缺氧预处理神经干细胞移植对大鼠急性脊髓损伤后神经胶质细胞凋亡及脊髓空洞形成的影响

目的:研究缺氧预处理神经干细胞(NSCs)移植对大鼠急性脊髓损伤(ASCI)后神经胶质细胞凋亡及脊髓空洞形成的影响。方法:将30只SD大鼠分为假手术对照组;脊髓损伤组;去铁敏组;普通NSCs组;缺氧预处理NSCs组。制成脊髓损伤模型,移植后观察脊髓神经胶质细胞凋亡情况及脊髓空洞形成情况。结果:经去铁敏缺氧预处理培养的NSCs与常规培养的NSCs无明显形态学变化。移植术后7d,缺氧预处理NSCs移植能显著减少脊髓损伤周围区神经胶质细胞的凋亡数量,减少脊髓空洞形成。结论:缺氧预处理NSCs移植能明显抑制大鼠急性脊髓损伤后神经胶质细胞凋亡,减少脊髓空洞的形成。     

缺氧预处理神经干细胞移植对大鼠急性脊髓损伤后神经胶质细胞凋亡及脊髓空洞形成的影响

目的:研究缺氧预处理神经干细胞(NSCs)移植对大鼠急性脊髓损伤(ASCI)后神经胶质细胞凋亡及脊髓空洞形成的影响.方法:将30只SD大鼠分为假手术对照组;脊髓损伤组;去铁敏组;普通NSCs组;缺氧预处理NSCs组.制成脊髓损伤模型,移植后观察脊髓神经胶质细胞凋亡情况及脊髓空洞形成情况.结果:经去铁敏缺氧预处理培养的NSCs与常规培养的NSCs无明显形态学变化.移植术后7d,缺氧预处理NSCs移植能显著减少脊髓损伤周围区神经胶质细胞的凋亡数量,减少脊髓空洞形成.结论:缺氧预处理NSCs移植能明显抑制大鼠急性脊髓损伤后神经胶质细胞凋亡,减少脊髓空洞的形成.     

小鼠脊髓损伤模型的建立及神经干细胞移植后运动功能恢复情况的研究

目的制作小鼠脊髓损伤打击模型,观察神经干细胞(NSCs)移植对脊髓损伤小鼠运动功能恢复及Nestin表达的影响。方法将50只小鼠随机分为空白组(5只)、模型组(15只)、对照组(15只)、治疗组(15只),运用改良Allen's法制备小鼠T10脊髓损伤模型并立即在损伤节段进行NSCs移植,于损伤后1、3、7、14、21d进行BBB评分,并通过免疫荧光法及荧光定量PCR检测Nestin的表达情况。结果所有脊髓打击后小鼠均出现双后肢瘫痪,但随时间延长运动功能可有不同程度恢复,NSCs移植14d后治疗组较模型组及对照组BBB评分显著增高(P0.05),且治疗组Nestin表达量也高于模型组及对照组。结论成功建立了小鼠脊髓损伤打击模型;移植的外源性神经干细胞在脊髓损伤处存活并促进损伤后小鼠运动功能的恢复。     

NAAGS基因修饰神经干细胞在创伤性颅脑损伤治疗中的研究

目的：探讨移植NAAG合酶（NAAG synthetase,NAAGS）基因修饰的神经干细胞（Neural Stem Cells,NSCs）能否促进创伤性颅脑损伤大鼠神经功能的恢复。方法：利用电穿孔转染大鼠NSCs,通过脑立体定向仪分别将PBS（模型组）、NSCs（NSCs组）、转基因NSCs（NAAGS＋NSCs组）移植到创伤性颅脑损伤（Traumatic Brain Injury,TBI）大鼠局部损伤灶边缘,通过NSS评分评价移植后大鼠神经功能的变化以及用TUNEL法检测NSCs的凋亡情况,并采用放射免疫法分析脑组织中促炎因子水平。结果：Nss评分结果显示NAAGs＋NSCs组和NSCs组在第7、14、21天神经功能评分均低于模型组（P〈0．05）;NAAGS＋NSCs组在第14和21天神经功能评分低于NSCs组（P〈0．05）;在各时间点细胞移植组比模型组的神经细胞凋亡数明显减少;转基因NSCs移植能明显降低TBI脑组织中促炎因子水平。结论：转基因NSCs移植后可以合成NAAGS促进TBI大鼠神经功能的恢复。     

骨髓间充质干细胞移植治疗大鼠脊髓损伤的神经分化研究

目的探讨骨髓间充质干细胞(BMSC)对移植脊髓损伤(SCI)模型神经再生修复的作用及其机制。方法 (1)分离原代SD大鼠BMSC;(2)体外诱导BMSC向神经分化,应用免疫荧光技术检测神经诱导分化后的BMSC神经标志Nestin、Neu N的表达;(3)运用改良的Allen撞击装置制备SD大鼠SCI模型,成年雌性SD大鼠12只,随机分组:损伤对照(n=6),BMSC细胞移植组(n=6),并选择在SCI后半小时内在蛛网膜下腔原位移植1×106 BMSC细胞注射治疗,对照组原位注射10μl PBS作为对照。每周对SCI大鼠进行BBB运动功能行为学评价。(4)在治疗后1个月处死SCI大鼠取脊髓样本进行冰冻切片检测Nestin、NeuN神经标记物表达情况,从而评判BMSC对SCI的治疗效果。计量资料结果服从正态分布、方差齐性时,采用t检验;若不服正态分布,采用KruskalWallis H秩和检验。结果 (1)分离的BMSC纯度高、生物学特征稳定。(2)BMSC在体外神经诱导环境下可分化为神经细胞,对比正常对照组,神经诱导组Nestin与Neu N的表达具有统计学差异(t=11.49、6.76,P0.05)。(3)BMSC移植治疗SCI大鼠运动行为学功能显著改善,移植组比对照组治疗5周后的BBB评分具有统计学意义(t=5.59,P0.05);损伤导致组织形态学出现脊髓白质灰质结构性损毁,神经细胞大量丢失,而BMSC移植组Nestin、GFAP与Neu N表达细胞均较损伤组差异有统计学意义(t=4.74、6.59、15.46,均P0.05)。结论 BMSC移植可促进SCI后神经细胞的存活与再生分化,在一定程度上促进SCI脊髓组织功能的恢复。     

在大鼠创伤性脑损伤模型中神经干细胞移植对突触素表达的影响

目的探讨神经干细胞（NSCs）移植对创伤性脑损伤（TBI）模型大鼠感觉运动功能的恢复作用及其对损伤脑组织中突触素（SYP）表达的影响。方法体外培养大鼠胚胎皮质NSCs;采用Feeney法制备TBI模型,于造模后72h,移植组采用PKH26荧光示踪剂标记的NSCs直接移植于脑损伤区,对照组以等量生理盐水代替NSCs;分别于移植后不同时间点,采用Gridwalk和Latency试验检测TBI大鼠的感觉运动功能;荧光显微镜下计数移植细胞的存活数量;采用免疫印迹和RT-PCR技术检测脑损伤区及周围组织中SYP的表达。结果 NSCs移植大鼠前、后肢功能分别在移植后第2w和4w恢复至手术前水平,而直到第8w,对照组大鼠后肢功能和通过平板移动时间与NSCs移植组和基线比较仍有显著性差异（P〈0.05）。移植的NSCs随移植时间延长存活数量减少,移植后第4w和8w的存活数分别为6.3%±1.0%和4.1%±0.9%。在移植后的8w期间,移植组脑损伤区及周围组织中SYP的表达均明显高于对照组（P〈0.05）。结论移植的NSCs在TBI脑内能够存活,并明显改善了TBI大鼠对侧肢体的感觉运动功能;NSCs移植促进了脑损伤区及周围组织中SYP的表达,这可能是NSCs移植促进功能恢复的机理之一。     

人源胚胎神经干细胞移植可对抗大鼠的脑缺血/再灌注损伤

本文旨在研究人源胚胎神经干细胞(human embryonic neural stem cells,h NSCs)移植到脑缺血/再灌注损伤大鼠脑内后的迁移、分化,以及对大鼠脑卒中的疗效。我们在大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)1 h的大鼠模型上,于血流再灌注后第7天注射h NSCs到缺血侧侧脑室,通过焦油紫染色测量大鼠的脑梗死体积,通过检测大鼠的感觉运动行为评估其神经功能的恢复水平,通过免疫荧光共标观察移植后的h NSCs在脑内的迁移与分化。结果显示,h NSCs移植后能够显著减小脑卒中大鼠脑梗死体积,并改善脑卒中大鼠的转棒、错步和转角等运动行为能力;侧脑室注射的h NSCs优先向胼胝体以及梗死区周边迁移,迁移到胼胝体的h NSCs可以分化成少突胶质细胞和星形胶质细胞,迁移到梗死区周边的细胞能够分化成神经元。以上这些结果提示,侧脑室移植的h NSCs可能通过向特定脑区的迁移和分化发挥对脑缺血/再灌注损伤大鼠的保护作用。     

GDNF对神经干细胞增殖、分化的影响

神经干细胞(neural stem cells,NSCs)具有如下特点:(1)可以向神经组织分化或源自神经系统的一部分。(2)具备维持和更新的自主能力。(3)可通过细胞分裂增殖。以上特点决定了它的应用价值,被公认为治疗阿尔茨海默氏病,帕金森氏症,脊髓损伤,中风等神经退行性疾病的最佳方案。用干细胞治疗癌症,免疫相关性疾病,和其他疾病被认为是很有创新的新疗法,可能有一天会扩展到修复和补充大脑损伤。胶质细胞源性神经营养因子(glial Cell line一derived neurotrophic factor,GDNF)为TGF一β超家族的一员,具有很强神经保护作用,大量实验研究证实GDNF可促进帕金森病大鼠模型的中脑神经干细胞定向分化为多巴胺能神经元,同时大量实验发现其可促进神经干细胞增殖及分化,为神经干细胞的应用奠定了基础。     

免疫球蛋白G(IgG)对急性颈髓损伤大鼠模型神经炎症反应的抑制效应

目的:探讨免疫球蛋白(immunoglobulinG,IgG)对大鼠脊髓损伤(spinal cord injury,SCI)模型神经炎症的抑制效应.方法:60只雌性Wistar大鼠接受C7-T2椎板切除术后随机分为三组,空白对照组,盐水安慰剂组,IgG治疗组,每组各20只动物.创建盐水安慰剂组和IgG治疗组动物的C7-T1节段脊髓损伤模型,空白对照组动物不进行SCI模型的创建.盐水安慰剂组以单剂量0.4g/kg体重在SCI模型重建后15 min静脉注射入动物体内,而安慰剂组动物静脉注射lmL无菌盐水.实验动物SCI模型创建后4h处死动物进行细胞因子和趋化因子的酶联免疫检测,24 h处死动物采用westem blot技术进行MMP-9的蛋白表达检测分析.结果:SCI后4h,相比空白对照组,盐水安慰剂组动物的TNF-α,IL-1β及IL-6等炎性介质的表达水平显著升高,IgG治疗组的炎性介质水平也有一定程度升高,但均显著低于盐水安慰剂组,差异有统计学意义(P＜0.05);盐水安慰剂组和IgG治疗组的MCP-1和CINC-1的蛋白表达水平均显著高于空白对照组(P＜0.05),但相比IgG治疗组,盐水安慰剂组的MCP-1的蛋白表达水平显著更高(P＜0.05)而CINC-1的蛋白表达水平在安慰剂组和IgG治疗组间差异无统计学意义(P＞0.05).SCI后24 h,安慰剂组动物的MMP-9水平显著升高,而空白对照组动物标本中未检测到MMP-9表达.而IgG治疗组动物的MMP-9水平显著低于盐水安慰剂组,差异有统计学意义(P＜0.05).结论:SCI大鼠采用IgG治疗后会显著降低TNF-α,IL-1β,IL-6,MCP-1以及MMP-9等介质的表达水平,表明IgG在SCI中可能具有神经保护效应.     

氧化铁标记神经干细胞对大鼠放射性脑损伤的磁共振成像研究

目的 探讨用超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)标记的神经干细胞(NSCs)在放射性脑损伤大鼠脑的磁共振(MR)显像及迁移。方法 培养NSCs,制作大鼠放射损伤模型。用SPIO标记NSCs并植入大鼠脑内,行MR检查,了解干细胞的成像情况及在放射后大鼠脑内的分布迁移。结果 标记的NSCs可以在MR显像,移植术后可以发现干细胞向放射部位的迁移。结论 MR可以显示干细胞向放射损伤部位的定向迁移。     

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.     

Viability-dependent promoting action of adult neural precursors in spinal cord injury

The aim of the study was the assessment of the effects of adult neural stem cell (NSC) transplantation in a mouse model of spinal cord injury (SCI). The contusion injury was performed by means of the Infinite Horizon Device to allow the generation of reproducible traumatic lesion to the cord. We administered green fluorescent-labeled (GFP-)NSCs either by intravenous (i.v.) injection or by direct transplantation into the spinal cord (intraspinal route). We report that NSCs significantly improved recovery of hind limb function and greatly attenuated secondary degeneration. The i.v. route of NSC administration yielded better recovery than the intraspinal route of administration. About 2% of total i.v.-administered NSCs homed to the spinal cord injury site, and survived almost undifferentiated; thus the positive effect of NSC treatment cannot be ascribed to damaged tissue substitution. The NSCs homing to the injury site triggered, within 48 h, a large increase of the expression of neurotrophic factors and chemokines. One wk after transplantation, exogenous GFP-NSCs still retained their proliferation potential and produced neurospheres when recovered from the lesion site and cultured in vitro. At a later time, GFP-NSC were phagocytated by macrophages. We suggest that the process of triggering the recovery of function might be strongly related to the viability of GFP-NSC, still capable ex vivo of producing neurospheres, and their ability to modify the lesion environment in a positive fashion.     

Application of the sodium hyaluronate-CNTF scaffolds in repairing adult rat spinal cord injury and facilitating neural network formation

The present study aimed to explore the potential of the sodium hyaluronate-CNTF (ciliary neurotrophic factor) scaffold in activating endogenous neurogenesis and facilitating neural network re-formation after the adult rat spinal cord injury (SCI). After completely cutting and removing a 5-mm adult rat T8 segment, a sodium hyaluronate-CNTF scaffold was implanted into the lesion area. Dil tracing and immunofluorescence staining were used to observe the proliferation, differentiation and integration of neural stem cells (NSCs) after SCI. A planar multielectrode dish system (MED64) was used to test the electrophysiological characteristics of the regenerated neural network in the lesioned area. Electrophysiology and behavior evaluation were used to evaluate functional recovery of paraplegic rat hindlimbs. The Dil tracing and immunofluorescence results suggest that the sodium hyaluronate-CNTF scaffold could activate the NSCs originating from the spinal cord ependymal, and facilitate their migration to the lesion area and differentiation into mature neurons, which were capable of forming synaptic contact and receiving glutamatergic excitatory synaptic input. The MED64 results suggest that functional synapsis could be established among regenerated neurons as well as between regenerated neurons and the host tissue, which has been evidenced to be glutamatergic excitatory synapsis. The electrophysiology and behavior evaluation results indicate that the paraplegic rats’ sensory and motor functions were recovered in some degree. Collectively, this study may shed light on paraplegia treatment in clinics.     

Assessment of Glial Scar,Tissue Sparing,Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion

Objective and Methods

This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo.

Results

Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord.

Conclusion

Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.     

Establishment of a Spinal Cord Injury Model in Adult Rats by an Electrocircuit-Controlled Impacting Device and its Pathological Observations

One of the crucial challenges in medicine is the treatment and rehabilitation of spinal cord injury (SCI). In this study, we established a stable and reproducible acute spinal cord injury model in adult rats. The SCI was inflicted by our self-innovated spinal cord impact device controlled by electrical circuit. The Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB) score, electrophysiology, histological, and immunohistochemical changes after SCI were observed. The BBB score of the injured rats began to increase from the 3rd day of SCI and reached at the score 7.2 ± 1.3 at the 28th day. The latency of cortical somatosensory evoked potentials (CSEP) was not observed 2 and 6 h after injury, but appeared 24 h after injury which was significantly prolonged. It recovered from day 3 gradually to 27.3 ± 2.7 ms on day 28. H&E staining showed that the structure of gray and white matter was disrupted after the SCI. The result also showed dramatic neuron degenerations, cellular swelling, and the proliferation of glial cells. The immunohistochemical analysis showed that the expression of neuron specific enolase (NSE) and neurofilament 200 (NF200) started lowering at 2 h and dropped to the bottom at 24 h. Their expression rebound from day 3 and yet to the original level at day 28 (P < 0.05). The number of cells expressing glial fibrillary acidic protein (GFAP) hiked from day 3, peaked at day 14, and began recovering from day 28 (P < 0.05). The changes of NSE, NF200, GFAP, and CSEP were significantly associated with the BBB score (P < 0.05). In conclusion, our self-innovated device can reproduce the injury model stably. The changes of NSE, NF, and GFAP after spinal cord injury reflect the characteristics of pathological change, which are closely associated with the functional recovery from the spinal cord injury.     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

The neuronal differentiation microenvironment is essential for spinal cord injury repair

Spinal cord injury (SCI) often leads to substantial disability due to loss of motor function and sensation below the lesion. Neural stem cells (NSCs) are a promising strategy for SCI repair. However, NSCs rarely differentiate into neurons; they mostly differentiate into astrocytes because of the adverse microenvironment present after SCI. We have shown that myelin-associated inhibitors (MAIs) inhibited neuronal differentiation of NSCs. Given that MAIs activate epidermal growth factor receptor (EGFR) signaling, we used a collagen scaffold-tethered anti-EGFR antibody to attenuate the inhibitory effects of MAIs and create a neuronal differentiation microenvironment for SCI repair. The collagen scaffold modified with anti-EGFR antibody prevented the inhibition of NSC neuronal differentiation by myelin. After transplantation into completely transected SCI animals, the scaffold-linked antibodies induced production of nascent neurons from endogenous and transplanted NSCs, which rebuilt the neuronal relay by forming connections with each other or host neurons to transmit electrophysiological signals and promote functional recovery. Thus, a scaffold-based strategy for rebuilding the neuronal differentiation microenvironment could be useful for SCI repair.     

Intrathecal Epigallocatechin Gallate Treatment Improves Functional Recovery After Spinal Cord Injury by Upregulating the Expression of BDNF and GDNF

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague–Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI.     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．