Transplantation of human bone marrow stromal cell‐derived Schwann cells reduces cystic cavity and promotes functional recovery after contusion injury of adult rat spinal cord

The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell‐derived Schwann cells (hBMSC‐SC) promotes functional recovery after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from bone marrow of adult human patients and induced into Schwann cells (hBMSC‐SC) in vitro. Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC‐SC were detected using cytokine antibody array. Immunosppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC‐SC (hBMSC‐SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl‐violet staining revealed that the area of cystic cavity was smaller in the hBMSC‐SC group than that in the control group. Immunohistochemstry revealed that the number of anti‐growth‐associated protein‐43‐positive nerve fibers was significantly larger in the hBMSC‐SC group than that in the control group. At the same time, the number of tyrosine hydroxylase‐ or serotonin‐positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC‐SC group than that in the control group. In electron microscopy, formation of peripheral‐type myelin was recognized near the lesion epicenter in the hBMSC‐SC group. Hind limb function recovered significantly in the hBMSC‐SC group compared with the control group. In conclusion, the functions of hBMSC‐SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury.     

Motor axonal regeneration after partial and complete spinal cord transection

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.     

Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat

The goal of this study was to ascertain whether olfactory ensheathing cells (OECs) were able to promote axonal regeneration and functional recovery when transplanted 45 days after complete transection of the thoracic spinal cord in adult rats. OECs promoted partial restitution of supraspinal pathways evaluated by motor evoked potentials and modest recovery of hindlimb movements. In addition, OEC grafts reduced lumbar reflex hyperexcitability from the first month after transplantation. Histological results revealed that OECs facilitated corticospinal and raphespinal axons regrowth through the injury site and into the caudal spinal cord segments. Interestingly, raphespinal but not corticospinal fibers regenerated long distances through the gray matter and reached the lower lumbar segments (L5) of the spinal cord. However, delayed OEC grafts failed to reduce posttraumatic astrogliosis. In conclusion, the beneficial effects found in the present study further support the use of OECs for treating chronic spinal cord injuries.     

Transplantation of olfactory ensheathing cells promotes axonal regeneration in a rat model of spinal cord injury

BACKGROUND:Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to promote axonal regeneration and functional recovery.However,the mechanisms underlying the effects of OEC transplantation remain controversial.OBJECTIVE:To observe fibrotic scar formation and axonal regeneration in the damaged spinal cord following OEC transplantation,and to determine whether OEC transplantation promotes neural regeneration by attenuating fibrotic scar formation.DESIGN,TIME AND SETTING:A randomized,controlled animal experiment was performed at the Department of Developmental Morphology,Tokyo Metropolitan Institute for Neuroscience,Fuchu,Japan and at the Department of Human Anatomy,College of Basic Medical Sciences,China Medical University,China between April 2007 and May 2009.MATERIALS:OECs were obtained from olfactory nerves and olfactory bulbs of male,4-week-old,Sprague Dawley rats.Rabbit anti-serotonin polyclonal antibody,rabbit anti-calcitonin gene-related peptide polyclonal antibody,rabbit anti-glial fibrillary acidic protein polyclonal antibody,rabbit anti-type IV collagen polyclonal antibody,and mouse anti-rat endothelial cell antigen-1 monoclonal antibody were used.METHODS:Male,Sprague Dawley rats aged 8 weeks were randomly divided into three groups:sham-surgery (n = 3),surgery (n = 9),and OEC transplantation (n = 11).Spinal cord transection at the T9-10 level was performed and the rats were transplanted with a 2-μL (1 × 105 cells) cell suspension.MAIN OUTCOME MEASURES:Formation of glial and fibrotic scars was examined using immunohistochemistry for glial fibrillary acidic protein and type IV collagen.Serotonin-positive and calcitonin gene-related peptide-positive axons were visualized by immunohistochemistry,respectively.Double immunofluorescence for type IV collagen and rat endothelial cell antigen-1 was also performed to determine co-localization of type IV collagen deposition and blood vessels.RESULTS:At 1 week after spinal cord injury,numerous glial cells were observed around the lesion site.Formation of fibrotic scar was determined by a large amount of type IV collagen deposition in the lesion center,and descending serotonin- or ascending calcitonin gene-related peptideconiaining axons stopped at the fibrotic scar that was formed in the lesion site.At week after transplantation,the formation of fibrotic scar was significantly inhibited.In addition,the fibrotic structure was partly formed and centralized in the blood vessel,and serotonergic and calcitonin gene-related peptide-containing axons were regenerated across the lesion site.CONCLUSION:OEC transplantation into the injured spinal cord attenuated fibrotic scar formation and promoted axon regeneration.     

骨髓间质细胞源性神经球移植对大鼠脊髓损伤的修复作用

目的探讨骨髓间质细胞源性神经球(BMSCs-NS)移植在大鼠脊髓损伤修复中的作用。方法体外原代培养大鼠BMSCs-NS。Wistar大鼠45只,玻璃器压迫法制备胸髓(T9-10)完全损伤模型,随机分成三组:BMSCs-NS移植组(A组,n=15)、BMSCs移植组(B组,n=15)和培养液组(C组,n=15)。BBB评分评估三组大鼠后肢运动功能在不同时间段的恢复及双下肢运动诱发电位(MEPs)情况。结果 BBB平均分值为:A组,7.8±1.42;B组,5.7±0.90;C组,1.4±0.29。移植修复21d后BBB绝对分值,A组较B组和C组明显改善(B组:p<0.001;C组:p<0.0001)。移植5w后B组少数大鼠可以诱发出MEPs,但潜伏期较长(20ms)且波幅较低。2w时A组未能诱发出MEPs,但5w时的MEPs接近正常。结论细胞移植治疗能够恢复大鼠后肢的承重运动功能并能成功诱导出理想的MEPs。     

Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection

Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT_2A receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.     

LINGO-1 antagonist promotes functional recovery and axonal sprouting after spinal cord injury

LINGO-1 is a CNS-specific protein and a functional component of the NgR1/p75/LINGO-1 and NgR1/TAJ(TROY)/LINGO-1 signaling complexes that mediate inhibition of axonal outgrowth. These receptor complexes mediate the axonal growth inhibitory effects of Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) via RhoA activation. Soluble LINGO-1 (LINGO-1-Fc), which acts as an antagonist of these pathways by blocking LINGO-1 binding to NgR1, was administered to rats after dorsal or lateral hemisection of the spinal cord. LINGO-1-Fc treatment significantly improved functional recovery, promoted axonal sprouting and decreased RhoA activation and increased oligodendrocyte and neuronal survival after either rubrospinal or corticospinal tract transection. These experiments demonstrate an important role for LINGO-1 in modulating axonal outgrowth in vivo and that treatment with LINGO-1-Fc can significantly enhance recovery after spinal cord injury.     

Acellular spinal cord scaffold seeded with bone marrow stromal cells protects tissue and promotes functional recovery in spinal cord‐injured rats

Therapy using scaffolds seeded with stem cells plays an important role in repair of spinal cord injury (SCI), with the transplanted cells differentiating into nerve cells to replace the lost tissue while releasing neurotrophic factors that contribute to repair following SCI and enhance the function of the damaged nervous system. The present study investigated the ability to extend the survival time of bone marrow stromal cells (BMSCs) to restore the damaged spinal cord and improve functional recovery by grafting acellular spinal cord (ASC) scaffold seeded or not with BMSCs in a rat model of acute hemisected SCI. BBB scores revealed that treatment with BMSCs seeded into ASC scaffold led to an obvious improvement in motor function recovery compared with treatment with ASC scaffold alone or untreated controls. This improvement was evident at 2 and 8 weeks after surgery (P < 0.05). When BMSCs labeled with 5‐bromodeoxyuridine were implanted together with ASC scaffold into the injured sites, they differentiated into glial cells, and some BMSCs could be observed within the graft by immunofluorescent staining at 8 weeks after implantation. Evaluation of caspase‐3 activation suggested that the graft group was able to reduce apoptosis compared with SCI alone at 8 weeks after operation (P < 0.05). This study suggests that ASC scaffolds have the ability to enhance BMSC survival and improve differentiation and could also reduce native damaged nerve tissue apoptosis, thus protecting host tissue as well as improving functional recovery after implantation. © 2013 Wiley Periodicals, Inc.     

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.     

Fractionated radiation facilitates repair and functional motor recovery after spinal cord transection in rat

Previous studies suggest that motor recovery does not occur after spinal cord injury because reactive glia abort the natural repair processes. A permanent wound gap is left in the cord and the brain-cord circuitry consequently remains broken. Single-dose x-irradiation destroys reactive glia at the damage site in transected adult rat spinal cord. The wound then heals naturally, and a partially functional brain-cord circuitry is reconstructed. Timing is crucial; cell ablation is beneficial only within the third week after injury. Data presented here point to the possibility of translating these observations into a clinical therapy for preventing the paralysis following spinal cord injury in the human. The lesion site (at low thoracic level) in severed adult rat spinal cord was treated daily, over the third week postinjury, with protocols of fractionated radiation similar to those for treating human spinal cord tumors. This resulted, as with the single-dose protocol, in wound healing and restoration of some hindquarter motor function; in addition, the beneficial outcome was augmented. Of the restored hindlimb motor functions, weight-support and posture in stance was the only obvious one. Recovery of this motor function was partial to substantial and its incidence was 100% instead of about 50% obtained with the single-dose treatment. None of the hindlimbs, however, regained frequent stepping or any weight-bearing locomotion. These data indicate that the therapeutic outcome may be further augmented by tuning the radiation parameters within the critical time-window after injury. These data also indicate that dose-fractionation is an effective strategy and better than the single-dose treatment for targeting of reactive cells that abort the natural repair, suggesting that radiation therapy could be developed into a therapeutic procedure for repairing injured spinal cord.     

Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats

Introduction: Olfactory ensheathing cells (OECs) hold promise for cell therapy because they may promote regeneration of the central nervous system. However, OECs have been less studied after peripheral nerve injury (PNI). The purpose of this investigation was to determine the effect of OEC transplantation on a severe sciatic nerve (SN) lesion. Methods: OECs were injected in rats after section and 2‐cm resection of the SN. Results: Three months after therapy, muscle strength and morphometric studies showed complete restoration of the contractile properties of the gastrocnemius and complete repair of the SN. Immunohistochemistry and RT‐PCR studies indicated an increase in the presence of neurotrophic factors. Interestingly, tracking of green fluorescent protein (GFP)‐positive OECs showed that no OECs were present in the SN. Discussion: Our results demonstrate that, after severe PNI, OECs have remarkable potential for nerve regeneration by creating a favorable microenvironment. Muscle Nerve, 2011     

Evidence for functional regeneration in the adult lamprey spinal cord following transection

We report here that following partial spinal transections in adult lampreys, the fibers of the spinal cords can regenerate and restore some intersegmental coordination to the central pattern generator for locomotion, as tested in the isolated cord preparation. However, the regeneration by this test is not successful in all animals.     

Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury

Trauma to the central nervous system (CNS) triggers intraparenchymal inflammation and activation of systemic immunity with the capacity to exacerbate neuropathology and stimulate mechanisms of tissue repair. Despite our incomplete understanding of the mechanisms that control these divergent functions, immune-based therapies are becoming a therapeutic focus. This review will address the complexities and controversies of post-traumatic neuroinflammation, particularly in spinal cord. In addition, current therapies designed to target neuroinflammatory cascades will be discussed.     

Major vault protein promotes locomotor recovery and regeneration after spinal cord injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor functions after spinal cord injury (SCI), in part due to axonal regrowth and regeneration permissivity of the central nervous system. Upregulation of major vault protein (MVP) expression after spinal cord injury in the brainstem of the adult zebrafish prompted us to probe for its contribution to recovery after SCI. MVP is a multifunctional protein expressed not only in many types of tumours but also in the nervous system, where its importance for regeneration is, however, unclear. Using an established zebrafish SCI model, we found that MVP mRNA and protein expression levels were increased in ependymal cells in the spinal cord caudal to the lesion site at 6 and 11 days after SCI. Double immunolabelling showed that MVP was co‐localised with Islet‐1 or tyrosine hydroxylase around the central canal of the spinal cord in sham‐injured control fish and injured fish 11 days after surgery. MVP co‐localised with the neural stem cell marker nestin in ependymal cells after injury. By using an in vivo morpholino‐based knock‐down approach, we found that the distance moved by MVP morpholino‐treated fish was reduced at 4, 5 and 6 weeks after SCI when compared to fish treated with standard control morpholino. Knock‐down of MVP resulted in reduced regrowth of axons from brainstem neurons into the spinal cord caudal to the lesion site. These results indicate that MVP supports locomotor recovery and axonal regrowth after SCI in adult zebrafish.     

大鼠骨髓间质干细胞静脉移植在脊髓损伤中的定向迁移

目的观察大鼠骨髓间质干细胞(ratbonemarrowstromalcells,rMSCs)静脉移植在体内的定向迁移。方法分离培养rMSCs,流式细胞术检测其表面标志,运用改良Allen法制备大鼠T10脊髓损伤模型,随机分为假手术组、对照组、rMSCs静脉移植组。假手术组、rMSCs静脉移植组同时于大鼠损伤后72小时经尾静脉移植溴脱氧尿苷(BrdU)标记MSCs。免疫组化技术检测rMSCs在体内迁移、存活以及分化情况。结果rMSCs体外分离培养扩增5代,细胞数可达1～2×1011个,具有多态性和贴壁生长特性,流式细胞术检测CD34、CD45阴性,CD29、CD90表达阳性。移植的rMSCs在宿主损伤脊髓中聚集并存活,3～5W后即有部分移植细胞表达神经元特异性烯醇化酶(neuronspecificenolase,NSE)、神经丝蛋白(neurofilament,NF)、微管相关蛋白(microtubuleassociatedprotein2,MAP2)。结论rMSCs体外扩增迅速,具有干细胞特性,经静脉移植在宿主体内可向损伤区脊髓聚集存活分化。     

Chondroitinase ABC promotes axonal regeneration of Clarke's neurons after spinal cord injury

We examined whether enzymatic digestion of chondroitin sulfate (CS) promoted the axonal regeneration of neurons in Clarke's nucleus (CN) into a peripheral nerve (PN) graft following injury of the spinal cord. After hemisection at T11, a segment of PN graft was implanted at the lesion site. Either vehicle, brain-derived neurotrophic factor (BDNF) or chondroitinase ABC was applied at the implantation site. The postoperative survival period was 4 weeks. Treatment with vehicle or BDNF did not promote the axonal regeneration of CN neurons into the PN graft. Application of 2.5 unit/ml chondroitinase ABC resulted in a significant increase (12.8%) in the number of regenerated CN neurons. Double labeling with Fluoro-Gold and NADPH-diaphorase histochemistry showed that the regenerated CN neurons did not express nitric oxide synthase (NOS). Our results suggest that CS is inhibitory to the regeneration of CN neurons following injury of the spinal cord.     

骨髓基质细胞移植促进脊髓全横断损伤区神经元轴突的再生变化☆○

背景：近年来,部分学者证明骨髓基质细胞移植可促进轴突再生,改善脊髓损伤引起的运动功能障碍,但目前关于移植骨髓基质细胞如何促进轴突再生,移植细胞与再生轴突的关系尚不清楚。 目的：通过免疫荧光组织化学和免疫电镜的方法,探讨移植骨髓基质细胞促进脊髓全横断损伤区轴突再生的机制。 设计、时间及地点：随机对照动物实验,细胞学体内观察,于2006-03/2007-06在新加坡国立大学解剖系完成。 材料：清洁级Wistar新生大鼠1只,用于骨髓基质细胞培养。清洁级成年雌性Wistar大鼠36只,无菌条件下显露、切断脊髓T10,制备脊髓全横断损伤模型。 方法：通过传代法培养、纯化骨髓基质细胞。36只成年Wistar雌性大鼠随机投币法分为移植组和对照组,每组18只。移植组大鼠脊髓全横断损伤9 d后以1×1011 L-1的密度移植骨髓基质细胞,缺损区5 μL,损伤区上、下1 mm处各2.5 μL,对照组动物在相同部位注射等量DMEM完全培养基,注射速度1 μL/min。 主要观察指标：①移植骨髓基质细胞存活、分化情况。②轴突再生情况。③移植组和对照组宿主自身的nestin、NF200、GFAP和CNP阳性细胞在脊髓损伤区存活情况。④内源性CNP阳性细胞和再生纤维关系。 结果：骨髓基质细胞移植2周时,脊髓损伤区可见大量CFDA-SE标记的移植细胞,随时间延长,存活的移植细胞数目逐渐降低,考虑脊髓损伤区内大量的OX42阳性吞噬细胞/激活小胶质细胞及空洞可能影响移植细胞的存活。虽然骨髓基质细胞数目逐渐降低,骨髓基质细胞移植可促进损伤区轴突的再生,而且还可促进宿主自身的nestin、NF200、GFAP和CNP阳性细胞在脊髓损伤区存活。宿主自身CNP和许旺细胞促进损伤轴突的再生和髓鞘形成。 结论：移植骨髓基质细胞移植可促进宿主自身CNP和许旺细胞在脊髓损伤区存活,后者具有促进损伤轴突再生和髓鞘形成的作用。     

移植人羊膜间充质干细胞促进脊髓损伤大鼠神经功能恢复

目的研究移植人羊膜间充质干细胞（hAMSCs）是否促进脊髓损伤大鼠神经功能恢复,探索其可能作用机制。 方法60只雌性SD大鼠按照随机数字表法分为磷酸盐缓冲液（PBS）治疗组（30只）和hAMSCs治疗组（30只）。脊髓损伤采用脊髓撞击损伤模型,hAMSCs或PBS立刻移植到离脊髓损伤中心2 mm的头尾两端。免疫荧光检测细胞分化,血管再生和轴突再生。酶联免疫吸附剂测定试剂盒检测脑源性神经营养因子（BDNF）和血管内皮生长因子（VEGF）含量,BBB运动功能评分检测行为学。 结果在脊髓损伤后14 d、21 d和28 d,hAMSCs治疗组BBB评分分别为（8.75±0.701）、（10.375±0.532）和（12.125±0.350）,高于PBS组（6.0±0.463）、（7.25±0.412）和（9.125±0.440）,差异具有统计学意义（P<0.05）。在第7天和第14天,hAMSCs治疗组BDNF表达水平分别为（75.138±4.367）pg/mg和（66.483±4.099）pg/mg,高于PBS组（43.901±3.607）pg/mg和（41.108±3.848）pg/mg,差异具有统计学意义（P<0.05）。在第7天,第14天和第28天,hAMSCs治疗组VEGF表达水平分别为（23.328±2.463）pg/mg,（22.301±2.223）pg/mg和（14.855±1.282）pg/mg,高于PBS组（9.978±1.572）pg/mg,（9.271±1.496）pg/mg和（7.113±1.123）pg/mg,差异具有统计学意义（P<0.05）。hAMSCs治疗组血管数目（17.5±2.102）高于PBS组（6.25±1.750）,差异具有统计学意义（P<0.05）。hAMSCs治疗组小鼠抗5羟色胺阳性神经纤维面积（3486±203.643）和GAP43阳性神经纤维面积（4568.25±253.881）高于PBS组（2070.25±156.344）和（2455.725±314.475）,差异具有统计学意义（P<0.05）。 结论移植hAMSCs能促进脊髓损伤大鼠神经功能恢复,其作用机制可能是通过增加神经营养因子表达,促进血管再生和轴突再生。因此hAMSCs移植是治疗脊髓损伤的理想方法。