An integrin inhibiting molecule decreases oxidative damage and improves neurological function after spinal cord injury

Our previous studies have shown that treatment with an α4β1 integrin blocking antibody after spinal cord injury (SCI) in rats decreases intraspinal inflammation and oxidative damage, improving neurological function. Here, we studied effects of a high affinity small molecule α4β1 inhibitor, BIO5192. First, rats were treated intravenously with BIO5192 (10 mg/kg) or with vehicle (controls) to assess effects of integrin blockade for 24 h or 72 h after thoracic clip-compression SCI. BIO5192 treatment significantly decreased the MPO enzymatic activity (neutrophil infiltration) and ED-1 expression (macrophage density) by 40% and 38% at 24 h and by 52% and 25% at 72 h post injury, respectively. In cord homogenates, BIO5192 treatment decreased expression of the oxidative enzymes gp91^phox, inducible nitric oxide and cyclooxygenase-2 by  40% at both times of analysis. Free radical concentration decreased by 30% and lipid peroxidation decreased by 34% and 46%, respectively, at 24 h and 72 h after SCI. Next, after blockade by BIO5192 for 72 h, neurological outcomes were analyzed for 1–6 weeks after SCI. Motor function significantly improved when assessed by an open-field test. Treated rats planter placed their hind paws and/or dorsal stepped, with weight support, whereas controls only swept their hindlimbs. BIO5192 treatment also decreased mechanical allodynia elicited from the trunk and hind paw by up to 35%. This improved function correlated with decreased lesion size and spared myelin-containing white matter. The neurological improvement offered by this neuroprotective strategy supports the potential for an anti-integrin treatment for SCI.     

α4β1 integrin blockade after spinal cord injury decreases damage and improves neurological function

The extent of disability caused by spinal cord injury (SCI) relates to secondary tissue destruction arising partly from an intraspinal influx of neutrophils and monocyte/macrophages after the initial injury. The integrin α4β1, expressed by these leukocytes, is a key to their activation and migration into/within tissue. Therefore, blocking this integrin's functions may afford significant neuroprotection. Rats were treated intravenously with a blocking monoclonal antibody (mAb) to the α4 subunit of α4β1 at 2 and 24 h after thoracic clip-compression SCI. Anti-α4β1 treatment significantly decreased neutrophil and monocyte/macrophage influx at 3 d by 47% and 53%, respectively, and decreased neutrophil influx by 61% at 7 d after SCI. Anti-α4β1 treatment also significantly reduced oxidative activity in injured cord homogenates at 3 d. For example, myeloperoxidase activity decreased by 38%, inducible nitric oxide by 44%, dichlorofluorescein (marking free radicals) by 33% and lipid peroxidation (malondialdehyde) by 42%. At 2–8 weeks after SCI, motor function improved by up to 2 points on an open-field locomotor scale. Treated rats supported weight with their hind paws instead of sweeping. At 2–4 weeks after SCI, anti-α4β1 treatment decreased blood pressure responses during autonomic dysreflexia by as much as 43% and, at 2–8 weeks, decreased mechanical allodynia elicited from the trunk and hind paw by up to 54% and 40%, respectively. This improved functional recovery correlated with spared myelin-containing white matter and > 10-fold more bulbospinal serotonergic axons below the injury than were in controls. The significant neurological improvement offered by this neuroprotective strategy underscores the potential for an anti-integrin treatment for SCI.     

Ephedra sinica inhibits complement activation and improves the motor functions after spinal cord injury in rats

The activation of complement system has been known as an important and significant reaction against the secondary injury after spinal cord injury (SCI). In the present study, we investigated the effect of Ephedra sinica to the inflammation or complement system of injured spinal cord and the influence to the functional recovery after spinal cord injury in rats. We prepared the complement-inhibiting component from E. sinica. Contusive spinal cord injury was induced to Sprague-Dawley rats. We administrated the product from E. sinica to E. sinica group, while distilled water was administered to the control group by gavage after SCI. Complement hemolytic activity (CH50), expression of C3 and C9, myeloperoxidase activity, and motor function were evaluated in E. sinica group and control group. The CH50, complement depositions, and myeloperoxidase activity in the E. sinica group were significantly reduced as compared to the control group. The motor function of E. sinica group was significantly improved from the 7th day as compared with the control group. The results demonstrated that E. sinica might reduce inflammation and improve motor function in rats after spinal cord injury by inhibiting complement activation. The present study has shown that complement system is playing an important role in spinal cord injury, and the possibility of a new therapy strategy, inhibiting or controlling the complement activation and inflammation, for spinal cord injury.     

Expression and regulatory network of long noncoding RNA in rats after spinal cord hemisection injury

Long noncoding RNAs (lncRNAs) participate in a variety of biological processes and diseases. However, the expression and function of lncRNAs after spinal cord i...     

FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury

After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.     

Recombinant human erythropoietin decreases myeloperoxidase and caspase-3 activity and improves early functional results after spinal cord injury in rats.

Inflammatory response and apoptosis have been proposed as mechanisms of secondary injury of the spinal cord after primary insult. Recent studies have shown that erythropoietin (EPO) has neuroprotective properties. In this study, we assessed the efficacy of recombinant human erythropoietin (r-Hu-EPO) in the treatment of acute spinal cord injury (SCI) in rats. Rats were divided into five groups of eight rats each. Controls (Group 1) received laminectomy only. The trauma-only group (Group 2) underwent 40 g/cm contusion injury and had no medication. In group 3, 30 mg/kg of methylprednisolone (MPSS) was administered. Group 4 received 1000 IU/kg body weight of r-Hu-EPO. The vehicle group (Group 5) received a vehicle solution containing human serum albumin, which is the solvent for r-Hu-EPO. Twenty-four hours after trauma, animals were functionally evaluated and a spinal cord samples were obtained for the assessment of caspase-3 and myeloperoxidase (MPO) activities. The results showed that MPO and caspase-3 activities increased to statistically significant higher levels in the spinal cord after contusion injury comparing to the control group. MPO and caspase-3 enzyme activity levels were significantly reduced in animals treated either with r-Hu-EPO or MPSS. In addition, we observed significant early functional recovery in EPO-treated rats. EPO has anti-apoptotic and anti-inflammatory effects, and improves early clinical results after SCI.     

急性CO中毒迟发脑病大鼠海马中CDK5、p35及p25的表达

目的检测细胞周期素依赖性蛋白激酶5(CDK5)、p35及p25在急性一氧化碳(CO)中毒迟发脑病大鼠(DEACMP)海马组织中的表达,初步探讨它们的可能作用。方法 220～280g雄性SD大鼠60只,随机分为CO组和空气组,CO组给予腹腔追加注射纯品CO气体并动态监测动脉血碳氧血红蛋白(HbCO),空气组同法给予等量空气注射。分别于1周后、3周后采用Morris水迷宫实验筛选DEACMP大鼠模型。应用实时荧光定量聚合酶链反应(RT-qPCR)、反转录聚合酶链反应(RT-PCR)方法检测DEACMP及空气组大鼠海马CDK5、p35及p25的相对表达水平。结果 CO组大鼠急性期动脉血HbCO为(55.07±3.98)%,空气组为(0.64±0.19)%;DEACMP大鼠海马CDK5、p35及p25的表达均高于空气组(P<0.01)。结论 CDK5、p35及p25可能在DEACMP的发病中起重要作用。     

Effect of methylprednisolone on motor function and spinal cord blood flow after spinal cord compression in rats

The effect of methylprednisolone (MP) on neurologic recovery and spinal cord blood flow (SCBF) was investigated up to 4 days after a spinal cord compression injury in rats. The injury was produced at midthoracic level by applying a load of 35 g on a 2.2 x 5.0 mm compression plate for 5 min, which resulted in transient paraparesis. MP was given as a bolus dose of 30 mg/kg i.v. 60 min after injury (n = 20) and controls were given saline (n = 10). The motor performance was assessed daily as the capacity angle on the inclined plane and SCBF was measured by 14C-iodoantipyrine autoradiography on Days 1 or 4. On Day 1 the capacity angle was reduced from about 63 degrees preoperatively to 33 +/- 2 degrees (mean +/- SEM) in the control group and to 50 +/- 1 degrees in the group treated with MP (p less than 0.05). Thereafter there was a slight improvement in both groups, but the difference persisted throughout the observation period. On Day 4 both gray and white matter SCBF was better preserved in MP-treated animals than in the control group (59 +/- 4 versus 49 +/- 3 ml/min/100 g tissue for gray matter and 13.6 +/- 0.6 versus 10.7 +/- 0.8 ml/min/100 g tissue for white matter). Posttraumatic treatment with MP, thus, improved both the neurologic recovery during the first 4 days and SCBF as measured on Day 4. It is speculated that the effect of MP is at least partly exerted on the vascular bed.     

Systemic hypothermia improves histological and functional outcome after cervical spinal cord contusion in rats

Hypothermia has been employed during the past 30 years as a therapeutic modality for spinal cord injury (SCI) in animal models and in humans. With our newly developed rat cervical model of contusive SCI, we investigated the therapeutic efficacy of transient systemic hypothermia (beginning 5 minutes post‐injury for 4 hours, 33°C) with gradual rewarming (1°C per hour) for the preservation of tissue and the prevention of injury‐induced functional loss. A moderate cervical displacement SCI was performed in female Fischer rats, and behavior was assessed for 8 weeks. Histologically, the application of hypothermia after SCI resulted in significant increases in normal‐appearing white matter (31% increase) and gray matter (38% increase) volumes, greater preservation (four‐fold) of neurons immediately rostral and caudal to the injury epicenter, and enhanced sparing of axonal connections from retrogradely traced reticulospinal neurons (127% increase) compared with normothermic controls. Functionally, a faster rate of recovery in open field locomotor ability (BBB score, weeks 1–3) and improved forelimb strength, as measured by both weight‐supported hanging (43% increase) and grip strength (25% increase), were obtained after hypothermia. The current study demonstrates that mild systemic hypothermia is effective for retarding tissue damage and reducing neurological deficits following a clinically relevant contusive cervical SCI. J. Comp. Neurol. 514:433–448, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of inflammatory cytokines after early decompression may mediate recovery of neurological function in rats with spinal cord injury

A variety of inflammatory cytokines are involved in spinal cord injury and influence the recovery of neuronal function. In the present study, we established a rat model of acute spinal cord injury by cerclage. The cerclage suture was released 8 or 72 hours later, to simulate decompression surgery. Neurological function was evaluated behaviorally for 3 weeks after surgery, and tumor necrosis factor α immunoreactivity and apoptosis were quantified in the region of injury. Rats that underwent decompression surgery had significantly weaker immunoreactivity of tumor necrosis factor α and significantly fewer apoptotic cells, and showed faster improvement of locomotor function than animals in which decompression surgery was not performed. Decompression at 8 hours resulted in significantly faster recovery than that at 72 hours. These data indicate that early decompression may improve neurological function after spinal cord injury by inhibiting the expression of tumor necrosis factor α.     

5-HT precursor loading, but not 5-HT receptor agonists, increases motor function after spinal cord contusion in adult rats

Serotonergic (5-HT) receptors are upregulated following spinal cord transection. Stimulation by administration of serotonergic receptor agonists has been successful in improving hindlimb function. We tested whether this strategy would be successful in incomplete injury models (moderate or severe thoracic contusion) where descending projections are partially spared which should produce less denervation-induced receptor upregulation. Adult rats received midthoracic moderate (MOD: 25 mm drop) or severe (SEV: 50 mm drop) contusion injuries. Distribution of 5-HT and its transporter and expression of 5-HT_2C receptors were evaluated in lumbar spinal cord and motor response to 5-HT receptor activation was assessed using open field locomotion (BBB) score, percent weight supported treadmill stepping (%WS) and evaluation of hindlimb muscle activation (tremor and serotonin syndrome).5-HT immunostaining 3 months post-contusion revealed few 5-HT fibers caudal to the severe contusion, and more spared caudal to the moderate contusion. The distribution of 5-HT transporter paralleled 5-HT staining, but was more greatly reduced. Thus serotonin reuptake may be less efficient in the injured spinal cord. Immunostaining for the 5-HT_2C receptor in the dorsal and ventral horns at L5 showed significant upregulation in SEV, compared to sham or MOD rats.Neither 5-HT_2C nor 5-HT_1A receptor agonists, alone or in combination, nor the serotonin transporter inhibitor d-fenfluramine modified BBB scores or %WS in either group. Despite the increased sensitivity of post-synaptic targets, agonist treatment did not improve function in SEV rats. We conclude that selective 5-HT_2C or 5-HT_1A receptor activation was not effective in improving hindlimb function after incomplete lesions. In contrast, the 5-HT precursor 5-hydroxytryptophan (l-5-HTP), which leads to activation of all classes of 5-HT receptors, increased both %WS and hindlimb activity in the MOD group. While no side effects were observed in normal or MOD rats, SEV rats displayed hindlimb tremors and 33% mortality, indicating hypersensitivity to the precursor.     

Retraction Note to: Triptolide (TPL) improves locomotor function recovery in rats and reduces inflammation after spinal cord injury

Neurological Sciences -     

The relationships among the severity of spinal cord injury, residual neurological function, axon counts, and counts of retrogradely labeled neurons after experimental spinal cord injury

Substantial residual neurological function may persist after spinal cord injury (SCI) with survival of as few as 5–10% of the original number of axons. A detailed understanding of the relationships among the severity of injury, the number and origin of surviving axons at the injury site, and the extent of neurological recovery after SCI is of importance in understanding the pathophysiology of SCI and in designing treatment strategies. In the present study, these relationships were examined in rats with graded severity of clip compression injury of the cord at T1. The rats were randomly assigned to one of the following injury groups (n = 5 each): normal (laminectomy only), 2-, 18-, 30-, 50-, and 98-g clip injuries. Neurological function was assessed by the inclined plane method and by the modified Tarlov technique. A morphometric assessment of axons at the injury site was performed by a computer-assisted line sampling technique. The origin of descending lions at the injury site was determined by retrograde labeling with horseradish peroxidase. The inclined plane scores varied as a negative linear function of the closing force of the clip used to inflict SCI(r = −0.93; P < 0.0001). The mean axon count was 367,000 ± 529,000 in normal rats and decreased as a negative exponential function of injury force (r = −0.92; P < 0.0001). As well, SCI caused preferential destruction of large axons as reflected by the change in mean axon diameter from 1.74 ± 0.06 μm in normal cords to 1.46 ± 0.04 μm in injured cords (pooled mean for all injuries). Inclined plane scores varied logarithmically with the number of axons at the injury site (r = 0.90; P < 0.001) and were highly correlated with the integrity of nonpyramidal tracts, in particular the rubrospinal (R = 0.89), Vestibulospinal (r = 0.87), and raphespinal (R = 0.84) tracts. These data demonstrate a precise relationship between the number of surviving axons at the injury site and residual neurological function and confirm the importance of nonpyramidal tracts in modulating gross motor performance.     

Early profiles of axonal growth and astroglial response after spinal cord hemisection and implantation of Schwann cell-seeded guidance channels in adult rats

We previously demonstrated that transplantation of Schwann cell-seeded channels promoted the regrowth of injured axons in the adult spinal cord. It is not clear, however, whether injured axons recapitulate the developmental scenarios to accomplish regeneration. In the present study, we investigated the early events associated with axonal regrowth after spinal cord hemisection at the eighth thoracic level and implantation of a Schwann cell-seeded minichannel in adult rats. Animals were sacrificed at postoperative days (PO) 2, 4, 7, and 14. Anterograde tracing with fluoro-ruby showed that regenerating axons grew into the graft prior to PO2 and reached the distal end of the channel at PO7. These axons expressed both embryonic neural cell adhesion molecule (E-NCAM) and growth associated protein-43 (GAP-43). Although the expression of E-NCAM decreased by PO7, that of GAP-43 remained high throughout the first 2 weeks after implantation. A close relation of vimentin-positive astroglia to the growing axons in the host tissue suggested a contact-mediated role of these cells in axon guidance. Aggregation of glial fibrillary acidic protein (GFAP)-positive astrocytes together with the increased expression of chondroitin sulfate proteoglycans (CSPGs) starting at PO7 appeared to inhibit axonal growth at the host-graft interface. Thus, adult regenerating axons and astroglia do express developmentally related molecules that may facilitate axonal growth into a permissive graft at the early phase of injury and regeneration. These results suggest that molecules and astroglia essential to development are both important in influencing axonal regrowth in the adult spinal cord.     

Inhibition of ROS-induced p38MAPK and ERK activation in microglia by acupuncture relieves neuropathic pain after spinal cord injury in rats

Acupuncture (AP) is currently used worldwide to relieve pain. However, little is known about its mechanisms of action. We found that after spinal cord injury (SCI), AP inhibited the production of superoxide anion (O(2)·), which acted as a modulator for microglial activation, and the analgesic effect of AP was attributed to its anti-microglial activating action. Direct injection of a ROS scavenger inhibited SCI-induced NP. After contusion injury which induces the below-level neuropathic pain (NP), Shuigou and Yanglingquan acupoints were applied. AP relieved mechanical allodynia and thermal hyperalgesia, while vehicle and simulated AP did not. AP also decreased the proportion of activated microglia, and inhibited both p38MAPK and ERK activation in microglia at the L4-5. Also, the level of prostaglandin E(2) (PGE2), which is produced via ERK signaling and mediates the below-level pain through PGE2 receptor, was reduced by AP. Injection of p38MAPK or ERK inhibitors attenuated NP and decreased PGE2 production. Furthermore, ROS produced after injury-induced p38MAPK and ERK activation in microglia, and mediated mechanical allodynia and thermal hyperalgesia, which were inhibited by AP or a ROS scavenger. AP also inhibited the expression of inflammatory mediators. Therefore, our results suggest that the analgesic effect of AP may be partly mediated by inhibiting ROS-induced microglial activation and inflammatory responses after SCI and provide the possibility that AP can be used effectively as a non-pharmacological intervention for SCI-induced chronic NP in patients.     

Fluoxetine,a selective serotonin reuptake inhibitor used clinically,improves bladder function in a mouse model of moderate spinal cord injury

After spinal cord injury,the upward conduction of the spinal cord is lost,resulting in the loss of micturition control,which manifests as detrusor sphincter dyssynergia and insufficient micturition.Studies have shown that serotonergic axons play important roles in the control of the descending urination tract.In this study,mouse models of moderate spinal cord contusions were established.The serotonin agonists quipazine(0.2 mg/kg),8-hydroxy-2-(di-n-propylamino)tetralin(8-OH-DAPT,0.1 mg/kg),buspirone(1 mg/kg),sumatriptan(1 mg/kg),and rizatriptan(50 mg/kg),the serotonin reuptake inhibitors fluoxetine(20 mg/kg)and duloxetine(1 mg/kg),and the dopamine receptor agonist SKF-82197(0.1 mg/kg)were intraperitoneally administered to the model mice 35 days post-injury in an acute manner.The voided stain on paper method and urodynamics revealed that fluoxetine reduced the amount of residual urine in the bladder and decreased bladder and external urethral sphincter pressure in a mouse model of moderate spinal cord injury.However,fluoxetine did not improve the micturition function in a mouse model of severe spinal cord injury.In contrast,the other serotonergic drugs had no effects on the micturition functions of spinal cord injury model mice.This study was ethically approved by the Institutional Animal Care and Use Committee of Jiangsu Province Hospital of Chinese Medicine(approval No.2020DW-20-02)on September 11,2020.