Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice

Chondroitin sulphate proteoglycans (CSPGs) are extracellular matrix molecules whose inhibitory activity is attenuated by the enzyme chondroitinase ABC (ChABC). Here we assess whether CSPG degradation can promote compensatory sprouting of the intact corticospinal tract (CST) following unilateral injury and restore function to the denervated forelimb. Adult C57BL/6 mice underwent unilateral pyramidotomy and treatment with either ChABC or a vehicle control. Significant impairments in forepaw symmetry were observed following pyramidotomy, with injured mice preferentially using their intact paw during spontaneous vertical exploration of a cylinder. No recovery on this task was observed in vehicle‐treated mice. However, ChABC‐treated mice showed a marked recovery of function, with forelimb symmetry fully restored by 5 weeks post‐injury. Functional recovery was associated with robust sprouting of the uninjured CST, with numerous axons observed crossing the midline in the brainstem and spinal cord and terminating in denervated grey matter. CST fibres in the denervated side of the spinal cord following ChABC treatment were closely associated with the synaptic marker vGlut1. Immunohistochemical assessment of chondroitin‐4‐sulphate revealed that CSPGs were heavily digested around lamina X, alongside midline crossing axons and in grey matter regions where sprouting axons and reduced peri‐neuronal net staining was observed. Thus, we demonstrate that CSPG degradation promotes midline crossing and reinnervation of denervated target regions by intact CST axons and leads to restored function in the denervated forepaw. Enhancing compensatory sprouting using ChABC provides a route to restore function that could be applied to disorders such as spinal cord injury and stroke.     

Degradation of chondroitin sulfate proteoglycans potentiates transplant-mediated axonal remodeling and functional recovery after spinal cord injury in adult rats

Transplantation of growth-permissive cells or tissues was used to bridge a lesion cavity and induce axonal growth in experimental spinal cord injury (SCI). Axonal interactions between host and transplant may be affected by upregulation of inhibitory chondroitin sulfate proteoglycans (CSPGs) following various transplantation strategies. The extent of axonal growth and functional recovery after transplantation of embryonic spinal cord tissue decreases in adult compared to neonatal host. We hypothesized that CSPGs contribute to the decrease in the extent to which transplant supports axonal remodeling and functional recovery. Expression of CSPGs increased after overhemisection SCI in adult rats but not in neonates. Embryonic spinal cord transplant was surrounded by CSPGs deposited in host cord, and the interface between host and transplant seemed to contain a large amount of CSPGs. Intrathecally delivered chondroitinase ABC (C'ase) improved recovery of distal forelimb usage and skilled motor behavior after C4 overhemisection injury and transplantation in adults. This behavioral recovery was accompanied by an increased amount of raphespinal axons growing into the transplant, and raphespinal innervation to the cervical motor region was promoted by C'ase plus transplant. Moreover, C'ase increased the number of transplanted neurons that grew axons to the host cervical enlargement, suggesting that degradation of CSPGs supports remodeling not only of host axons but also axons from transplanted neurons. Our results suggest that CSPGs constitute an inhibitory barrier to prevent axonal interactions between host and transplant in adults, and degradation of the inhibitory barrier can potentiate transplant-mediated axonal remodeling and functional recovery after SCI.     

Lentiviral‐mediated silencing of glial fibrillary acidic protein and vimentin promotes anatomical plasticity and functional recovery after spinal cord injury

In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)‐mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral‐mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral‐mediated RNAi in SCI. © 2014 Wiley Periodicals, Inc.     

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.     

Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine

It is well established that axons of the adult mammalian CNS are capable of regrowing only a limited amount after injury. Astrocytes are believed to play a crucial role in the failure to regenerate, producing multiple inhibitory proteoglycans, such as chondroitin sulphate proteoglycans (CSPGs). After spinal cord injury (SCI), astrocytes become hypertrophic and proliferative and form a dense network of astroglial processes at the site of lesion constituting a physical and biochemical barrier. Down-regulations of astroglial proliferation and inhibitory CSPG production might facilitate axonal regeneration. Recent reports indicated that aberrant activation of cell cycle machinery contributed to overproliferation and apoptosis of cells in various insults. In the present study, we sought to determine whether a cell cycle inhibitior, olomoucine, would decrease neuronal cell death, limit astroglial proliferation and production of inhibitory CSPGs, and eventually enhance the functional compensation after SCI in rats. Our results showed that up-regulations of cell cycle components were closely associated with neuronal cell death and astroglial proliferation as well as the production of CSPGs after SCI. Meanwhile, administration of olomoucine, a selective cell cycle kinase (CDK) inhibitor, has remarkably reduced the up-regulated cell cycle proteins and then decreased neuronal cell death, astroglial proliferation, and accumulation of CSPGs. More importantly, the treatment with olomoucine has also increased expression of growth-associated proteins-43, reduced cavity formation, and improved functional deficits. We consider that suppressing astroglial cell cycle in acute SCIs is beneficial to axonal growth. In the future, therapeutic strategies can be designed to achieve efficient axonal regeneration and functional compensation after traumatic CNS injury.     

Neuregulin‐1 positively modulates glial response and improves neurological recovery following traumatic spinal cord injury

Spinal cord injury (SCI) results in glial activation and neuroinflammation, which play pivotal roles in the secondary injury mechanisms with both pro‐ and antiregeneration effects. Presently, little is known about the endogenous molecular mechanisms that regulate glial functions in the injured spinal cord. We previously reported that the expression of neuregulin‐1 (Nrg‐1) is acutely and chronically declined following traumatic SCI. Here, we investigated the potential ramifications of Nrg‐1 dysregulation on glial and immune cell reactivity following SCI. Using complementary in vitro approaches and a clinically‐relevant model of severe compressive SCI in rats, we demonstrate that immediate delivery of Nrg‐1 (500 ng/day) after injury enhances a neuroprotective phenotype in inflammatory cells associated with increased interleukin‐10 and arginase‐1 expression. We also found a decrease in proinflammatory factors including IL‐1β, TNF‐α, matrix metalloproteinases (MMP‐2 and 9) and nitric oxide after injury. In addition, Nrg‐1 modulates astrogliosis and scar formation by reducing inhibitory chondroitin sulfate proteoglycans after SCI. Mechanistically, Nrg‐1 effects on activated glia are mediated through ErbB2 tyrosine phosphorylation in an ErbB2/3 heterodimer complex. Furthermore, Nrg‐1 exerts its effects through downregulation of MyD88, a downstream adaptor of Toll‐like receptors, and increased phosphorylation of Erk1/2 and STAT3. Nrg‐1 treatment with the therapeutic dosage of 1.5 μg/day significantly improves tissue preservation and functional recovery following SCI. Our findings for the first time provide novel insights into the role and mechanisms of Nrg‐1 in acute SCI and suggest a positive immunomodulatory role for Nrg‐1 that can harness the beneficial properties of activated glia and inflammatory cells in recovery following SCI.     

Blockade of interleukin-6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury

Endogenous neural stem/progenitor cells (NSPCs) have recently been shown to differentiate exclusively into astrocytes, the cells that are involved in glial scar formation after spinal cord injury (SCI). The microenvironment of the spinal cord, especially the inflammatory cytokines that dramatically increase in the acute phase at the injury site, is considered to be an important cause of inhibitory mechanism of neuronal differentiation following SCI. Interleukin-6 (IL-6), which has been demonstrated to induce NSPCs to undergo astrocytic differentiation selectively through the JAK/STAT pathway in vitro, has also been demonstrated to play a critical role as a proinflammatory cytokine and to be associated with secondary tissue damage in SCI. In this study, we assessed the efficacy of rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) in the treatment of acute SCI in mice. Immediately after contusive SCI with a modified NYU impactor, mice were intraperitoneally injected with a single dose of MR16-1 (100 microg/g body weight), the lesions were assessed histologically, and the functional recovery was evaluated. MR16-1 not only suppressed the astrocytic diffentiation-promoting effect of IL-6 signaling in vitro but inhibited the development of astrogliosis after SCI in vivo. MR16-1 also decreased the number of invading inflammatory cells and the severity of connective tissue scar formation. In addition, we observed significant functional recovery in the mice treated with MR16-1 compared with control mice. These findings suggest that neutralization of IL-6 signaling in the acute phase of SCI represents an attractive option for the treatment of SCI.     

Sustained release of neurotrophin‐3 via calcium phosphate‐coated sutures promotes axonal regeneration after spinal cord injury

Because of the dynamics of spinal cord injury (SCI), the optimal treatment will almost certainly be a combination approach to control the environment and promote axonal growth. This study uses peripheral nerve grafts (PNGs) as scaffolds for axonal growth while delivering neurotrophin‐3 (NT‐3) via calcium phosphate (CaP) coatings on surgical sutures. CaP coating was grown on sutures, and NT‐3 binding and release were characterized in vitro. Then, the NT‐3‐loaded sutures were tested in a complete SCI model. Rats were analyzed for functional improvement and axonal growth into the grafts. The CaP‐coated sutures exhibited a burst release of NT‐3, followed by a sustained release for at least 20 days. Functionally, the rats with PNGs + NT‐3‐loaded sutures and the rats treated with PNGs scored significantly higher than controls on day 56 postoperatively. However, functional scores in rats treated with PNGs + NT‐3‐loaded suture were not significantly different from those of rats treated with PNGs alone. Cholera toxin subunit B (CTB) labeling rostral to the graft was not observed in any controls, but CTB labeling rostral to the graft was observed in almost all rats that had had a PNG. Neurofilament labeling on transverse sections of the graft revealed that the rats treated with the NT‐3‐loaded sutures had significantly more axons per graft than rats treated with an NT‐3 injection and rats without NT‐3. These data demonstrate that PNGs serve as scaffolds for axonal growth after SCI and that CaP‐coated sutures can efficiently release NT‐3 to increase axonal regeneration. © 2016 Wiley Periodicals, Inc.     

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.     

Combination treatment with anti‐Nogo‐A and chondroitinase ABC is more effective than single treatments at enhancing functional recovery after spinal cord injury

Anti‐Nogo‐A antibody and chondroitinase ABC (ChABC) enzyme are two promising treatments that promote functional recovery after spinal cord injury (SCI). Treatment with them has encouraged axon regeneration, sprouting and functional recovery in a variety of spinal cord and central nervous system injury models. The two compounds work, in part, through different mechanisms, so it is possible that their effects will be additive. In this study, we used a rat cervical partial SCI model to explore the effectiveness of a combination of anti‐Nogo‐A, ChABC, and rehabilitation. We found that spontaneous recovery of forelimb functions reflects the extent of the lesion on the ipsilateral side. We applied a combination treatment with acutely applied anti‐Nogo‐A antibody followed by delayed ChABC treatment starting at 3 weeks after injury, and rehabilitation starting at 4 weeks, to accommodate the requirement that anti‐Nogo‐A be applied acutely, and that rehabilitation be given after the cessation of anti‐Nogo‐A treatment. We found that single treatment with either anti‐Nogo‐A or ChABC, combined with rehabilitation, produced functional recovery of similar magnitude. The combination treatment, however, was more effective. Both single treatments produced increases in sprouting and axon regeneration, but the combination treatment produced greater increases. Anti‐Nogo‐A stimulated growth of a greater number of axons with a diameter of > 3 μm, whereas ChABC treatment stimulated increased growth of finer axons with varicosities. These results point to different functions of Nogo‐A and chondroitin sulfate proteoglycans in axonal regeneration. The combination of anti‐Nogo‐A, ChABC and rehabilitation shows promise for enhancing functional recovery after SCI.     

Combined transplantation of bone marrow mesenchymal stem cells and pedicled greater omentum promotes locomotor function and regeneration of axons after spinal cord injury in rats*★

BACKGROUND： According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells （BMSCs） into neural cells. OBJECTIVE： Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETFING： A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007. MATERIALS： Fifteen male inbred Wistar rats, weighing （200±20） g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS： Cell culture： We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention： The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury （SCI） only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the Tl0 level for 30 seconds to produce the SCI model. 5 μ L PBS containing 10^5 BMSCs was injected in     

神经营养素-3基因非病毒载体转染的嗅鞘细胞移植促进脊髓损伤大鼠轴突再生及功能恢复

目的将神经营养素-3（neurotrophin-3,NT-3）基因转染的嗅鞘细胞（olfactory ensheathing glia,OEG）移植到脊髓损伤大鼠体内,以期促进大鼠胸脊髓损伤的恢复。方法将自行构建的质粒pcDNA3．1（＋）-NT3,应用脂质体介导的方法导入体外培养的OEG,将其移植入急性脊髓损伤大鼠体内,连续观察12周,与接受单纯OEG移植和空白质粒转染OEG移植及无OEG移植的脊髓损伤大鼠进行比较。结果pcDNA3.1（＋）-NT3转染的OEG移植后能在体内长期存活,表达NT-3基因,并较对照组更能促进脊髓损伤区轴突的再生和后肢功能的恢复。结论OEG是脊髓损伤基因治疗较好的受体细胞。转染OEG移植后可以在体内较长时间存活。能明显促进急性脊髓挫伤神经纤维再生和功能恢复的作用,为基因修饰嗅鞘细胞在脊髓损伤治疗的应用提供了实验和理论依据。