纯横断组（n=10）、GDNF治疗组（n=10）。于大鼠脊髓损伤术后不同时间点进行行为学评估。24周时行生物素葡聚糖胺（BDA）顺行示踪处理，取材前行电生理检测。所取脊髓标本作神经中丝（NF-200）、生长相关肽-43（GAP-43）、胶质原纤维生长蛋白（GFAP）免疫组化检查，并应用图像分析系统进行定量分析。结果：行为学评分表明，3周后，GDNF组好于单纯横断组（P〈O．05），术后24周，GDNF组和单纯脊髓横断组中均未记录到SEP波形，BDA示踪也未见伤区及远段蓝染的神经纤维，但GDNF组空泡样变较单纯横断组轻。免疫组化图像分析GDNF组的NF-200和GAP-43染色结果与单纯横断组间无统计学差异（P〉0．05）：但GDNF组GFAP染色明显弱于单纯横断组（P〈0．05）。结论：GDNF能一定程度上改善脊髓损伤区及两端的神经细胞功能，但没有功能意义上的神经纤维再生。     

基于神经搭桥的中枢神经-周围神经直接连接技术治疗脊髓损伤的实验研究

目的 研究通过自体周围神经移植桥接中枢神经系统和周围神经系统,观察中枢神经系统轴突能否再生并通过移植的周围神经重建脊髓损伤大鼠的股四头肌神经通路,恢复脊髓损伤大鼠肢体部分运动功能. 方法 取200～220 g SD雌性大鼠33只,随机分成3组.A组(18只):脊髓左侧半横断,周围神经移植加远端吻合股神经;B组(10只):脊髓左侧半横断组,周围神经假移植加远端吻合股神经;C组(5只):假手术组,不做任何处理.通过动物行为学观察、肌肉湿重、组织学检查和生物素葡聚糖胺(BDA)顺行示踪评价中枢神经-周围神经直接连接对脊髓损伤的修复效果.结果 术后14周,A组大鼠左下肢有逐渐恢复的运动,股四头肌肌肉力量评分为2.40±1.12,较第4周(0.80±0.41)和第8周( 1.20±0.67)差异有统计学意义(P＜0.05);B组大鼠左下肢未见任何恢复.第14周时,A组动物左下肢股四头肌肌肉湿重占对侧股四头肌的比值(31.0±8.0)较B组(15.0±4.0)差异有统计学意义(P＜0.05).在移植的周围神经中,出现BDA阳性及NF200阳性的神经纤维. 结论 初步结果显示自体周围神经桥接中枢神经-周围神经能起到修复脊髓损伤的作用.     

氯化锂联合自体激活雪旺细胞治疗脊髓损伤

目的 观察氯化锂联合移植自体激活雪旺细胞(AASCs)在治疗脊髓损伤(SCI)中的作用.方法 Wistar成鼠48只建立T10急性SCI模型.随机均分为4组:AASCs移植组、氯化锂移植组、氯化锂联合AASCs组和DMEM移植组.术后行体感诱发电位(SEP),运动诱发电位(MEP)检查和BBB评分.12周后进行10%BDA神经顺行示踪标记,切片后,行Cy3荧光探针染色、免疫组织化学及苏木素-伊红(HE)染色.结果 术后4周BBB评分各组之间差异开始有统计学意义(F=46.897,P<0.05).实验结束时,SEP、MEP波幅值各组间差异有统计学意义(F-SEP=15.47,P<0.05;F-MEP=8.974,P<0.05);BDA示踪,HE和免疫组织化学染色各组间差异有统计学意义(P<0.05).结论 氯化锂联合AASCs移植可以显著改善SCI后的功能恢复.     

显微操作在脐血干细胞移植修复大鼠脊髓损伤中的应用

[目的]建立大鼠脊髓全横断损伤模型,应用显微操作方法和人脐血干细胞移植修复大鼠脊髓损伤,观察显微操作技术对大鼠受损脊髓功能恢复的影响.[方法]取足月健康顺产新生儿脐血,分离、培养脐血干细胞.60只SD雌性大鼠,随机分为假手术对照组(A组,n=10)、实验对照组(B组,n=10)、常规治疗组(C组,n=20)和显微治疗组(D组,n=20).A组大鼠只打开椎板;B、C组大鼠采用常规方法行T 8-9,平面脊髓全横断,造成急性脊髓损伤,C组大鼠于脊髓两断端用1 μl注射器分别注射脐血干细胞悬液1μl(干细胞浓度为6×10 9/L～7×109/L),B组同法注射等量PBS液;D组采用显微操作技术行脊髓横断,并以口径50 μm的毛细玻璃针用相同方法注射脐血干细胞.术后1～8周内每周进行1次后肢BBB评分,术后第8周处死大鼠,对比观察术中和术后第8周各组大鼠脊髓大体标本外形、颜色、质地和体积大小改变.[结果]A组大鼠手术前后运动功能、脊髓大体标本未见明显变化.B、C、D组大鼠术后双侧后肢完全性瘫痪,B组大鼠无明显恢复.而C、D组大鼠,从术后第2周开始逐渐恢复部分后肢运动功能.第3周以后,D组大鼠后肢运动功能BBB评分明显优于C组,具有显著性差异(P＜0.05).术后第8周,所有大鼠脊髓横断处均瘢痕愈合,B组大鼠远侧段脊髓干瘪、皱缩;C组大鼠远侧段脊髓直径轻度变细,外形尚饱满;D组大鼠横断处两侧脊髓直径无明显差异,外形圆润,但局部可见明显瘢痕粘连.[结论]人脐血干细胞移植对脊髓横断性损伤具有较好的治疗作用,显微操作技术的应用能最大可能地减少人为造成的脊髓损伤,有利于脊髓损伤的功能修复.     

神经干细胞-多肽自组装凝胶复合体移植治疗脊髓损伤

目的 观察神经干细胞( NSCs)复合多肽自组装凝胶移植对大鼠脊髓损伤(SCI)后功能修复的影响.方法 36只SD大鼠造模后1周随机分为3组,分别为DMEM/F12对照组(n＝12)、NSCs移植组(n=12)和NSCs-凝胶移植组(n=12).通过不同时间点BBB评分、病理组织学、免疫荧光技术评价脊髓损伤的修复.结果 移植后2周开始3组大鼠各时间点评分差异有统计学意义(P＜0.01),且组间差异均有统计学意义(P＜0.01);移植后6周,病理切片示C组大量再生的神经纤维桥接脊髓断端,胶质瘢痕不明显;免疫荧光染色示C组5-溴脱氧尿嘧啶核苷(5-BrdU)/NF-200双标阳性细胞比例(24.83±1.47)％明显多于B组(6.83±1.47)％(P＜0.01),但B组BrdU/GFAP双标阳性细胞比例(42.17±2.71)％明显多于C组(34.33±4.63)％ (P＜0.01).结论 自组装多肽凝胶能提高神经干细胞向神经元分化的比例,复合移植能更有效地促进脊髓功能恢复.     

肋间神经转位脊髓神经根桥接联合应用GDNF促进大鼠脊髓损伤后功能恢复

目的：肋间神经转位脊髓神经根桥接联合应用胶质源性神经营养因子(GDNF)恢复大鼠脊髓损伤的功能。方法：将成年大鼠分为脊髓半切洞损伤组(A组)、脊髓半切洞损伤 肋间神经转位脊髓神经根桥接组(B组)、脊髓半切洞 肋间神经转位脊髓神经根桥接 胶质源性神经营养因子(C组)。手术后应用联合行为评分(CBS)。感觉诱发电位(SEP)和运动诱发电位(MEP)检查，测定脊髓功能恢复情况。结果：3组CBS得分A组＞B组＞C组，SEP和MEP潜峰时，均A组＞B组＞C组，统计分析均有显著差异性(P＜0.05)。结论：肋间神经转位脊髓神经根桥接联合应用胶质源性神经营养因子能促进损伤脊髓功能的恢复。     

电针联合嗅鞘细胞移植对大鼠脊髓损伤神经轴突再生及走向的影响

目的:探讨电针对嗅鞘细胞(OECs)移植大鼠轴突再生的影响及作用机制。方法:2.5月龄Sprague Dawley(SD)雄性大鼠72只,体重(220±20)g,采用挫伤加全横断的造模方法造成T9脊髓损伤模型后随机分成模型组、电针组、嗅鞘细胞组和电针加嗅鞘细胞组。各组动物于造模后4周和8周注射5%荧光金水溶液(flurosecentgold,FG) 0.5 μl进行逆行标记,后进行荧光金逆行示踪观察以及动物行为学观察(BBB评分).结果:(1)BBB评分结果示术后第1天至第1周,各组间比较差异无统计学意义(P>0.05);从第3周开始,电针+OECs组高于模型组、OECs组、电针组3组(P<0.05).(2) 荧光金逆行示踪结果显示:术后4、8周各组脊髓内均可观察到有FG阳性神经纤维再生;在脊髓损伤区,电针加OECs组通过脊髓损伤区荧光金标记的阳性神经纤维数量多于其他3组,走行较其他3组规则。结论:电针联合OECs移植治疗能够极大的促进脊髓损伤大鼠神经纤维的再生以及大鼠后肢功能的恢复,能够恢复神经传导通路,并对再生的神经纤维生长方法有一定的导向性。     

Peripheral nerve grafts and aFGF restore partial hindlimb function in adult paraplegic rats

The purpose of this study was to evaluate the degree of functional recovery in adult rats with completely transected spinal cord following experimental treatment regimens that include implantation of peripheral nerve segments and local application of acidic fibroblast growth factor (aFGF). Rats were randomly divided to five groups: (1) spinal cord transection, (2) spinal cord transection and aFGF treatment, (3) spinal cord transection and peripheral nerve grafts, (4) spinal cord transection, aFGF treatment, and peripheral nerve grafts, and (5) sham control (laminectomy only). The locomotor behavior of all rats was analyzed by the Basso, Beattie and Bresnahan (BBB) open field locomotor test over the six months survival time. Immunohistochemisty for neurofilament protein, and somatosensory (SSEP) and motor evoked potentials (MEP) were used to evaluate axon growth across the damage site following the different treatments. The results show four principal findings: (1) Only the combination of peripheral nerve grafts and aFGF treatment improved hindlimb locomotor function after spinal cord transection. (2) The SSEP and MEP demonstrated electrophysiological evidence of both sensory and motor information crossing the damaged site, but only in the combined nerve grafts and aFGF treatment rats. (3) Immunostaining demonstrated neurofilament positive axons extending through the graft area and into distal end of spinal cord, but only in the group with combined nerve grafts and aFGF treatment. (4) Retransection of group 4 rats eliminated the behavioral recovery, MEP, and SSEP responses, indicating that the improvement of hindlimb locomotor activity came from supraspinal control. These results demonstrate the ability of the repair strategy combining peripheral nerve grafts and aFGF treatment to facilitate the regeneration of spinal ascending and descending tracts and also recovery of motor behavior following spinal cord injury.     

高压氧与带血供周围神经移植对成鼠损伤脊髓诱发电位的影响

目的：研究高压氧对带血供周围神经移植修复成鼠损伤脊髓功能恢复的作用。方法：将40只成年Wistar大鼠作成脊髓半切损伤模型,随机分为A、B两组,A组单纯带血供周围神经移植修复脊髓损伤,B组为带血供周围神经移植后给予高压氧治疗。手术后1、2、4、8、10周进行感觉诱发电位（SEP）和运动诱发电位（MEP）检查。结果：SEP和MEP潜峰时的恢复B组优于A组。结论：高压氧与带血供周围神经移植对成鼠损伤脊髓功能恢复有较好的促进作用。     

Adenovirus vector-mediated in vivo gene transfer of brain-derived neurotrophic factor (BDNF) promotes rubrospinal axonal regeneration and functional recovery after complete transection of the adult rat spinal cord

Neurotrophins have been shown to promote axonal regeneration, but the techniques available for delivering neurotrophins have limited effectiveness. The aim of this study was to evaluate the effect of adenovirus vector mediated gene transfer of brain-derived neurotrophic factor (BDNF) on axonal regeneration after spinal cord injury. We prepared adenovirus vectors encoding either beta-galactosidase (AxCALacZ) or BDNF (AxCABDNF). AxCALacZ was used to assess infection levels of the adenovirus BDNF produced by AxCABDNF was detected by Western blotting and its bioactivity was confirmed by bioassay. As a model of spinal cord injury, the rat spinal cord was completely transected at the T8 level. Immediately after transection, the vectors were injected into both stumps of the spinal cord. Axonal regeneration after transection was assessed by retrograde and anterograde tracing. In AxCALacZ-injected rats, adenovirus-infected cells were observed not only at the injected site but also in brainstem nuclei, as shown by LacZ expression. After the injection of the retrograde tracer fluorogold (FG) distal portion to the transection, AxCABDNF-injected rats showed FG-labeled neurons in the red nucleus. The anterograde tracer biotinylated dextran amine (BDA) injected into the red nucleus was also found in regenerating rubrospinal fibers distal to the transection. These tracing experiments demonstrated the regeneration of descending axons. In addition, rats of the AxCABDNF group showed significant locomotor recovery of hindlimb function, which was completely abolished by re-transection. These results indicate that the recovery was caused by regeneration of rubrospinal axons, not by simple enhancement of the central pattern generator.     

Soluble cell adhesion molecule L1-Fc promotes locomotor recovery in rats after spinal cord injury

Previous studies suggest that the cell adhesion molecule L1 promotes neurite growth by neutralizing white matter associated inhibitors of axonal growth. We made a soluble chimeric dimer by linking mouse L1 to human Fc. This L1-Fc construct (40 microg/mL) markedly facilitated neurite outgrowth, as well as neuronal adhesion to white matter on frozen sections of spinal cord. We applied L1-Fc intrathecally (200 microg/mL at 0.5 microL/h) to rat spinal cords for 2 weeks after a 25-mm weight drop contusion of the T13 spinal cord. Initial experiments indicated that L1-Fc is present in the spinal cord after 2 weeks of intrathecal infusion and significantly improved locomotor recovery by 6-12 weeks after injury. We then randomized 45 rats to intrathecal infusion of L1-Fc (L1), phosphate-buffered saline controls (PBS), and a mouse monoclonal IgM antibody (M1). By 12 weeks after injury, L1-treated rats recovered significantly (p < 0.005) better locomotor function (BBB score 10.57 +/- 0.25, n = 14) than PBS-treated rats (BBB score 9.00 +/- 0.33, n = 14) or M1-treated (BBB score 8.71 +/- 0.16, n = 14). Only two rats of 22 treated with saline recovered weight-supported ambulation. Of 20 L1-Fc-treated rats, however, 18 recovered weight-supported walking by 12 weeks. The L1-Fc-treated rats also showed more consistent hindlimb contact placing than saline controls. We injected biotinylated dextran amine (BDA) into the motor cortices of 14 rats treated with L1-Fc to label corticospinal axons, comparing these with 13 rats treated with saline. In saline-treated rats, BDA-labeled corticospinal axons often grew up to the impact edge and occasionally into the impact site. L1-treated rats showed longer corticospinal tract growth at the injury site. Three rats had BDA-labeled axons that extended beyond the impact center. One L1-Fc-treated rat showed axonal extension and synapse formation in cord distal to the injury. These results indicate that soluble L1-Fc promotes axonal growth and functional recovery after spinal cord injury. However, the limited corticospinal tract growth across the injury site cannot account for the observed locomotor recovery. Thus, L1 may be stimulating growth of other motor tracts or protecting axons and neurons. More studies are required to elucidate the mechanisms of L1-Fc-induced locomotor recovery.     

GDNF基因修饰的嗅鞘细胞移植联合IN-1注射修复大鼠急性脊髓损伤

目的 研究胶质细胞源性神经营养因子(GDNF)基因修饰的嗅鞘细胞(OECs)移植联合轴突生长抑制蛋白抗体(IN-1)局部持续注射对大鼠急性横断性脊髓损伤(SCI)的修复作用.方法 构建载有GDNF基因的慢病毒(Lentivirus)载体并体外转染OECs,Western Blot检测GDNF的表达.用50只成年雌性SD大鼠建立胸脊髓全横断损伤模型,随机分为A(对照组)、B(IN-1微泵注射组)、C(OECs组)、D(GDNF-OECs组)和E(GDNF-OECs+IN-1组)5组各10只.应用神经丝蛋白200(NF200)单抗免疫组化、生物素化的葡聚糖胺(BDA),顺行神经追踪对SCI区神经纤维再生进行形态学观察.采用BBB评分评估大鼠后肢功能恢复情况.结果 术后共有13只大鼠死亡.术后8周可观察到Hoechst标记的OECs在体内存活并在脊髓内迁移;E组和D组可见SCI区杂乱无序的再生轴突,有连续性神经纤维通过损伤区;C组可见少量无序的再生轴突,可疑连续性神经纤维通过损伤区;B组和A组脊髓残端萎缩,未见轴突再生.A、B、C、D和E组后肢功能运动平均BBB评分分别为7.70±0.24、7.89±0.15、10.50±0.25、11.43±0.23和12.81±0.40.结论 GDNF-OECs移植联合IN-1抗体注射可有效促进损伤脊髓神经轴突的存活、再生,促进损伤脊髓的修复.     

Transplantation of preconditioned schwann cells in peripheral nerve grafts after contusion in the adult spinal cord. Improvement of recovery in a rat model

BACKGROUND: Recovery after injury to the peripheral nervous system is based on the pro-regenerative relationship between axons and the extracellular matrix, a relationship established by Schwann cells. As mechanical conditioning of Schwann cells has been shown to stimulate their regenerative behavior, we sought to determine whether transplantation of these cells to the central nervous system (i.e., the spinal cord), with its limited regenerative capacity after injury, would improve axonal regeneration and functional recovery. METHODS: A moderate contusion injury of the spinal cord was created with a force-directed impactor in forty-eight adult Sprague-Dawley rats, and, at one week postinjury, the spinal cords were reexposed in all animals. In twenty-four of these animals, peripheral nerve grafts with Schwann cells that had been obtained from the sciatic nerves of donor animals, and had been either untreated or subjected to mechanical conditioning, were transplanted to the contused area of the cords following resection of the glial scar. Another group of animals was treated with glial scar excision only, and a fourth group had the contusion injury but neither glial excision nor transplantation. Scores according to the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale were assigned preoperatively and weekly thereafter. Tract tracing of descending and ascending spinal cord tracts was performed at six weeks postoperatively for quantitative histological evaluation of axonal regeneration. RESULTS: While the recovery following glial scar excision without peripheral nerve transplantation was significantly worse than the recovery in the other groups, both transplantation groups had significantly higher BBB scores than the controls (no transplantation) in the early postoperative period (p < 0.05). Moreover, histological analysis showed markedly increased axonal regeneration at the lesional sites in the animals treated with the mechanically conditioned grafts than in the other groups (p < 0.05). CONCLUSIONS: Functional recovery after spinal cord contusion improved following glial scar excision with transplantation of Schwann cells in peripheral nerve grafts to the contusion areas. Although recovery did not differ significantly between the transplantation groups, only the preconditioned grafts led to axonal regeneration at and past the lesional site. These grafts may further enhance functional recovery as the descending tracts eventually reach their target end-organs.     

AFGF promotes axonal growth in rat spinal cord organotypic slice co-cultures

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15-P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.     

BBB评分评估脊髓损伤大鼠后肢运动功能的探讨

目的：探讨大鼠脊髓损伤后和修复中如何评估人鼠后肢运动的BBB评分。方法：对4组大鼠分别行T10脊髓背侧半切断(A组)、T10脊髓全切断(B组)、T10脊髓节段全切除(C组)、T10以下脊髓全切除(D组)，制成不同损伤程度的大鼠脊髓损伤模型，对所有动物的后肢运动功能进行BBB评分和脊髓组织学观察。结果：A组大鼠BBB评分存损伤后5崩达到20分或21分，B组和C组大鼠存术后2周以后BBB评分维持在8分．D组大鼠BBB评分维持在0。B组和C组大鼠脊髓顺行追踪显示脊髓损伤区和尾侧无追踪剂分布．连续矢状冰冻切片抗神经丝(NF)染色未见连续NF通过损伤区，结论：大鼠脊髓损伤模型的后肢运动功能BBB评分如果在8分以下，就需要慎重评价，这种运动有可能完全是或包括有自发的后肢运动。     

Dissociated predegenerated peripheral nerve transplants for spinal cord injury repair: a comprehensive assessment of their effects on regeneration and functional recovery compared to schwann cell transplants

Abstract Several recent studies suggest that predegenerated nerves (PDNs) or dissociated PDNs (dPDNs) can improve behavioral and histological outcomes following transplantation into the injured rat spinal cord. In the current study we tested the efficacy of dPDN transplantation by grafting cells isolated from the sciatic nerve 7 days after crush. We did not replicate one study, but rather assessed what appeared, based on five published reports, to be a reported robust effect of dPDN grafts on corticospinal tract (CST) regeneration and locomotor recovery. Using a standardized rodent spinal cord injury model (200 kD IH contusion) and transplantation procedure (injection of GFP(+) cells 7 days post-SCI), we demonstrate that dPDN grafts survive within the injured spinal cord and promote the ingrowth of axons to a similar extent as purified Schwann cell (SC) grafts. We also demonstrate for the first time that while both dPDN and SC grafts promote the ingrowth of CGRP axons, neither graft results in mechanical or thermal hyperalgesia. Unlike previous studies, dPDN grafts did not promote long-distance axonal growth of CST axons, brainstem spinal axons, or ascending dorsal column sensory axons. Moreover, using a battery of locomotor tests (Basso Beattie Bresnahan [BBB] score, BBB subscore, inked footprint, Catwalk, and ladderwalk), we failed to detect any beneficial effects of dPDN transplantation on the recovery of locomotor function after SCI. We conclude that dPDN transplants are not sufficient to promote CST regeneration or locomotor recovery after SCI.