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.     

Treadmill step training promotes spinal cord neural plasticity after incomplete spinal cord injury

A large body of evidence shows that spinal circuits are significantly affected by training,and that intrinsic circuits that drive locomotor tasks are located in lumbosacral spinal segments in rats with complete spinal cord transection.However,after incomplete lesions,the effect of treadmill training has been debated,which is likely because of the difficulty of separating spontaneous stepping from specific training-induced effects.In this study,rats with moderate spinal cord contusion were subjected to either step training on a treadmill or used in the model(control) group.The treadmill training began at day 7 post-injury and lasted 20 ± 10 minutes per day,5 days per week for 10 weeks.The speed of the treadmill was set to 3 m/min and was increased on a daily basis according to the tolerance of each rat.After 3 weeks of step training,the step training group exhibited a significantly greater improvement in the Basso,Beattie and Bresnahan score than the model group.The expression of growth-associated protein-43 in the spinal cord lesion site and the number of tyrosine hydroxylase-positive ventral neurons in the second lumbar spinal segment were greater in the step training group than in the model group at 11 weeks post-injury,while the levels of brain-derived neurotrophic factor protein in the spinal cord lesion site showed no difference between the two groups.These results suggest that treadmill training significantly improves functional recovery and neural plasticity after incomplete spinal cord injury.     

Methylprednisolone inhibits Nogo-A protein expression after acute spinal cord injury

Oligodendrocyte-produced Nogo-A has been shown to inhibit axonal regeneration. Methylprednisolone plays an effective role in treating spinal cord injury, but the effect of methylprednisolone on Nogo-A in the injured spinal cord remains unknown. The present study established a rat model of acute spinal cord injury by the weight-drop method. Results showed that after injury, the motor behavior ability of rats was reduced and necrotic injury appeared in spinal cord tissues, which was accompanied by increased Nogo-A expression in these tissues. After intravenous injection of high-dose methylprednisolone, although the pathology of spinal cord tissue remained unchanged, Nogo-A expression was reduced, but the level was still higher than normal. These findings implicate that methylprednisolone could inhibit Nogo-A expression, which could be a mechanism by which early high dose methylprednisolone infusion helps preserve spinal cord function after spinal cord injury.     

Electrical stimulation modulates injury potentials in rats after spinal cord injury

An injury potential is the direct current potential difference between the site of spinal cord injury and the healthy nerves.Its initial amplitude is a significant indicator of the severity of spinal cord injury,and many cations,such as sodium and calcium,account for the major portion of injury potentials.This injury potential,as well as injury current,can be modulated by direct current field stimulation;however,the appropriate parameters of the electrical field are hard to define.In this paper,injury potential is used as a parameter to adjust the intensity of electrical stimulation.Injury potential could be modulated to slightly above 0 mV(as the anode-centered group) by placing the anodes at the site of the injured spinal cord and the cathodes at the rostral and caudal sections,or around -70 mV,which is resting membrane potential(as the cathode-centered group) by reversing the polarity of electrodes in the anode-centered group.In addition,rats receiving no electrical stimulation were used as the control group.Results showed that the absolute value of the injury potentials acquired after 30 minutes of electrical stimulation was higher than the control group rats and much lower than the initial absolute value,whether the anodes or the cathodes were placed at the site of injury.This phenomenon illustrates that by changing the polarity of the electrical field,electrical stimulation can effectively modulate the injury potentials in rats after spinal cord injury.This is also beneficial for the spontaneous repair of the cell membrane and the reduction of cation influx.     

Optimal time for subarachnoid transplantation of neural progenitor cells in the treatment of contusive spinal cord injury

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.     

BMS-345541对脊髓损伤后大鼠NF-κB表达及运动功能的影响

This study sought to elucidate the changes of nuclear factor kappa B (NF-κB) expression and locomotor function of hind limb after subdural injection of BMS-345541 was applied in rats with acute spinal cord injury.The results indicated that BMS-345541 treatment reduced the expression of NF-kB at 24 hours after injury,compared with normal saline-tre ated rats.This treatment also led to a significant improvement in locomotor functional recovery at 14 days after injury.Overall,the findings demonstrated that BMS-345541 significantly ameliorated spinal cord injury-induced hind limb dysfunction by inhibiting the expression of NF-kB after spinal cord injury.     

Valproic acid reduces autophagy and promotes functional recovery after spinal cord injury in rats

Secondary damage is a critical determinant of the functional outcome in patients with spinal cord injury (SCI), and involves multiple mechanisms of which the most important is the loss of nerve cells mediated by multiple factors. Autophagy can result in cell death, and plays a key role in the development of SCI. It has been recognized that valproic acid (VPA) is neuroprotective in certain experimental animal models, however, the levels of autophagic changes in the process of neuroprotection by VPA treatment following SCI are still unknown. In the present study, we determined the extent of autophagy after VPA treatment in a rat model of SCI. We found that both the mRNA and protein levels of Beclin-1 and LC3 were significantly increased at 1, 2, and 6 h after SCI and peaked at 2 h; however, Western blot showed that autophagy was markedly decreased by VPA treatment at 2 h post-injury. Besides, post-SCI treatment with VPA improved the Basso-Beattie-Bresnahan scale, increased the number of ventral horn motoneurons, and reduced myelin sheath damage compared with vehicle-treated animals at 42 days after SCI. Together, our results demonstrated the characteristics of autophagy expression following SCI, and found that VPA reduced autophagy and enhanced motor function.     

Combination therapy targeting Akt and mammalian target of rapamycin improves functional outcome after controlled cortical impact in mice

Akt and mammalian target of rapamycin (mTOR) are both activated after traumatic brain injury (TBI), however complex interplay between the two hampers deciphering their functional implications in vivo. We examined the effects of single and combination inhibitors of Akt/mTOR in a mouse controlled cortical impact (CCI) model. Following CCI, phospho-Akt-473 (p-Akt) and -S6 ribosomal protein (p-S6RP), a downstream substrate of mTOR, were increased in cortical and hippocampal brain homogenates (P<0.05 versus sham). At 24 hours, p-S6RP was detected in neurons and was robustly induced in microglia and astrocytes in injured hippocampus. In vivo activity of Akt and mTOR inhibitors administered separately was confirmed by reduced expression of p-GSK3β (P<0.01) or p-S6RP (P<0.05), respectively, after CCI. Importantly, administration of Akt and mTOR inhibitors together (but not of either alone) improved postinjury motor (P=0.02) and cognitive deficits (hidden platform trials, P=0.001; probe trials, P<0.05), decreased propidium iodide-positive cells in CA1 and CA3 (P<0.005), and unexpectedly increased p-GSK3β in hippocampus. Although the roles of Akt and mTOR in the pathogenesis of TBI remain to be fully elucidated, dual inhibition of Akt and mTOR may have therapeutic potential for TBI.     

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.     

Monitoring somatosensory evoked potentials in spinal cord ischemia-reperfusion injury

It remains unclear whether spinal cord ischemia-reperfusion injury caused by ischemia and other non-mechanical factors can be monitored by somatosensory evoked potentials. Therefore, we monitored spinal cord ischemia-reperfusion injury in rabbits using somatosensory evoked potential detection technology. The results showed that the somatosensory evoked potential latency was significantly prolonged and the amplitude significantly reduced until it disappeared during the period of spinal cord ischemia. After reperfusion for 30-180 minutes, the amplitude and latency began to gradually recover; at 360 minutes of reperfusion, the latency showed no significant difference compared with the pre-ischemic value, while the somatosensory evoked potential amplitude in- creased, and severe hindlimb motor dysfunctions were detected. Experimental findings suggest that changes in somatosensory evoked potentia~ ~atency can reflect the degree of spinat cord ischemic injury, while the amplitude variations are indicators of the late spinal cord reperfusion injury, which provide evidence for the assessment of limb motor function and avoid iatrogenic spinal cord injury.     

Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury

Classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) macrophages populate the local microenvironment after spinal cord injury (SCI). The former type is neurotoxic while the latter has positive effects on neuroregeneration and is less toxic. In addition, while the M1 macrophage response is rapidly induced and sustained, M2 induction is transient. A promising strategy for the repair of SCI is to increase the fraction of M2 cells and prolong their residence time. This study investigated the effect of M2 macrophages induced from bone marrow-derived macrophages on the local microenvironment and their possible role in neuroprotection after SCI. M2 macrophages produced anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor β and infiltrated into the injured spinal cord, stimulated M2 and helper T (Th)2 cells, and produced high levels of IL-10 and -13 at the site of injury. M2 cell transfer decreased spinal cord lesion volume and resulted in increased myelination of axons and preservation of neurons. This was accompanied by significant locomotor improvement as revealed by Basso, Beattie and Bresnahan locomotor rating scale, grid walk and footprint analyses. These results indicate that M2 adoptive transfer has beneficial effects for the injured spinal cord, in which the increased number of M2 macrophages causes a shift in the immunological response from Th1- to Th2-dominated through the production of anti-inflammatory cytokines, which in turn induces the polarization of local microglia and/or macrophages to the M2 subtype, and creates a local microenvironment that is conducive to the rescue of residual myelin and neurons and preservation of neuronal function.     

NG2 expression in rats with acute T10 spinal cord injury

Rat models of T10 spinal cord injury were established with a clamp method.NG2 expression was detected with immunohistochemical staining and western blot.Ten days after spinal cord injury,the number of NG2-positive cells in the damaged areas and NG2 absorbance were both significantly increased.The findings indicate that acute T10 spinal cord injury in rats can lead to upregulation of NG2 protein expression in damaged areas.     

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

Emerging studies of treating spinal cord injury （SCI） with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibdssa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2＇-deoxyuridine （BrdU） labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes （receptor-interacting protein positive cells） and neuronal-like cells （~lll-tubulin positive cells） at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. The results demon- strate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.     

Effects of Jisuikang on hemorheology and inflammatory factors in rats following spinal cord injury

BACKGROUND: Trauma can damage the spinal cord or cauda equina to different degrees. Previous studies have verified that traditional Chinese medicine has effects on spinal cord injury via a variety of pathways. OBJECTIVE: To observe changes in hemorheology and inflammatory factors in spinal cord injury rats following treatment with the Chinese medicine Jisuikang, to verify the dose-dependent effect of Jisuikang, and to compare its effects with the effects of prednisone. DESIGN, TIME AND SETTING: A randomized study was performed at the Research Institute of Orthopedics, and Experimental Center of First Clinical Medical College, Nanjing University of Traditional Chinese Medicine, China from September 2007 to March 2008. MATERIALS: Jisuikang powdered extract, composed of milkvetch root (30 g), Chinese angelica (12 g), red peony root (12 g), earthworm (10 g), szechwan lovage rhizome (10 g), peach seed (10 g) and safflower (10 g), was provided by the Experimental Center, First Clinical Medical College, Nanjing University of Traditional Chinese medicine. Each gram of powdered extract was equivalent to 6.47 g crude drug. METHODS: A total of 72 Sprague Dawley rats were randomly assigned into 6 groups (n = 12). Rat models of spinal cord injury were established using the occlusion method. Rats in the model group were treated with distilled water. Rats in the 25 g/kg, 12.5 g/kg, and 6.25 g/kg Jisuikang groups were given 25 g/kg, 12.5 g/kg, or 6.25 g/kg Jisuikang by gavage, for 14 days. Rats in the prednisone group received 0.06 g/kg prednisone by gavage, for 7 days. Rats in the normal group were given the same volume of distilled water. The volume of administration was 15 mL/kg.MAIN OUTCOME MEASURES: Rat serum interleukin-10, tumor necrosis factor-α (TNF-α), nitric oxide, nitric oxide synthase levels, malondialdehyde content, superoxide dismutase activity and whole blood viscosity were measured in each group. Spinal cord around the site of the model was collected. Half the spinal cord was used for histopathologic examination. The other half was used for measurement of nitric oxide and NOS levels, malondialdehyde contents, and superoxide dismutase activity. RESULTS: Superoxide dismutase activity was higher in the 25 g/kg Jisuikang group than in the model group. Malondialdehyde contents, nitric oxide and NOS levels were lower in the 25 g/kg and 12.5 g/kg Jisuikang groups compared with the model group. Whole blood viscosity was lower in the 25 g/kg and 12.5 g/kg Jisuikang groups compared with the model group (P < 0.05-0.01). Serum TNF-α content was lower in each Jisuikang group compared with the model group (P < 0.05-0.01). Serum interleukin-10 levels were greater in the prednisone group and each Jisuikang group compared with the model group (P < 0.01). Mild hemorrhage and necrosis in the rat spinal cord, and unclear neural cell swelling were seen in the 25 g/kg Jisuikang group. Severe hemorrhage and necrosis in the rat spinal cord, and distinct neural cell swelling were seen in the 12.5 g/kg Jisuikang group. Edema in the white matter was found in the 6.25 g/kg Jisuikang group. Pathological changes in the prednisone group were identical to the 25 g/kg and 12.5 g/kg Jisuikang groups. CONCLUSION: Jisuikang inhibits nitric oxide synthase expression, reduces nitric oxide and TNF-α levels, decreases malondialdehyde content, increases interleukin-10 levels and superoxide dismutase activity, improves indices of hemorheology, and prevents secondary changes in spinal cord injury, resulting in relieving pathological changes in spinal cord tissue. The outcome was significant in the 25 g/kg Jisuikang group compared with the 12.5 g/kg Jisuikang group.     

Effects of Jisuikang on hemorheology and inflammatory factors in rats following spinal cord injury*☆

BACKGROUND： Trauma can damage the spinal cord or cauda equina to different degrees. Previous studies have verified that traditional Chinese medicine has effects on spinal cord injury via a variety of pathways. OBJECTIVE： To observe changes in hemorheology and inflammatory factors in spinal cord injury rats following treatment with the Chinese medicine Jisuikang, to verify the dose-dependent effect of Jisuikang, and to compare its effects with the effects of prednisone. DESIGN, TIME AND SETTING： A randomized study was performed at the Research Institute of Orthopedics, and Experimental Center of First Clinical Medical College, Nanjing University of Traditional Chinese Medicine, China from September 2007 to March 2008. MATERIALS： Jisuikang powdered extract, composed of milkvetch root （30 g）, Chinese angelica （12 g）, red peony root （12 g）, earthworm （10 g）, szechwan lovage rhizome （10 g）, peach seed （10 g） and safflower （10 g） was provided by the Experimental Center, First Clinical Medical College, Nanjing University of Traditional Chinese medicine. Each gram of powdered extract was equivalent to 6.47 g crude drug. METHODS： A total of 72 Sprague Dawley rats were randomly assigned into 6 groups （n = 12）. Rat models of spinal cord injury were established using the occlusion method. Rats in the model group were treated with distilled water. Rats in the 25 g/kg, 12.5 g/kg, and 6.25 g/kg Jisuikang groups were given 25 g＆g, 12.5 g/kg, or 6.25 g/kg Jisuikang by gavage, for 14 days. Rats in the prednisone group received 0.06 g/kg prednisone by gavage, for 7 days. Rats in the normal group were given the same volume of distilled water. The volume of administration was 15 mL/kg. MAIN OUTCOME MEASURES： Rat serum interleukin-10, tumor necrosis factor- α （TNF-α ）, nitric oxide, nitric oxide synthase levels, malondialdehyde content, superoxide dismutase activity and whole blood viscosity were measured in each group. Spinal cord around the site of the model was collected. Half the     

Combination of methylprednisolone and rosiglitazone promotes recovery of neurological function atfer spinal cord injury

Methylprednisolone exhibits anti-inlfammatory antioxidant properties, and rosiglitazone acts as an anti-inlfammatory and antioxidant by activating peroxisome proliferator-activated receptor-γ in the spinal cord. Methylprednisolone and rosiglitazone have been clinically used during the early stages of secondary spinal cord injury. Because of the complexity and diversity of the inlfammatory process atfer spinal cord injury, a single drug cannot completely inhibit inlfammation. hTerefore, we assumed that a combination of methylprednisolone and rosiglitazone might promote recovery of neurological function atfer secondary spinal cord injury. In this study, rats were intraperitoneally injected with methylprednisolone (30 mg/kg) and rosiglitazone (2 mg/kg) at 1 hour atfer injury, and methylprednisolone (15 mg/kg) at 24 and 48 hours atfer injury. Rosiglitazone was then administered once every 12 hours for 7 consecutive days. Our results demonstrated that a combined treatment with methylprednisolone and rosiglitazone had a more pronounced effect on attenuation of inlfammation and cell apoptosis, as well as increased functional recovery, compared with either single treatment alone, indicating that a combination better pro-moted recovery of neurological function atfer injury.     

Role of endogenous Schwann cells in tissue repair after spinal cord injury

Schwann cells are glial cells of peripheral nervous system, responsible for axonal myelination and ensheathing, as well as tissue repair following a peripheral nervous system injury. They are one of several cell types that are widely studied and most commonly used for cell transplantation to treat spinal cord injury, due to their intrinsic characteristics including the ability to secrete a variety of neurotrophic factors. This mini review summarizes the recent findings of endogenous Schwann cells after spinal cord injury and discusses their role in tissue repair and axonal regeneration. After spinal cord injury, numerous endogenous Schwann cells migrate into the lesion site from the nerve roots, involving in the construction of newly formed repaired tissue and axonal myelination. These invading Schwann cells also can move a long distance away from the injury site both rostrally and caudally. In addition, Schwann cells can be induced to migrate by minimal insults (such as scar ablation) within the spinal cord and integrate with astrocytes under certain circumstances. More importantly, the host Schwann cells can be induced to migrate into spinal cord by transplantation of different cell types, such as exogenous Schwann cells, olfactory ensheathing cells, and bone marrow-derived stromal stem cells. Migration of endogenous Schwann cells following spinal cord injury is a common natural phenomenon found both in animal and human, and the myelination by Schwann cells has been examined effective in signal conduction electrophysiologically. Therefore, if the inherent properties of endogenous Schwann cells could be developed and utilized, it would offer a new avenue for the restoration of injured spinal cord.     

Synaptosomal-associated protein 25 may be an intervention target for improving sensory and locomotor functions after spinal cord contusion

Synaptosomal-associated protein 25 kDa (SNAP-25) is localized on the synapse and participates in exocytosis and neurotransmitter release. Decreased expression of SNAP-25 is associated with Alzheimer's disease and attention deficit/hyperactivity disorder. However, the expres-sion of SNAP-25 in spinal cord contusion injury is still unclear. We hypothesized that SNAP-25 is associated with sensory and locomotor functions after spinal cord injury. We established rat models of spinal cord contusion injury to detect gene changes with a gene array. A de-creased level of SNAP-25 was detected by quantitative real time-polymerase chain reaction and western blot assay at 1, 3, 7, 14 and 28 days post injury. SNAP-25 was localized in the cytoplasm of neurons of the anterior and posterior horns, which are involved in locomotor and sensory functions. Our data suggest that reduced levels of SNAP-25 are associated with sensory and locomotor functions in rats with spinal cord contusion injury.