Clemastine Enhances Myelination,Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.

     

Oncostatin M Reduces Lesion Size and Promotes Functional Recovery and Neurite Outgrowth After Spinal Cord Injury

The family of interleukin (IL)-6 like cytokines plays an important role in the neuroinflammatory response to injury by regulating both neural as well as immune responses. Here, we show that expression of the IL-6 family member oncostatin M (OSM) and its receptor is upregulated after spinal cord injury (SCI). To reveal the relevance of increased OSM signaling in the pathophysiology of SCI, OSM was applied locally after spinal cord hemisection in mice. OSM treatment significantly improved locomotor recovery after mild and severe SCI. Improved recovery in OSM-treated mice was associated with a reduced lesion size. OSM significantly diminished astrogliosis and immune cell infiltration. Thus, OSM limits secondary damage after CNS trauma. In vitro viability assays demonstrated that OSM protects primary neurons in culture from cell death, suggesting that the underlying mechanism involves direct neuroprotective effects of OSM. Furthermore, OSM dose-dependently promoted neurite outgrowth in cultured neurons, indicating that the cytokine plays an additional role in CNS repair. Indeed, our in vivo experiments demonstrate that OSM treatment increases plasticity of serotonergic fibers after SCI. Together, our data show that OSM is produced at the lesion site, where it protects the CNS from further damage and promotes recovery.     

Photomechanical Wave-Driven Delivery of siRNAs Targeting Intermediate Filament Proteins Promotes Functional Recovery after Spinal Cord Injury in Rats

The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.     

Sema4D Knockdown in Oligodendrocytes Promotes Functional Recovery After Spinal Cord Injury

Semaphorin4D (Sema4D) belongs to Semaphorins family and is secreted and membrane-bound protein. Its function on angiogenesis and axon regeneration makes it an ideal therapeutic target for spinal cord injury (SCI). Here we examined Sema4D expression profile by real-time PCR and western blot and found Sema4D was upregulated after SCI. In vitro study showed Sema4D was not only expressed in oligodendrocytes but also in endothelial cells (ECs). Hypoxia can mimic Sema4D upregulation in both cell lines. Moreover, overexpression of Sema4D through lentivirus in ECs promoted tube formation. However, Sema4D overexpression in oligodendrocytes precursor cells (OPCs) inhibited neuron myelination in neuron-oligodendrocyte co-culture system. Therefore, Sema4D knockdown in OPCs was applied in SCI rats. The results indicated that Sema4D knockdown significantly promoted functional recovery with blood–brain barrier score. Taken together, our data suggest that specific Sema4D knockdown in oligodendrocytes without disturbing its angiogenesis effect can be a beneficial strategy for SCI treatment.     

Recombinant DNA Vaccine Against Inhibition of Neurite Outgrowth Promotes Functional Recovery Associated with Endogeous NGF Expression in Spinal Cord Hemisected Adult Rats

Axonal regeneration across the site of spinal cord lesion is often aborted in adult mammalian species. The use of DNA vaccine to nullify the inhibitory molecules has been shown to be effective in promoting axonal regeneration in injured spinal cord. The possible molecular mechanisms, however, remain to be elucidated. The present study showed that the administration of recombinant DNA vaccine encoding multiple domains, Nogo-66, Nogo-N, TnR, and MAG, significantly improved hindlimb locomotor functions in rats subjected to ablation of the dorsal halves of the cord. Western blot analysis demonstrated that nerve growth factor (NGF) levels in the spinal cord of immunized rats were significantly upregulated than those of control rats. Immunohistochemistry as well as in situ hybridization confirmed that NGF was expressed in neurons of the spinal cord. These findings indicated that functional recovery in immunized rats could be correlated with endogeous NGF expression in hemisected rat spinal cords. Y. Zhang and C.-G. Hao contributed equally to this work.     

IT Delivery of ChABC Modulates NG2 and Promotes GAP-43 Axonal Regrowth After Spinal Cord Injury

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8–2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.     

Identification of Up-Regulated Genes After Complete Spinal Cord Transection in Adult Rats

Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.     

Granulocyte Colony-Stimulating Factor (G-CSF) Protects Oligpdendrocyte and Promotes Hindlimb Functional Recovery after Spinal Cord Injury in Rats

Background

Granulocyte colony-stimulating factor (G-CSF) is a protein that stimulates differentiation, proliferation, and survival of cells in the granulocytic lineage. Recently, a neuroprotective effect of G-CSF was reported in a model of cerebral infarction and we previously reported the same effect in studies of murine spinal cord injury (SCI). The aim of the present study was to elucidate the potential therapeutic effect of G-CSF for SCI in rats.

Methods

Adult female Sprague-Dawley rats were used in the present study. Contusive SCI was introduced using the Infinite Horizon Impactor (magnitude: 200 kilodyne). Recombinant human G-CSF (15.0 µg/kg) was administered by tail vein injection at 1 h after surgery and daily the next four days. The vehicle control rats received equal volumes of normal saline at the same time points.

Results

Using a contusive SCI model to examine the neuroprotective potential of G-CSF, we found that G-CSF suppressed the expression of pro-inflammatory cytokine (IL-1 beta and TNF- alpha) in mRNA and protein levels. Histological assessment with luxol fast blue staining revealed that the area of white matter spared in the injured spinal cord was significantly larger in G-CSF-treated rats. Immunohistochemical analysis showed that G-CSF promoted up-regulation of anti-apoptotic protein Bcl-Xl on oligpodendrocytes and suppressed apoptosis of oligodendrocytes after SCI. Moreover, administration of G-CSF promoted better functional recovery of hind limbs.

Conclusions

G-CSF protects oligodendrocyte from SCI-induced cell death via the suppression of inflammatory cytokines and up-regulation of anti-apoptotic protein. As a result, G-CSF attenuates white matter loss and promotes hindlimb functional recovery.     

Erratum to: Recombinant DNA Vaccine Against Inhibition of Neurite Outgrowth Promotes Functional Recovery Associated with Endogeous NGF Expression in Spinal Cord Hemisected Adult Rats

Neurochemical Research -     

Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins α-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

Regulation of Autophagy and Ubiquitinated Protein Accumulation by bFGF Promotes Functional Recovery and Neural Protection in a Rat Model of Spinal Cord Injury

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.     

Peripheral Nerve Transplantation Combined with Acidic Fibroblast Growth Factor and Chondroitinase Induces Regeneration and Improves Urinary Function in Complete Spinal Cord Transected Adult Mice

The loss of lower urinary tract (LUT) control is a ubiquitous consequence of a complete spinal cord injury, attributed to a lack of regeneration of supraspinal pathways controlling the bladder. Previous work in our lab has utilized a combinatorial therapy of peripheral nerve autografts (PNG), acidic fibroblast growth factor (aFGF), and chondroitinase ABC (ChABC) to treat a complete T8 spinal cord transection in the adult rat, resulting in supraspinal control of bladder function. In the present study we extended these findings by examining the use of the combinatorial PNG+aFGF+ChABC treatment in a T8 transected mouse model, which more closely models human urinary deficits following spinal cord injury. Cystometry analysis and external urethral sphincter electromyograms reveal that treatment with PNG+aFGF+ChABC reduced bladder weight, improved bladder and external urethral sphincter histology, and significantly enhanced LUT function, resulting in more efficient voiding. Treated mice’s injured spinal cord also showed a reduction in collagen scaring, and regeneration of serotonergic and tyrosine hydroxylase-positive axons across the lesion and into the distal spinal cord. Regeneration of serotonin axons correlated with LUT recovery. These results suggest that our mouse model of LUT dysfunction recapitulates the results found in the rat model and may be used to further investigate genetic contributions to regeneration failure.     

Plasminogen Deficiency Causes Reduced Corticospinal Axonal Plasticity and Functional Recovery after Stroke in Mice

Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg^-/-) mice and their Plg-native littermates (Plg^+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg^+/+ and Plg^-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg^+/+ and Plg^-/- embryos. In Plg^+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg^-/- mice was significantly impaired compared to Plg^+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg^-/- mice compared with Plg^+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R²>0.82, p<0.01). Plg^-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.     

超早期高压氧对脊髓完全横断损伤大鼠血液生化及后肢运动功能的影响

目的探讨超早期高压氧（HBO）治疗对脊髓完全横断损伤模型血液生化及后肢运动功能的影响。方法 55只SD大鼠随机分为A组（假手术组,15只）、B组（模型组,20只）及C组（高压氧组,20只）,A组仅行椎板切除术,其余2组均行T10椎板水平脊髓完全横向切断术。B、C组均予常规护理,C组于术后3 h置于动物舱内开始高压氧治疗,10 d一疗程,共3疗程。分别于建立模型后第1～6周末,用BBB运动功能评分法评价并比较两组大鼠后肢运动功能恢复程度,术后第6周过量麻醉处死大鼠,以40 g/L多聚甲醛行心室-主动脉灌注,取脊髓损伤区标本,光镜观察损伤脊髓的组织病理学改变。检测血钙（Ca）、血磷（P）、血清碱性磷酸酶（ALP）改变情况。结果 B、C两组大鼠术后第1～6周BBB运动功能评分逐渐增高,C组在3～6周末的BBB运动功能评分均明显高于B组。B组、C组血钙、血磷在术后1、3周高于A组,血清碱性磷酸酶（ALP）术后1、3周低于A组;C组血钙、血磷在术后5、6周低于B组。病理组织切片观察C组较B组组织水肿减轻,炎性细胞浸润减轻。结论超早期高压氧治疗能促进脊髓完全横断损伤大鼠后肢运动功能的部分恢复,降低血钙、血磷含量,对脊髓完全横断损伤大鼠具有保护和治疗作用。