Novel combination strategies to repair the injured mammalian spinal cord

Due to the varied and numerous changes in spinal cord tissue following injury, successful treatment for repair may involve strategies combining neuroprotection (pharmacological prevention of some of the damaging intracellular cascades that lead to secondary tissue loss), axonal regeneration promotion (cell transplantation, genetic engineering to increase growth factors, neutralization of inhibitory factors, reduction in scar formation), and rehabilitation. Our goal has been to find effective combination strategies to improve outcome after injury to the adult rat thoracic spinal cord. Combination interventions tested have been implantation of Schwann cells (SCs) plus neuroprotective agents and growth factors administered in various ways, olfactory ensheathing cell (OEC) implantation, chondroitinase addition, or elevation of cyclic AMP. The most efficacious strategy in our hands for the acute complete transection/SC bridge model, including improvement in locomotion [Basso, Beattie, Bresnahan Scale (BBB)], is the combination of SCs, OECs, and chondroitinase administration (BBB 2.1 vs 6.6, 3 times more myelinated axons in the SC bridge, increased serotonergic axons in the bridge and beyond, and significant correlation between the number of bridge myelinated axons and functional improvement). We found the most successful combination strategy for a subacute spinal cord contusion injury (12.5-mm, 10-g weight, MASCIS impactor) to be SCs and elevation of cyclic AMP (BBB 10.4 vs 15, significant increases in white matter sparing, in myelinated axons in the implant, and in responding reticular formation and red and raphe nuclei, and a significant correlation between the number of serotonergic fibers and improvement in locomotion). Thus, in two injury paradigms, these combination strategies as well as others studied in our laboratory have been found to be more effective than SCs alone and suggest ways in which clinical application may be developed.     

Designing cell- and gene-based regeneration strategies to repair the injured spinal cord

There is an array of new and promising strategies being developed to improve function after spinal cord injury (SCI). The targeting of a diversity of deleterious processes within the tissue after SCI will necessitate a multi-factorial intervention, such as the combination of cell- and gene-based approaches. To ensure proper development and design of these experiments, many issues need to be addressed. It is the purpose of this review to consider the strategies involved in testing the efficacy of these new combinations to improve axonal regeneration. For cell-based therapy, issues are choosing a SCI model, the time of cell implantation, placement of cells and their subsequent migration, fluid versus solid grafts, use of agents to prevent immune rejection, and tracking of implanted cells. Grafting is also discussed in view of improving function, reducing secondary damage, bridging the injured spinal cord, supporting axonal regrowth, replacing lost neurons, facilitating myelination, and promoting axonal growth from the implant into the cord. The choice of a gene delivery system, gene-based therapies in vivo to provide chemoattractant and guidance cues, altering the intrinsic regenerative capacity of neurons, enhancing endogenous non-neuronal cell functions, and targeting the synthesis of growth inhibitory molecules are also discussed, as well as combining ex vivo gene and cell therapies.     

Biodegradable polymer grafts for surgical repair of the injured spinal cord

PURPOSE: Biodegradable polymers have been used in the surgical repair of peripheral nerves, but their potential for use in the central nervous system has not been exploited adequately. This article discusses concepts related to the engineering of a biodegradable polymer graft for surgical repair of the injured spinal cord and explores the potential means by which such a device might promote axon regeneration and functional recovery after spinal cord injury. CONCEPT: A biodegradable polymer implant with controlled microarchitecture can be engineered, and its composition can be optimized for implantation in the spinal cord. RATIONALE: The use of a biodegradable polymer implant has the dual advantages of providing a structural scaffold for axon growth and a conduit for sustained-release delivery of therapeutic agents. As a scaffold, the microarchitecture of the implant can be engineered for optimal axon growth and transplantation of permissive cell types. As a conduit for the delivery of therapeutic agents that may promote axon regeneration, the biodegradable polymer offers an elegant solution to the problems of local delivery and controlled release over time. Thus, a biodegradable polymer graft would theoretically provide an optimal structural, cellular, and molecular framework for the regrowth of axons across a spinal cord lesion and, ultimately, neurological recovery. CONCLUSION: Biodegradable polymer grafts may have significant therapeutic potential in the surgical repair of the injured spinal cord. Further research should be focused on the bioengineering, characterization, and experimental application of these devices.     

Nogo in the injured spinal cord

Myelin of the adult mammalian central nervous system (CNS) has been attributed to suppress structural plasticity and to impede regenerating nerve fibers. Nogo-A is possibly the best characterized of a variety of neurite growth inhibitors present in CNS myelin. Neutralizing its activity results in improved axon regrowth and functional recovery in experimental CNS lesion models of adult rodents and primates. While Nogo-A has become a major target for therapeutic intervention to promote axon regeneration in the CNS, it is realized that such an approach will likely have to be combined with other therapeutic strategies to maximize functional recovery after spinal cord injury (SCI).     

Gifts from the molecular revolution: protection and repair of the injured spinal cord

Progress in molecular neurobiology is occurring at an accelerating rate, and the newer results carry with them an almost unprecedented degree of specificity. This, together with the development of techniques that permit, at least in principle, the targeting of DNA, RNA, and proteins in vivo, opens up a spectrum of possible new interventions, which almost certainly will have significant impact on the search for new therapies, and ultimately cures, for spinal cord injury and its consequences. This article outlines several areas of progress, based on progress in the author's laboratory, which supports the suggestion that, in the foreseeable future, it will be possible to protect and repair the injured spinal cord. In particular, this article focuses on progress in 3 interrelated areas: (a) neuroprotection (ie, the search for treatments that will protect vulnerable axons within the descending and ascending tracts within the spinal cord, so that they do not die after the initial traumatic insult); (b) restoration of conduction in spinal cord axons that survive the traumatic insult but fail to conduct impulses due to damage to their myelin; and (c) the targeting of molecules along the pain-signaling pathway, so as to reduce neuronal hyperexcitability that produces pain following SCI. Although much work remains to be done, we are coming much closer to our goals in each of these areas, and the overall objective--of protecting and repairing the injured spinal cord--appears achievable.     

胎鼠脊髓对成鼠急性脊髓损伤的修复作用——光镜CB—HRP示踪与还原银染色

把Ｅ１４胎鼠脊髓植入成鼠损伤脊髓后，分别在术后７、 １５、 ３０、 ４５、 ６０、 １２０天，用 ＣＢ－ＨＲＰ和还原银染色显示了宿主脊髓和移植物内的神经元及神经纤维。结果表明，虽然术后７天就可见宿主神经纤维再生进入移植物，但两种方法均不能显示移植的胚胎神经元及其突起。自术后３０天起，移植物内神经元可被标志，但神经元的体积小，突起数目也短而少。随着神经元体积的逐渐增大和突起数目的不断增多延长，可见宿主神经元向移植物内投射突起，并和后者神经元相互联系。作者认为，胚胎脊髓虽可在术后早期机械修补成鼠脊髓损伤，但并不能与宿主脊髓组织发生广泛的有机纤维联系，这和胚胎脊髓移植后，神经元的缓慢分化发育有关。     

Strategies to promote regeneration and recovery in the injured spinal cord

The damaged spinal cord has only a limited capacity for anatomical and functional recovery. However, a growing body of experimental work is demonstrating a potential for enhanced axonal regeneration and recovery following injury. Important pathophysiologic processes are discussed, along with strategies designed to improve outcome through the reconstruction of damaged pathways and the modulation of the spinal cord's response to the primary injury.     

胚胎脊髓组织移植联合应用神经生长因子对损伤脊髓再生修复的形态学影响

选择胎龄为１４～１５天的大鼠胚胎脊髓植入成年大鼠半横断的脊髓损伤腔，同时局部应用神经生长因子。术后２～８个月，用组织学、免疫细胞化学、辣根过氧化酶示踪及超微结构的检查方法证实，移植组织在宿主脊髓内的存活率为９０％，并且分化成熟，具有正常的神经组织结构特征，大多数移植物充满损伤腔，与宿主脊髓形成良好的融合，移植组织与宿主组织出现新的纤维联系，使脊髓损伤的两端恢复了解剖的连续性。     

Micturition in thoracic spinal cord injured cats with autografting of the adrenal medulla to the sacral spinal cord.

PURPOSE: The role of noradrenergic projection from the pontine micturition center to the sacral spinal cord during micturition was examined in thoracic spinal cord injured cats after autografting the adrenal medulla to the sacral spinal cord. MATERIALS AND METHODS: In 13 female cats the lower thoracic cord was transected and the right adrenal gland was removed under halothane anesthesia. The resected adrenal medulla was divided into several small pieces, which were subsequently autografted to the sacral spinal cord in 7 cats. Another 6 cats underwent sham operation and served as controls. Continuous cystometry and electromyography of the external urethral sphincter were performed every 2 weeks postoperatively without anesthesia. At week 8 the sacral spinal cord was removed and immunohistochemical testing was done to assess tyrosine hydroxylase immunoreactivity. RESULTS: At week 6 the relative mean duration of detrusor-external sphincter coordination plus or minus standard error during bladder contraction was 62.4% +/- 4.9% in adrenal grafted cats, which was significantly (p = 0.0485) longer than in controls (34.2% +/- 12.6%). However, maximum bladder contraction pressure, bladder contraction duration and post-void residual urine volume were not significantly different in the 2 groups. Tyrosine hydroxylase immunoreactive cells were observed in and on the sacral spinal cord in adrenal grafted animals but not in controls. CONCLUSIONS: Autografting the adrenal medulla to the sacral spinal cord prolonged detrusor-external sphincter coordination during bladder contraction in thoracic spinal cord injured cats, although other urodynamic parameters did not change. Therefore, noradrenergic projections to the sacral spinal cord may relax the external urethral sphincter during bladder contraction.     

脊髓损伤大鼠的阴茎海绵体肌电图研究

目的探讨海绵体肌电图诊断脊髓性勃起功能障碍的价值。方法将24只成年雄性SD大鼠(300～400 g)分成:对照组、T9和L6损伤组(每组8只)。损伤脊髓1周后,用肌电图仪采集注射阿朴吗啡前后阴茎肌电数据,采集频率20～3 000 Hz、扫描速度5 ms／d、灵敏度10 μV／d。用t检验方法分析统计数据。结果对照组使用阿朴吗啡10 min均方根振幅为(5．60±0．89)μV, 大于T9损伤组(3．60±1．14)μV(P<0．05);使用阿朴吗啡前、使用后5 min和10 min高／低功率比均为0．05±0．03,小于L6损伤组0．13±0．04、0．15±0．07、0．13±0．07(P<0．05)。T9损伤组使用阿朴吗啡后5 min和10 min平均频率分别为(122．40±47．99)、(151．80±76．42)Hz,较L6损伤组(278．83±118．66)、(265．00±81．35)Hz低(P<0．05)。结论海绵体肌电图对脊髓性勃起功能障碍有诊断价值。     

Functional characterization of NGF-secreting cell grafts to the acutely injured spinal cord

Previously we reported that grafts of cells genetically modified to produce human nerve growth factor (hNGF) promoted specific and robust sprouting of spinal sensory, motor, and noradrenergic axons. In the present study we extend these investigations to assess NGF effects on corticospinal motor axons and on functional outcomes after spinal cord injury. Fibroblasts from adult rats were transduced to express human NGF; control cells were not genetically modified. Fibroblasts were then grafted to sites of midthoracic spinal cord dorsal hemisection lesions. Three months later, recipients of NGF-secreting grafts showed deficits on conditioned locomotion over a wire mesh that did not differ in extent from control-lesioned animals. On histological examination, NGF-secreting grafts elicited specific sprouting from spinal primary sensory afferent axons, local motor axons, and putative cerulospinal axons as previously reported, but no specific responses from corticospinal axons. Axons responding to NGF robustly penetrated the grafts but did not exit the grafts to extend to normal innervation territories distal to grafts. Grafted cells continued to express NGF protein through the experimental period of the study. These findings indicate that 1) spinal cord axons show directionally sensitive growth responses to neurotrophic factors, 2) growth of axons responding to a neurotrophic factor beyond an injury site and back to their natural target regions will likely require delivery of concentration gradients of neurotrophic factors toward the target, 3) corticospinal axons do not grow toward a cellular source of NGF, and 4) functional impairments are not improved by strictly local sprouting response of nonmotor systems.     

Review: Infertility in the spinal cord injured male

Summary A review of the current theories regarding altered testicular function in the spinal cord injured male is presented. Therapy aimed at converting anejaculatory patients to antegrade or retrograde ejaculation is summarized. The current concepts of medical stimulation, electrovibration and electroejaculation are reviewed in depth. Side effects, including autonomic dysreflexia occurring in patients with high spinal lesions, limit but rarely prohibit these techniques. Other treatment options noted are vasal aspiration or implantation of alloplastic spermatoceles and subsequent use of in vitro fertilization techniques.     

Testis biopsy findings in the spinal cord injured patient

PURPOSE: Azoospermia after electroejaculation in spinal cord injured men may be due to testicular failure or obstruction. These men can initiate pregnancy with assisted reproductive techniques, such as intracytoplasmic sperm injection, but only if sperm are present in the testis biopsy. We analyzed the histopathology of testis biopsies from spinal cord injured men and assessed whether patient factors were predictive of testis biopsy pathology. MATERIALS AND METHODS: A total of 50 paraplegic men undergoing testis biopsy were divided into 2 groups based on normal or abnormal testis histopathology. Patient age, post-injury years, level of lesion, hormonal status and semen analysis results were compared. RESULTS: Spermatogenesis was normal in 28 of the 50 patients. Hypospermatogenesis was exhibited in 15, maturation arrest at the spermatid stage in 6 and maturation arrest at the spermatocyte stage in 1 of the 22 abnormal cases. Nevertheless, mature sperm were identified in 43 of 50 biopsies (normal spermatogenesis and hypospermatogenesis). Men with normal spermatogenesis had better forward progression of sperm and a higher testosterone-to-luteinizing hormone ratio. Otherwise, there was no statistically significant correlation between study variables and testis biopsy results. No factors were predictive of testis biopsy histopathology. CONCLUSIONS: The documentation of mature sperm in 43 of 50 biopsies from spinal cord injured patients suggests that a high rate of sperm retrieval is possible using testicular sperm extraction if sperm cannot be retrieved from the ejaculate. With intracytoplasmic sperm injection techniques the majority of spinal cord injured men retain fertility potential, even if azoospermic following electroejaculation.     

Improving outcome for the injured brain and spinal cord

Although injury to the brain and spinal cord can have varied etiology and mechanisms, the common pathway appears to be mediated by occurrence of ischemia and secondary injury. Because the pathophysiology in traumatic brain injury is heterogeneous, improvement in outcome will come from better diagnosis and monitoring, so that targeted therapy can be tailored to the individual patient. This review focuses on traumatic injury to the brain and spinal cord, and highlights recent developments in this area.     

人脐血单个核细胞促进脊髓损伤后的轴突再生

[目的] 讨论人脐血单个核细胞(human cord blood mononuclear cells,HCMNCs)移植治疗大鼠脊髓损伤后损伤区域轴突再生情况和功能恢复.[方法] 利用 Impactor Model Ⅱ打击器制成30例T10脊髓损伤模型,分组为:实验对照组(DMEM细胞培养基),损伤后3 d HCMNCs移植组,损伤后14 d HCMNCs移植组,每组10例.以HE染色和免疫组化染色以及BDA顺行示踪染色观察脊髓损伤处轴突再生情况,结合对各组实验动物脊髓损伤后肢体功能的恢复情况进行行为学评分(BBB 评分),综合评估脊髓功能恢复程度.[结果] 与对照组比较,HCMNCs移植治疗能够明显促进神经轴突再生,改善功能恢复,损伤后14 d HCMNCs移植组优于损伤后3 d移植组,各组间疗效差异具有统计学意义(P<0.01).[结论] HCMNCs在体内向神经元及神经胶质细胞分化,促进神经轴突再生和功能恢复.损伤后14 d是移植的较为理想的时间.     

Recovery of spinal cord function induced by direct current stimulation of the injured rat spinal cord

Direct current stimulation has been shown by others to enhance the regeneration of several types of tissues, including nervous tissue in some species. The purpose of the present experiment was to assess the value of direct current stimulation for enhancing the recovery of spinal cord function after clip compression injury of the rat spinal cord. Twenty Wistar rats underwent a 1-minute, 50-g clip compression injury at T-1, after which electrodes were placed epidurally with the anode proximal and the cathode distal to the injury site. These electrodes were attached to a stimulator implanted subcutaneously. Ten animals received stimulators that produced a constant current of 14 microA, and the remainder received stimulators with no electrical output and served as controls. Assignment of stimulators was random, and the treatment group was not identified until sacrifice. Neurological function was tested weekly for 15 weeks by the inclined plane technique, after which the animals were killed and the injured cords were examined for histological evidence of regeneration. The mean inclined plane result for the treatment group (39 +/- 5 degrees) was significantly better than that for the control group (31 +/- 6 degrees) (P less than 0.02), although there was no significant difference in histological findings between the two groups. Thus, direct current stimulation of the injured mammalian spinal cord produced improvement in neurological function and warrants further investigation.