Recovery Of Hindlimb Function Research Articles

Transplantation of cryopreserved rat fetal neural cells in suspension and in multicellular aggregates into rats with spinal cord injury

Today cell transplantation is one of the promising approaches of spinal cord injuries treatment. The aim of the work was to study the effect of cryopreserved fetal neural cell transplantation in suspensions and cell aggregates for motor activity recovery of rats with experimental spinal cord injury.Materials and methods. Cells were isolated from the brain tissue of rat fetuses 15-16 days of gestation. The formation of aggregates was performed during short-term cultivation at a concentration of 8·106cells/mL in medium with 10 % adult rat serum. Cell transplantation was performed into the damaged area of spinal cord in aggregates or suspension. To fix transplanted cells in the damaged area we used alginate gel.Results. Transplantation of cryopreserved fetal neural cells in alginate gel had the positive effect on dynamics of rats’ motor activity recovery. That was manifested in the extensive mobility of three joints of one limb and the limited mobility of two joints of the other with simultaneous recovery of the sensitivity of the hind limbs.Conclusion. Cryopreserved fetal neural cells aggregates had a high therapeutic potential on rat traumatic spinal cord injury compared with cell suspension by improving the structure of forming nervous tissue and significantly increasing the rate of hind limb function recovery.

Conditional Sox9 ablation improves locomotor recovery after spinal cord injury by increasing reactive sprouting

The absence of axonal regeneration after spinal cord injury (SCI) has been attributed to the up-regulation of axon-repelling molecules, such as chondroitin sulfate proteoglycans (CSPGs) present in the glial scar that forms post-SCI. We previously identified the transcription factor SOX9 as a key up-regulator of CSPG production and also demonstrated that conditional Sox9 ablation leads to decreased CSPG levels and improved recovery of hind limb function after SCI. We herein demonstrate increased neural input onto spinal neurons caudal to the lesion in spinal cord injured Sox9 conditional knock out mice as indicated by increased levels of the presynaptic markers synaptophysin and vesicular glutamate transporter 1 (VGLUT1) compared to controls. Axonal sparing, long-range axonal regeneration and reactive sprouting were investigated as possible explanations for the increase in neural inputs caudal to the lesion and for the improved locomotor outcomes in spinal cord-injured Sox9 conditional knock out mice. Whereas retrograde tract-tracing studies failed to reveal any evidence for increased axonal sparing or for long-range regeneration in the Sox9 conditional knock out mice, anterograde tract-tracing experiments demonstrated increased reactive sprouting caudal to the lesion after SCI. Finally we demonstrate that application of a broad spectrum MMP inhibitor to reduce CSPG degradation in Sox9 conditional knock out mice prevents the improvements in locomotor recovery observed in untreated Sox9 conditional knock out mice. These results suggest that improved recovery of locomotor function in Sox9 conditional knock out mice after SCI is due to increased reactive sprouting secondary to reduced CSPG levels distal to the lesion.

Transplanted Peripheral Blood Stem Cells Mobilized by Granulocyte Colony-Stimulating Factor Promoted Hindlimb Functional Recovery after Spinal Cord Injury in Mice

Granulocyte colony-stimulating factor (G-CSF) mobilizes peripheral blood stem cells (PBSCs) derived from bone marrow. We hypothesized that intraspinal transplantation of PBSCs mobilized by G-CSF could promote functional recovery after spinal cord injury. Spinal cords of adult nonobese diabetes/severe immunodeficiency mice were injured using an Infinite Horizon impactor (60 kdyn). One week after the injury, 3.0 µl of G-CSF-mobilized human mononuclear cells (MNCs; 0.5 × 10(5)/µl), G-CSF-mobilized human CD34-positive PBSCs (CD34; 0.5 × 10(5)/µl), or normal saline was injected to the lesion epicenter. We performed immunohistochemistry. Locomotor recovery was assessed by Basso Mouse Scale. The number of transplanted human cells decreased according to the time course. The CD31-positive area was significantly larger in the MNC and CD34 groups compared with the vehicle group. The number of serotonin-positive fibers was significantly larger in the MNC and CD34 groups than in the vehicle group. Immunohistochemistry revealed that the number of apoptotic oligodendrocytes was significantly smaller in cell-transplanted groups, and the areas of demyelination in the MNC- and CD34-transplanted mice were smaller than that in the vehicle group, indicating that cell transplantation suppressed oligodendrocyte apoptosis and demyelination. Both the MNC and CD34 groups showed significantly better hindlimb functional recovery compared with the vehicle group. There was no significant difference between the two types of transplanted cells. Intraspinal transplantation of G-CSF-mobilized MNCs or CD34-positive cells promoted angiogenesis, serotonergic fiber regeneration/sparing, and preservation of myelin, resulting in improved hindlimb function after spinal cord injury in comparison with vehicle-treated control mice. Transplantation of G-CSF-mobilized PBSCs has advantages for treatment of spinal cord injury in the ethical and immunological viewpoints, although further exploration is needed to move forward to clinical application.

Attenuating spinal cord injury by conditioned medium from human umbilical cord blood-derived CD34+ cells in rats

ObjectiveIntravenous or intraspinal transplantation of human umbilical cord blood cells-derived CD34+ cells (human CD34+ cells) or mesenchymal stem cells after spinal cord injury (SCI) improved hind limb functional recovery in adult rats. The objective of this study is to ascertain whether SCI in rats can be attenuated by conditioned medium (CM) or secretome obtained from cultured human CD34+ stem cells. Materials and methodsSprague-Dawley rats were assigned to one of the following five groups: the sham group, the SCI group treated with vehicle solution (SCI + V), the SCI group treated with CM (SCI + CM), the SCI group treated with 17β-estradiol E2 (10 μg; SCI + E2), and the SCI group treated with CM plus E2 (SCI + CM + E2). A 0.5-mL volume of CM or vehicle solution was administered intravenously immediately after SCI. ResultsCompared with the sham group, the (SCI + V) group had significantly higher scores of neurological motor dysfunction as well as inflammation apoptosis, oxidative stress, and astrogliosis in the injured spinal cord. The neurological deficits, numbers of apoptotic cell, extent of inflammation, oxidative stress, and astrogliosis in the injured spinal cord were significantly attenuated by CM, E2, or CM plus E2, but not by the vehicle solution. In addition, the neuroprotective effect exerted by a combination of CM and E2 is superior to that exerted by CM- or E2-alone therapy. ConclusionThe neuroprotective effects of CM from cultured human CD34+ cells are similar to those of human CD34+ cells and the CM was found to enhance the neuroprotective effects of E2 in rat SCI.

Pre-Differentiated Embryonic Stem Cell versus Olfactory Ensheathing Cell for Spinal Cord Regeneration after Compressive Injury in Rat

Background: Transplantation approaches are interventions currently available to apply to the devastating problem of spinal cord injury (SCI). Olfactory ensheathing cell (OEC) and embryonic stem cell (ESC) are considered to be promising therapeutic strategies . In this study, we compared the potential use of OECs and neurally pre-differentiated ESCs in contusion spinal cord. Methods: OECs were harvested from olfactory bulb of rats and labeled with Hoescht 33342. ESCs were generated by using feeder free GFP positive CGR8 mouse ESCs and neurally pre-differentiation was induced by retinoic acid (RA) and characterized by different antibodies. SCI was induced by fogarty catheter at T8-T9 level in adult rats. Transplantations were performed 9 days after the injury. Rats were randomly divided into 3 main groups (neurally pre-differentiated ESC, OEC and media as control group). The recovery of gross motor function was evaluated using Basso-Beattie-Bresnahan (BBB) locomotor rating scale on the ninth day post injury and once per week thereafter for 4 weeks after cell transplantation. At 28 days after transplantation, histological assessment including transplanted cell detection in tissue, tissue sparing and myelinated axons was performed. Results: Following transplantation, a significant recovery of hindlimb function according to BBB scale was observed in rats in the transplanted groups compared to control and sham groups (p<0.05). There was no significant difference between transplanted groups four weeks after transplantation. OEC and ESC were found in the tissue after transplantation. In OEC group, many of OECs were detected around and within the cystic cavity that number of these cells was significantly higher in comparison with number of cells in ESC group (p< 0.001). In the site of injury, several cavities were produced that disrupted portions of the gray and white matters. The extent of tissue damage was more severe in the sham and control groups than the other groups. Significantly more spinal tissue was spared in OEC and ESC groups (P < 0.001). No significant difference in percentage of spared tissue was found between sham and control groups or transplanted groups. The percentage of myelinated area was greater in OEC group than in three other groups (p< 0.05) (Figure 5, A). Although the percentage of myelinated area was more in ESC group in comparison with non-treated groups, but this difference was not significant. Conclusion: It seems that using of combination of a myelinating cells like OEC or schwann cell and source of cells to replace dead cells like Mesenchymal or embryonic stem cells, better results can be obtained due to probable synergic effects of these cells.

Beneficial effects of local profound hypothermia and the possible mechanism after experimental spinal cord injury in rats

Objective: The primary focus of this study was to investigate the effects of local profound hypothermia and to explore the possible mechanism in adult rats with spinal cord injury.Study Design and Methods: Spinal cord injury models were established by placing aneurysm clips on T10. An epidural perfusion device was applied to maintain a steady temperature (18 °C) for 120 min with gradual rewarming to 37 °C Total hypothermic duration lasted up to about 170 min. The expression of axon regeneration inhibitors was tested by Western blot and real-time PCR. Luxol Fast Blue (LFB) stain and Bielschowsky silver stain were used to observe spinal cord morphology. Motor function of the hind limbs (BBB score) was monitored for 21 days.Results: The expressions of RhoA, ROCK-II, NG2, Neurocan, Brevican, and Nogo-A were downregulated by regional hypothermia (RH) after spinal cord injury. Subsequent observation showed that rats that had received RH had an alleviated demyelinating condition and a greater number of nerve fibers. Furthermore, the RH group achieved higher BBB scores than the spinal cord injury (SCI) group.Conclusions: Recovery of hind limb function in rats can be promoted by local profound hypothermia; this may be caused by the suppression of axon regeneration inhibitors.

Compensatory projections of primary sensory fibers in lumbar spinal cord after neonatal thoracic spinal transection in rats.

Complete spinal transection in adult rats results in poor recovery of hind limb function, whereas significant spontaneous recovery can occur following spinal cord transection in rat neonates. The mechanisms underlying the recovery, however, are poorly understood. Recent studies in rodents suggested that the recovery is not due to axonal regeneration, but rather due to reorganization of the neural circuits in the spinal cord below the injury site, including central pattern generators. Few studies have reported histological evidence for changes in the primary sensory fibers or terminals. Thus, in the present study, we transected spinal cords of rats at thoracic level 8 at postnatal day 5. Four weeks after the injury, biotinylated-dextran amine (BDA), an anterograde tracer, was injected into the dorsal root ganglion of the lumbar spinal cord to examine the localization of sensory fibers and their terminal buttons in the spinal cord. BDA-positive axons in the rat spinal cord following neonatal spinal transection (neo ST) were longer than those in sham-operated or normal rats. The number of terminal buttons was also higher in spinal cords of neo ST rats compared with sham-operated or normal rats. These findings suggest that sensory fibers project more strongly and make more synapses following neo ST to compensate for the lack of supraspinal projections.

Targeting PTEN with lentiviral shRNA promotes neurite outgrowth of cortical neurons and improves functional recovery in a rat spinal cord contusion model

Objective To observe the effects of PTEN silencing on axon regeneration and functional recovery after spinal cord injury (SCI). Methods Four groups were designed: DMEM、lenti-control、lenti-scramble、lenti-shRNA. Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans (CSPGs). Three days after lentivirus infection, the expression of PTEN were assessed by Western blot, and immunocytochemistry method were applied to examine the length, number and crossing behavior of neurites; Lentivirus was locally injected into the conical motor area of the rats with spinal cord injury. 1 week later, the expression of PTEN was assessed by Western blot. 6 weeks later, motor function of hind limbs was evaluated by Basso Beattie Bresnahan (BBB) score, while the expressions of green fluorescence in injured spinal cord were observed by fluorescence microscope, and immunohistochemical method was used to observe the expression of synaptophysin changes in spinal tissues. Results Less expression of PTEN was detected in lentivirus-shRNA, and neurites with PTEN silencing exhibited significant enhancements in elongation, initiation and crossing ability when they encountered CSPGs in vitro. Better hindlimb functional recovery, less expression of PTEN and more synaptophysin positive response area were detected in rats injected with lentivirus-shRNA compared with other groups. Moreover, green fluorescence was found in the caudal region of injured spinal cord of rats injected with lentivirus-shRNA. Conclusion Lentiviral-mediated PTEN-shRNA can significantly improve the axon regeneration and neurological functional recovery after spinal cord injury. Key words: Spinal cord injuries; RNA interference; Lentivirus; Axons; Regeneration

Exogenous Adult Postmortem Neural Precursors Attenuate Secondary Degeneration and Promote Myelin Sparing and Functional Recovery following Experimental Spinal Cord Injury

Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. Neural stem cells from the subventricular zone of the forebrain have been considered a potential tool for cell replacement therapies. We recently isolated a subclass of neural progenitors from the cadaver of mouse donors. These cells, named postmortem neural precursor cells (PM-NPCs), express both erythropoietin (EPO) and its receptor. Their EPO-dependent differentiation abilities produce a significantly higher percentage of neurons than regular NSCs. The cholinergic yield is also higher. The aim of the present study was to evaluate the potential repair properties of PM-NPCs in a mouse model of traumatic SCI. Labeled PM-NPCs were administered intravenously; then the functional recovery and the fate of transplanted cells were studied. Animals transplanted with PM-NPCs showed a remarkable improved recovery of hindlimb function that was evaluated up to 90 days after lesion. This was accompanied by reduced myelin loss, counteraction of the invasion of the lesion site by the inflammatory cells, and an attenuation of secondary degeneration. PM-NPCs migrate mostly at the injury site, where they survive at a significantly higher extent than classical NSCs. These cells accumulate at the edges of the lesion, where a reach neuropile is formed by MAP2- and β-tubulin III-positive transplanted cells that are also mostly labeled by anti-ChAT antibodies.

Transplantation of human bone marrow stromal cell-derived neuroregenrative cells promotes functional recovery after spinal cord injury in mice.

Transplantation of bone marrow stromal cells (BMSCs) for spinal cord injury (SCI) has been shown to improve functional outcome. BMSCs can be easily obtained from bone marrow aspirate and have fewer problems in the clinical application for human SCI from the ethical and legal points of view. Recently, we produced cells with neural stem and/or progenitor cell property and neural regeneration supporting capacity from human bone marrow stromal cells (human bone marrow stromal cell-derived neuroregenerative cells: hBMSC-NRs). The aim of the present study was to clarify the effectiveness of transplantation of hBMSC-NRs to injured spinal cord of severe combined immunodeficiency (NOD/SCID) mice. Neurite outgrowth assay of PC-12 cells was performed. One week after a T9-level contusion SCI, hBMSCs or hBMSC-NRs were transplanted into the spinal cord. After the transplantation, functional and histological examinations were performed. Conditioned media of hBMSC-NRs significantly promoted the neurite outgrowth of PC-12 cells in vitro. Transplanted hBMSC-NRs survived in the injured spinal cord 8 weeks after SCI. Immunohistochemistry revealed that the density of serotonin-positive fibers of the transplanted group was significantly higher than that of the control group at the epicenter and caudal segment to the injured site. The recovery of hind limb function of the hBMSC-NRs group was significantly better than that of the control group. In conclusion, hBMSC-NRs can be one of the realistic candidates for cell transplantation therapy for human SCI.

Effects of hypothermia combined with neural stem cell transplantation on recovery of neurological function in rats with spinal cord injury.

The microenvironment of the injured spinal cord is hypothesized to be involved in driving the differentiation and survival of engrafted neural stem cells (NSCs). Hypothermia is known to improve the microenvironment of the injured spinal cord in a number of ways. To investigate the effect of NSC transplantation in combination with hypothermia on the recovery of rat spinal cord injury, 60 Sprague-Dawley female rats were used to establish a spinal cord hemisection model. They were divided randomly into three groups: A, spinal cord injury group; B, NSC transplantation group; and C, NSC transplantation + hypothermia group. At 1, 2, 4, 6 and 8 weeks post-injury, the motor function of all animals was evaluated using the Basso, Beattie and Besnaham locomotor scoring system and the inclined plane test. At 4 weeks post-transplantation, histological analysis and immunocytochemistry were performed. At 8 weeks post-transplantation, horseradish peroxidase nerve tracing and transmission electron microscopy were conducted to observe axonal regeneration. The outcome of hind limb motor function recovery in group C significantly surpassed that in group B at 4 weeks post-injury (P<0.05). Recovery was also observed in group A, but to a lesser degree. For the pathological sections no neural axonal were observed in group A. A few axon-like structures were observed in group B and more in group C. Horseradish peroxidase-labeled neurofibers and bromodeoxyuridine-positive cells were observed in the spinal cords of group C. Fewer of these cells were found in group B and fewer still in group A. The differences among the three groups were significant (P<0.05). Using transmission electron microscopy, newly formed nerve fibers and myelinated nerve fibers were observed in the central transverse plane in groups B and C, although these nerve fibers were not evident in group A. In conclusion, NSC transplantation promoted the recovery of hind limb function in rats, and combination treatment with hypothermia produced synergistic effects.

Therapeutic effect of human adipose-derived stromal cells cluster in rat hind-limb ischemia.

We investigated whether transplantation of three-dimensional cell masses (3DCM) of human adipose-derived stromal cells (hASCs) cultured on a basic fibroblast growth factor-immobilized substrate improved hind limb functional recovery by stimulating angiogenesis in an immune-competent rat ischemic limb model. In vitro experiments confirmed that cells within 3DCMs differentiate toward the endothelial lineage one day after culture in normal medium. The therapeutic effect of 3DCMs was evaluated by transplanting hASCs, phosphate-buffered saline alone, and the 3DCM into rat ischemic hind limbs. Blood flow was enhanced in the ischemic hind limb in the 3DCM-injected group compared with the other groups. The ratio of human nuclear antigen (HNA) and hVEGF-positive cells was significantly higher in the 3DCM-injected group compared to hASC-injected group. Human VEGF was observed in most HNA-positive cells. Many hCD31 and hSMA-positive cells were observed in vessel-like structures in the 3DCM-injected group. The 3DCM transplantation improved cell retention and angiogenic effects compared with ASC transplantation. These findings suggest that transplantation of 3DCMs may be an effective stem cell therapy for hind limb ischemia.

Effects of an immunomodulatory therapy and chondroitinase after spinal cord hemisection injury.

Individually, immunomodulatory therapy and chondroitinases have demonstrated neuroprotective and potential neuroregenerative effects following spinal cord injury. To investigate the therapeutic potential of combined immunomodulatory and chondroitin sulfate-glycosaminoglycan degradation therapy in spinal cord injury. A combined immunomodulatory treatment using (1) liposome-encapsulated clodronate (selectively depletes peripheral macrophages), and (2) rolipram (a selective type 4 phosphodiesterase inhibitor), along with the chondroitin sulfate proteoglycan-glycosaminoglycan-degrading enzyme, chondroitinase ABC (ChABC), was assessed for its potential to promote axonal regrowth and improve locomotor recovery following midthoracic spinal cord hemisection injury in adult rats. We demonstrate that combined treatment with liposomal clodronate, rolipram, and ChABC attenuates macrophage accumulation at the site of injury, reduces axonal die-back of injured dorsal column axons, and produces the greatest improvement in locomotor recovery at 6 weeks postinjury compared with controls and noncombined therapy. Anterograde and retrograde tracing revealed that delivery of clodronate, rolipram, and ChABC did not promote substantial axonal regeneration through the site of injury, although the treatment did limit the extent of axonal die-back. Histological assessments revealed that combined treatment with clodronate/rolipram and/or ChABC resulted in a significant reduction in lesion size and cystic cavitation in comparison with injured controls. Combined clodronate, rolipram, and ChABC treatment reduced the accumulation of macrophages within the injured spinal cord 7 weeks after injury. The present data suggest that delivery of an immunomodulatory therapy consisting of clodronate and rolipram, in combination with ChABC, reduces axonal injury and enhances neuroprotection, plasticity, and hindlimb functional recovery after hemisection spinal cord injury in adult rats.

N-acetylcysteine amide preserves mitochondrial bioenergetics and improves functional recovery following spinal trauma

Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteine amide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague–Dawley rats (n=120) received either vehicle or NACA (75, 150, 300 or 600mg/kg) at 15min and 6h post-injury. After 24h the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.

CD36 deletion improves recovery from spinal cord injury

CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36−/− mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36+/+ mice. CD36−/− mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36−/− mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury.

Hyperbaric oxygen intervention on expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in spinal cord injury models in rats

Background Hyperbaric oxygen (HBO) intervention is a main therapeutic method and the curative effect has been certified for spinal cord injury (SCI), but the mechanisms of the neuroprotective effect of HBO on SCI remain elusive. This study aimed to observe the change in expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) after SCI at different time points and to investigate the neuroprotective mechanism of HBO on SCI in rats. Methods A total of 160 adult Sprague-Dawley rats, weighing between 250 and 300 g, were randomly assigned to four experimental groups (n=40 per group). SCI group: SCI was created with a special NYU impactor of Allen's by a 25 gram-centimeter impacting energy on T10 of the spinal cord. SCI+HBO group: HBO therapy after SCI model was established. Sham operation (SH) group: only laminectomy of T10 and no impact on the spinal cord was done. SH+HBO group: HBO therapy after sham operation. The hindlimb functional recovery was evaluated using Basso, Beattie, and Bresnahan (BBB) score and the expressions of HIF-1α and VEGF were observed with fluorescent quantitation PCR and Western blotting method of six rats picked randomly from each group at different time points of 1, 3, 7, and 14 days after operation. Results Rats in the SCI group and SCI+HBO group were paralyzed completely after operation with BBB 0-1 score. Rats in the SH group and SH+HBO group could walk after sham operation with BBB 20-21 score. The BBB score of rats in the SCI+HBO group (4.67±1.97 and 10.83±2.23) was higher than that in the SCI group (1.83±0.75 and 6.67±2.16) at 7 and 14 days time points obviously (P &lt;0.05). The expressions of HIF-1α and VEGF in the SCI group and SCI+HBO group were higher than in the SH group and SH+HBO group at any time point obviously (P &lt;0.05), while the SCI+HBO group presented the least expression of HIF-1α mRNA and protein (3.82±0.41 and 0.59±0.06; 2.26±0.41 and 0.37±0.05; 1.58±0.26 and 0.29±0.05) than that in the SCI group (6.36±0.58 and 0.76±0.07; 3.55±0.47 and 0.51±0.07; 2.27±0.39 and 0.40±0.06) respectively at 3, 7, and 14 days time points (P &lt;0.05) with significant difference and more expression of VEGF mRNA and protein (5.83±0.77 and 0.72±0.06; 4.59±0.51 and 0.63±0.06) than that in the SCI group (3.06±0.30 and 0.48±0.07; 2.25±0.24 and 0.39±0.09) respectively at 7 and 14 days time points (P &lt;0.05) with significant difference. Conclusions HBO could improve the hind limb functional recovery after SCI in rats. The elevation and duration of the expression of VEGF and the reduction of expression of HIF-1α by HBO intervention may be inversely related in the repair of damaged spinal cord and neuroprotective effect.

Cotransplantation of Olfactory Ensheathing Cells and Schwann Cells Combined with Treadmill Training Promotes Functional Recovery in Rats with Contused Spinal Cords

The present study investigated the ability of cotransplantation of Schwann cells (SCs) and olfactory ensheathing cells (OECs) combined with treadmill training in facilitating neuronal plasticity and promoting hindlimb function recovery of subacute moderate thoracic (T10) spinal cord contusion in rats. Two weeks postinjury, SCs were injected directly into the lesion, while OECs were injected into the adjacent tissues. The treadmill training with the rats began postinjury on day 7, with each session lasting 20 ± 10 min per day, 5 days per week, for 10 weeks. At the 11th week postinjury, OECs were found migrating longitudinally and laterally from the injection site to the injury site through the gray and white matter, while some traveled along the central canal or pia. The SCs remained densely packed and concentrated at the transplant site. The transplanted SCs supported ingrowth of numerous, densely populated neurofilament-positive (NF(+)), MBP(+) axons. The OECs promoted elongation of moderate NF(+), GAP-43(+) axons and a few MBP(+) axons in parallel with OEC processes. The GFAP immunoreactivity in the spared tissue surrounding the graft of SCs and OECs at the lesion site was less intense than that in the DMEM group. Treadmill training had no effect on GFAP immunoreactivity. Treadmill training increased the number of TH-immunoreactive neurons in the gray matter of L2 spinal cord. Moreover, cotransplantation of OECs and SCs significantly increased the BBB score during 5-8 weeks postinjury alongside treadmill training between 5 and 11 weeks. Cotransplantation of OECs and SCs combined with treadmill training resulted in the highest BBB score at 4 and 11 weeks. The study details the differential mechanisms of neuronal plasticity: (1) axon growth and remyelination induced by cotransplantation of OECs and SCs and (2) neuron plasticity below the lesion enhanced by treadmill training. The synergistic effects of the combined strategy enhance functional recovery. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Combined use of an epidural cooling catheter and systemic moderate hypothermia enhances spinal cord protection against ischemic injury in rabbits

Epidural placement of a cooling catheter can protect against ischemic spinal cord injury. With the use of rabbits, we investigated whether this epidural cooling technique, when combined with systemic moderate hypothermia, can protect the spinal cord against ischemic metabolic stress. New Zealand white rabbits (n = 28) were assigned to 1 of 4 different groups. Animals underwent abdominal aortic occlusion for 30 minutes using a 3F balloon catheter. Group 1 (n = 7) underwent epidural cooling by the catheter and systemic moderate hypothermia (35 °C) induced with a cooling blanket. Group 2 (n = 7) underwent epidural cooling under systemic normothermia (38.5 °C). Group 3 (n = 7) underwent systemic moderate hypothermia (35 °C) without epidural cooling. Group 4 (n = 7) underwent neither epidural nor blanket cooling as a negative control. Neurologic status of their hind limbs was graded according to the modified Tarlov scale at 1, 2, and 7 days after surgery. During infrarenal aortic ischemia, epidural temperature was significantly lower in group 1 (18.5 °C ± 0.8 °C) than in group 2 (28.6 °C ± 1.0 °C; P = .0001), group 3 (34.2 °C ± 0.06 °C; P = .0001), or group 4 (38.5 °C ± 0.2 °C; P = .0001). Hind limb function recovery was greater in group 1 (mean Tarlov score, 4.9 ± 0.057) than in group 2 (2.6 ± 0.3; P = .0028), group 3 (2.1 ± 0.34; P = .0088), or group 4 (0.0 ± 0.0; P = .0003). Epidural cooling catheter combined with systemic moderate hypothermia produced additive cooling ability and protected the spinal cord against ischemia in rabbits more effectively than either intervention alone.

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

BackgroundGranulocyte 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.MethodsAdult 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.ResultsUsing 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.ConclusionsG-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.

Tetramethylpyrazine accelerates the function recovery of traumatic spinal cord in rat model by attenuating inflammation

In the present study, we explored the effects of tetramethylpyrazine (TMP), an alkaloid extracted from the Chinese herbal medicine Ligusticum wallichii Franchat (chuanxiong), on a rat model of contusion spinal cord injury (SCI). The contusion SCI model was induced in rats by a modified Allen's weight-drop method with a severity of 5g×50mm impacting on the T10 segment. In the TMP treatment group, rats were injected intraperitoneally (i.p.) with TMP (200mg/kg), every 24h for 5days, starting half an hour after contusion SCI. The control group was treated with saline. Compared with the control group, the TMP group significantly ameliorated the recovery of hindlimb function of rats. TMP treatment significantly reduced the expression of macrophage migration inhibitory factor, nuclear factor κappa B, pro-inflammatory cytokine interleukin-18 and neutrophil infiltration. On the other hand, TMP enhanced the expression of inhibitor κappa B and anti-inflammation cytokine interleukin-10. In conclusion, our results demonstrate that TMP inhibits the development of inflammation and tissue injury associated with spinal cord contusion in rats which may improve the rats' hindlimb function.