The role of Rho/Rho-kinase pathway and the neuroprotective effects of fasudil in chronic cerebral ischemia

The Rho/Rho-kinase signaling pathway plays an important role in cerebral ischemia/reperfusion injury. However, very few studies have examined in detail the changes in the Rho/Rho-kinase signaling pathway in chronic cerebral ischemia. In this study, rat models of chronic cerebral ischemia were established by permanent bilateral common carotid artery occlusion and intragastrically administered 9 mg/kg fasudil, a powerful ROCK inhibitor, for 9 weeks. Morris water maze results showed that cognitive impairment progressively worsened as the cerebral ischemia proceeded. Immunohistochemistry, semi-quantitative RT-PCR and western blot analysis showed that the expression levels of Rho-kinase, its substrate myosin-binding subunit, and its related protein alpha smooth muscle actin, significantly increased after chronic cerebral ischemia. TUNEL staining showed that chronic cerebral ischemia could lead to an increase in neuronal apoptosis, as well as the expression level of caspase-3 in the frontal cortex of rats subjected to chronic cerebral ischemia. Fasudil treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, and decreased the expression level of Rho-kinase, myosin-binding subunit and alpha smooth muscle actin. Furthermore, fasudil could regulate cerebral injury by reducing cell apoptosis and decreasing caspase-3 expression in the frontal cortex. These findings demonstrate that fasudil can protect against cognitive impairment induced by chronic cerebral ischemia via the Rho/Rho-kinase signaling pathway and anti-apoptosis mechanism.     

Cyclooxygenase inhibition in nerve-injury- and TNF-induced hyperalgesia in the rat

After nerve injury, cyclooxygenase-2 (COX-2) is upregulated in spinal cord and peripheral nerve, the latter being dependent on tumor necrosis factor-alpha (TNF). Here we asked whether COX inhibitors attenuate pain behavior induced by chronic constrictive sciatic nerve injury (CCI) or intraneural injection of TNF (2.5 pg/ml). Rats received either 0.9% saline, the nonselective COX inhibitor ibuprofen (40 mg/kg) or the selective COX-2 inhibitor celecoxib (10 or 30 mg/kg) twice daily by gavage started 2 days before, 12 h or 7 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI was moderately, but consistently attenuated by early (day -2 or 12 h after CCI), but not late (7 days after CCI) ibuprofen and celecoxib treatment. Mechanical allodynia, but not thermal hyperalgesia induced by intraneural TNF, was reduced by ibuprofen, but not by celecoxib treatment 5 and 7 days after injection. Sciatic nerves, lumbar dorsal root ganglia (DRG) and spinal cords from rats with treatment started 12 h after surgery were analyzed for prostaglandin E2 (PGE2) levels 10 days after CCI. In injured nerves and ipsilateral DRG, PGE2 levels were increased. Ibuprofen treatment reversed PGE2 levels in injured nerves and DRG, whereas celecoxib blocked increased PGE2 levels only in nerves. In spinal cord, no change in PGE2 levels was observed. In contrast to the marked inhibition of nerve-injury-induced upregulation of PGE2 by COX inhibitors, the effect on pain behavior was modest. Nerve-injury- and TNF-induced pain-related behavior seem to be only partly dependent on peripheral prostaglandins.     

Upregulated Ras/Raf/ERK1/2 signaling pathway: a new hope in the repair of spinal cord injury

An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T₉. Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.     

Hyperbaric oxygen therapy combined with Schwann cell transplantation promotes spinal cord injury recovery

Schwann cell transplantation and hyperbaric oxygen therapy each promote recovery from spinal cord injury, but it remains unclear whether their combination improves therapeutic results more than monotherapy. To investigate this, we used Schwann cell transplantation via the tail vein, hyperbaric oxygen therapy, or their combination, in rat models of spinal cord contusion injury. The combined treatment was more effective in improving hindlimb motor function than either treatment alone; injured spinal tissue showed a greater number of neurite-like structures in the injured spinal tissue, somatosensory and motor evoked potential latencies were notably shorter, and their amplitudes greater, after combination therapy than after monotherapy. These findings indicate that Schwann cell transplantation combined with hyperbaric oxygen therapy is more effective than either treatment alone in promoting the recovery of spinal cord in rats after injury.     

Tamoxifen and Src kinase inhibitors as neuroprotective/neuroregenerative drugs after spinal cord injury

Spinal cord injury(SCI) is a devastating condition that produces significant changes in the lifestyle of patients. Many molecular and cellular events are triggered after the initial physical impact to the cord. Two major phases have been described in the field of SCI: an acute phase and late phase. Most of the therapeutic strategies are focused on the late phase because this provides an opportunity to target cellular events like apoptosis, demyelination, scar formation and axonal outgrowth. In this mini-review, we will focus on two agents(tamoxifen and a Src kinase family inhibitor known as PP2) that have been shown in our laboratory to produce neuroprotective(increase cell survival) and/or regenerative(axonal outgrowth) actions. The animal model used in our laboratory is adult female rat(~250 g) with a moderate contusion(12.5 mm) to the spinal cord at the T10 level, using the MASCIS impactor device. Tamoxifen or PP2 was administered by implantation of a 15 mg pellet(Innovative Research of America, Sarasota, FL, USA) or by intraperitoneal injections(1.5 mg/kg, every 3 days), respectively, to produce a long-term effect(28 days). Tamoxifen and the Src kinase inhibitor, PP2, are drugs that in rats with a moderate spinal cord injury promote functional locomotor recovery, increase spared white matter tissue, and stimulate axonal outgrowth. Moreover, tamoxifen reduces the formation of reactive oxygen species. Therefore, these drugs are possible therapeutic agents that have a neuroprotective/regenerative activity in vertebrates with SCI.     

Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui(GV14) and Mingmen(GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.     

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guidance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord injury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, caspase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental findings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.     

The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway

Rho-associated kinase(ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system.Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase(ERK) signaling pathway,but its effect on microglial migration was unknown.Therefore,in this study,we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord,and we examined the underlying mechanisms.The microglia were treated with Y27632,fasudil and/or the ERK inhibitor U0126.Cellular morphology was observed by immunofluorescence.Transwell chambers were used to assess cell migration.ERK levels were measured by incell western blot assay.Y27632 and fasudil increased microglial migration,and the microglia were irregularly shaped and had many small processes.These inhibitors also upregulated the levels of phosphorylated ERK protein.The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil.These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.     

Exercise promotes motor functional recovery in rats with corticospinal tract injury:anti-apoptosis mechanism

Studies have shown that exercise interventions can improve functional recovery after spinal cord injury, but the mechanism of action remains unclear. To investigate the mechanism, we established a unilateral corticospinal tract injury model in rats by pyramidotomy, and used a single pellet reaching task and horizontal ladder walking task as exercise interventions postoperatively. Functional recovery of forelimbs and forepaws in the rat models was noticeably enhanced after the exercises. Furthermore, TUNEL staining revealed significantly fewer apoptotic cells in the spinal cord of exercised rats, and western blot analysis showed that spinal cord expression of the apoptosis-related protein caspase-3 was significantly lower, and the expression of Bcl-2 was significantly higher, while the expression of Bax was not signifiantly changed after exercise, compared with the non-exercised group. Expression of these proteins decreased with time after injury, towards the levels observed in sham-operated rats, however at 4 weeks postoperatively, caspase-3 expression remained significantly greater than in sham-operated rats. The present findings indicate that a reduction in apoptosis is one of the mechanisms underlying the improvement of functional recovery by exercise interventions after corticospinal tract injury.     

Neuroprotective effects of electroacupuncture on early- and late-stage spinal cord injury

Previous studies have shown that the neurite growth inhibitor Nogo-A can cause secondary neural damage by activating Rho A. In the present study, we hypothesized that electroacupuncture promotes neurological functional recovery after spinal cord injury by inhibiting Rho A expression. We established a rat model of acute spinal cord injury using a modification of Allen's method. The rats were given electroacupuncture treatment at Dazhui(Du14), Mingmen(Du4), Sanyinjiao(SP6), Huantiao(GB30), Zusanli(ST36) and Kunlun(BL60) acupoints with a sparsedense wave at a frequency of 4 Hz for 30 minutes, once a day, for a total of 7 days. Seven days after injury, the Basso, Beattie and Bresnahan(BBB) locomotor scale and inclined plane test scores were significantly increased, the number of apoptotic cells in the spinal cord tissue was significantly reduced, and Rho A and Nogo-A m RNA and protein expression levels were decreased in rats given electroacupuncture compared with rats not given electroacupuncture. Four weeks after injury, pathological tissue damage in the spinal cord at the site of injury was alleviated, the numbers of glial fibrillary acidic protein- and neurofilament 200-positive fibers were increased, the latencies of somatosensory-evoked and motor-evoked potentials were shortened, and their amplitudes were increased in rats given electroacupuncture. These findings suggest that electroacupuncture treatment reduces neuronal apoptosis and decreases Rho A and Nogo-A m RNA and protein expression at the site of spinal cord injury, thereby promoting tissue repair and neurological functional recovery.     

Nogo-A expression dynamically varies after spinal cord injury

The mechanism involved in neural regeneration after spinal cord injury is unclear. The myelin-derived protein Nogo-A, which is specific to the central nervous system, has been identified to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth. In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mR NA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immunohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mR NA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mR NA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic variation of Nogo-A should be taken into account in the treatment of spinal cord injury.     

Feasibility of 3.0 T diffusion-weighted nuclear magnetic resonance imaging in the evaluation of functional recovery of rats with complete spinal cord injury

Diffusion tensor imaging is a sensitive way to reflect axonal necrosis and degeneration, glial cell regeneration and demyelination following spinal cord injury, and to display microstructure changes in the spinal cord in vivo. Diffusion tensor imaging technology is a sensitive method to diagnose spinal cord injury; fiber tractography visualizes the white matter fibers, and directly displays the structural integrity and resultant damage of the fiber bundle. At present, diffusion tensor imaging is restricted to brain examinations, and is rarely applied in the evaluation of spinal cord injury. This study aimed to explore the fractional anisotropy and apparent diffusion coefficient of diffusion tensor magnetic resonance imaging and the feasibility of diffusion tensor tractography in the evaluation of complete spinal cord injury in rats. The results showed that the average combined scores were obviously decreased after spinal cord transection in rats, and then began to increase over time. The fractional anisotropy scores after spinal cord transection in rats were significantly lower than those in normal rats(P 0.05); the apparent diffusion coefficient was significantly increased compared with the normal group(P 0.05). Following spinal cord transection, fractional anisotropy scores were negatively correlated with apparent diffusion coefficient values(r = –0.856, P 0.01), and positively correlated with the average combined scores(r = 0.943, P 0.01), while apparent diffusion coefficient values had a negative correlation with the average combined scores(r = –0.949, P 0.01). Experimental findings suggest that, as a non-invasive examination, diffusion tensor magnetic resonance imaging can provide qualitative and quantitative information about spinal cord injury. The fractional anisotropy score and apparent diffusion coefficient have a good correlation with the average combined scores, which reflect functional recovery after spinal cord injury.     

Propofol injection combined with bone marrow mesenchymal stem cell transplantation better improves electrophysiological function in the hindlimb of rats with spinal cord injury than monotherapy

The repair effects of bone marrow mesenchymal stem cell transplantation on nervous system damage are not satisfactory. Propofol has been shown to protect against spinal cord injury. Therefore, this study sought to explore the therapeutic effects of their combination on spinal cord injury. Rat models of spinal cord injury were established using the weight drop method. Rats were subjected to bone marrow mesenchymal stem cell transplantation via tail vein injection and/or propofol injection via tail vein using an infusion pump. Four weeks after cell transplantation and/or propofol treatment, the cavity within the spinal cord was reduced. The numbers of PKH-26-positive cells and horseradish peroxidase-positive nerve fibers apparently increased in the spinal cord. Latencies of somatosensory evoked potentials and motor evoked potentials in the hindlimb were noticeably shortened, amplitude was increased and hindlimb motor function was obviously improved. Moreover, the combined effects were better than cell transplantation or propofol injection alone. The above data suggest that the combination of propofol injection and bone marrow mesenchymal stem cell transplantation can effectively improve hindlimb electrophysiological function, promote the recovery of motor funtion, and play a neuroprotective role in spinal cord injury in rats.     

Propofol promotes spinal cord injury repair by bone marrow mesenchymal stem cell transplantation

Propofol is a neuroprotective anesthetic. Whether propofol can promote spinal cord injury repair by bone marrow mesenchymal stem cells remains poorly understood. We used rats to investigate spinal cord injury repair using bone marrow mesenchymal stem cell transplantation combined with propofol administration via the tail vein. Rat spinal cord injury was clearly alleviated; a large number of newborn non-myelinated and myelinated nerve fibers appeared in the spinal cord, the numbers of CM-Dil-labeled bone marrow mesenchymal stem cells and fluorogold-labeled nerve fibers were increased and hindlimb motor function of spinal cord-injured rats was markedly improved. These improvements were more prominent in rats subjected to bone marrow mesenchymal cell transplantation combined with propofol administration than in rats receiving monotherapy. These results indicate that propofol can enhance the therapeutic effects of bone marrow mesenchymal stem cell transplantation on spinal cord injury in rats.     

The effects of fasudil on the permeability of the rat blood-brain barrier and blood-spinal cord barrier following experimental autoimmune encephalomyelitis

Dysfunction of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) is a primary characteristic of multiple sclerosis (MS). We evaluated the protective effects of fasudil, a selective ROCK inhibitor, in a model of experimental autoimmune encephalomyelitis (EAE) that was induced by guinea-pig spinal cord. In addition, we studied the effects of fasudil on BBB and BSCB permeability. We found that fasudil partly alleviated EAE-dependent damage by decreasing BBB and BSCB permeability. These results provide rationale for the development of selective inhibitors of Rho kinase as a novel therapy for MS.     

Leuprolide acetate induces structural and functional recovery of injured spinal cord in rats

Gonadotropin-releasing hormone (GnRH) and its synthetic analog leuprolide acetate, a GnRH agonist, have neurotrophic properties. This study was designed to determine whether administration of leuprolide acetate can improve locomotor behavior, gait, micturition reflex, spinal cord morphology and the amount of microglia in the lesion epicenter after spinal cord injury in rats. Rats with spinal cord compression injury were administered leuprolide acetate or saline solution for 5 weeks. At the 5^th week, leuprolide acetate-treated rats showed locomotor activity recovery by 38%, had improvement in kinematic gait and exhibited voiding reflex recovery by 60%, as compared with the 1^st week. By contrast, saline solution-treated rats showed locomotor activity recovery only by 7%, but voiding reflex did not recover. More importantly, leuprolide acetate treatment reduced microglial immunological reaction and induced a trend towards greater area of white and gray matter in the spinal cord. Therefore, leuprolide acetate has great potential to repair spinal cord injury.     

visual bone marrow mesenchymal stem cell transplantation in the repair of spinal cord injury

An important factor in improving functional recovery from spinal cord injury using stem cells is maximizing the number of transplanted cells at the lesion site. Here, we established a contusion model of spinal cord injury by dropping a weight onto the spinal cord at T7–8. Superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells were transplanted into the injured spinal cord via the subarachnoid space. An outer magnetic field was used to successfully guide the labeled cells to the lesion site. Prussian blue staining showed that more bone marrow mesenchymal stem cells reached the lesion site in these rats than in those without magnetic guidance or superparamagnetic iron oxide labeling, and immunofluorescence revealed a greater number of complete axons at the lesion site. Moreover, the Basso, Beattie and Bresnahan(BBB) locomotor rating scale scores were the highest in rats with superparamagnetic labeling and magnetic guidance. Our data confirm that superparamagnetic iron oxide nanoparticles effectively label bone marrow mesenchymal stem cells and impart sufficient magnetism to respond to the external magnetic field guides. More importantly, superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells can be dynamically and non-invasively tracked in vivo using magnetic resonance imaging. Superparamagnetic iron oxide labeling of bone marrow mesenchymal stem cells coupled with magnetic guidance offers a promising avenue for the clinical treatment of spinal cord injury.     

Femoral nerve regeneration and its accuracy under different injury mechanisms

Surgical accuracy has greatly improved with the advent of microsurgical techniques. However, complete functional recovery after peripheral nerve injury has not been achieved to date. The mechanisms hindering accurate regeneration of damaged axons after peripheral nerve injury are in urgent need of exploration. The present study was designed to explore the mechanisms of peripheral nerve regeneration after different types of injury. Femoral nerves of rats were injured by crushing or freezing. At 2, 3, 6, and 12 weeks after injury, axons were retrogradely labeled using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(Dil) and True Blue, and motor and sensory axons that had regenerated at the site of injury were counted. The number and percentage of Dil-labeled neurons in the anterior horn of the spinal cord increased over time. No significant differences were found in the number of labeled neurons between the freeze and crush injury groups at any time point. Our results confirmed that the accuracy of peripheral nerve regeneration increased with time, after both crush and freeze injury, and indicated that axonal regeneration accuracy was still satisfactory after freezing, despite the prolonged damage.     

Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pc DNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the subarachnoid cavity to help repair spinal cord injury and promote the recovery of spinal cord function better than neural stem cell transplantation alone. These findings may lead to significant improvements in the clinical treatment of spinal cord injuries.