Activation of Neurogenesis in Multipotent Stem Cells Cultured In Vitro and in the Spinal Cord Tissue After Severe Injury by Inhibition of Glycogen Synthase Kinase-3

The inhibition of glycogen synthase kinase-3 (GSK-3) can induce neurogenesis, and the associated activation of Wnt/β-catenin signaling via GSK-3 inhibition may represent a means to promote motor function recovery following spinal cord injury (SCI) via increased astrocyte migration, reduced astrocyte apoptosis, and enhanced axonal growth. Herein, we assessed the effects of GSK-3 inhibition in vitro on the neurogenesis of ependymal stem/progenitor cells (epSPCs) resident in the mouse spinal cord and of human embryonic stem cell–derived neural progenitors (hESC-NPs) and human-induced pluripotent stem cell–derived neural progenitors (hiPSC-NPs) and in vivo on spinal cord tissue regeneration and motor activity after SCI. We report that the treatment of epSPCs and human pluripotent stem cell–derived neural progenitors (hPSC-NPs) with the GSK-3 inhibitor Ro3303544 activates β-catenin signaling and increases the expression of the bIII-tubulin neuronal marker; furthermore, the differentiation of Ro3303544-treated cells prompted an increase in the number of terminally differentiated neurons. Administration of a water-soluble, bioavailable form of this GSK-3 inhibitor (Ro3303544-Cl) in a severe SCI mouse model revealed the increased expression of bIII-tubulin in the injury epicenter. Treatment with Ro3303544-Cl increased survival of mature neuron types from the propriospinal tract (vGlut1, Parv) and raphe tract (5-HT), protein kinase C gamma–positive neurons, and GABAergic interneurons (GAD65/67) above the injury epicenter. Moreover, we observed higher numbers of newly born BrdU/DCX-positive neurons in Ro3303544-Cl–treated animal tissues, a reduced area delimited by astrocyte scar borders, and improved motor function. Based on this study, we believe that treating animals with epSPCs or hPSC-NPs in combination with Ro3303544-Cl deserves further investigation towards the development of a possible therapeutic strategy for SCI.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00928-0) contains supplementary material, which is available to authorized users.Key Words: Spinal cord injury, stem cells, neurogenesis, axonal growth, GSK3 inhibition     

EphB2 knockdown decreases the formation of astroglial-fibrotic scars to promote nerve regeneration after spinal cord injury in rats

AimsAt the beginning of spinal cord injury (SCI), the expression of EphB2 on fibroblasts and ephrin‐B2 on astrocytes increased simultaneously and their binding triggers the formation of astroglial‐fibrotic scars, which represent a barrier to axonal regeneration. In the present study, we sought to suppress scar formation and to promote recovery from SCI by targeting EphB2 in vivo.MethodsThe female rats SCI models were used in vivo experiments by subsequently injecting with EphB2 shRNA lentiviruses. The effect on EphB2 knockdown was evaluated at 14 days after injury. The repair outcomes were evaluated at 3 months by electrophysiological and morphological assessments to regenerated nerve tissue. The EphB2 expression and TGF‐β1 secretion were detected in vitro using a lipopolysaccharides (LPS)‐induced astrocyte injury model.ResultsRNAi decreased the expression of EphB2 after SCI, which effectively inhibited fibroblasts and astrocytes from aggregating at 14 days. The expression of EphB2 in activated astrocytes, in addition to fibroblasts, was significantly increased after SCI in vivo, in line with upregulated expression of EphB2 and increased secretion of TGF‐β1 in astrocyte culture treated with LPS. Compared to the scramble control, RNAi targeting with EphB2 could promote more nerve regeneration and better myelination.ConclusionsEphB2 knockdown may effectively inhibit the formation of astroglial‐fibrotic scars at the beginning of SCI. It is beneficial to eliminate the barrier of nerve regeneration.     

Cell transplantation for the treatment of spinal cord injury–bone marrow stromal cells and choroid plexus epithelial cells

Transplantation of bone marrow stromal cells(BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury(SCI).BMSCs did not survive long-term,disappearing from the spinal cord within 2–3 weeks after transplantation.Astrocyte-devoid areas,in which no astrocytes or oligodendrocytes were found,formed at the epicenter of the lesion.It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas.Regenerating axons were associated with Schwann cells embedded in extracellular matrices.Transplantation of choroid plexus epithelial cells(CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI.Although CPECs disappeared from the spinal cord shortly after transplantation,an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas,as in the case of BMSC transplantation.These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord,including axonal regeneration and reduced cavity formation.This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate.The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI.It should be emphasized that the generally anticipated long-term survival,proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.     

Combined treatment with αMSH and methylprednisolone fails to improve functional recovery after spinal injury in the rat

To date, relatively little progress has been made in the treatment of spinal cord injury (SCI)-related neurological impairments. Until now, methylprednisolone (MP) is the only agent with clinically proven beneficial effect on functional outcome after SCI. Although the mechanism of action is not completely clear, experimental data point to protection against membrane peroxidation and edema reduction. The melanocortin melanotropin is known to improve axonal regeneration following sciatic nerve injury, and to stimulate corticospinal outgrowth after partial spinal cord transection. Recently, we showed that intrathecally administered αMSH had beneficial effects on functional recovery after experimental SCI. Since both drugs have shown their value in intervention studies after (experimental) spinal cord injury (ESCI), we decided to study the effects of combined treatment. Our results again showed that αMSH enhances functional recovery after ESCI in the rat and that MP, although not affecting functional recovery adversely by itself, abolished the effects observed with αMSH when combined. Our data, thus, suggest that the mechanism of action of MP interferes with that of αMSH.     

Neuroprotective Effect of Anthocyanin on Experimental Traumatic Spinal Cord Injury

Objective

We investigated the neuroprotective effect of anthocyanin, oxygen radical scavenger extracted from raspberries, after traumatic spinal cord injury (SCI) in rats.

Methods

The animals were divided into two groups : the vehicle-treated group (control group, n=20) received an oral administration of normal saline via stomach intubation immediately after SCI, and the anthocyanin-treated group (AT group, n=20) received 400 mg/kg of cyanidin 3-O-β-glucoside (C3G) in the same way. We compared the neurological functions, superoxide expressions and lesion volumes in two groups.

Results

At 14 days after SCI, the AT group showed significant improvement of the BBB score by 16.7±3.4%, platform hang by 40.0±9.1% and hind foot bar grab by 30.8±8.4% (p<0.05 in all outcomes). The degree of superoxide expression, represented by the ratio of red fluorescence intensity, was significantly lower in the AT group (0.98±0.38) than the control group (1.34±0.24) (p<0.05). The lesion volume in lesion periphery was 32.1±2.4 µL in the control and 24.5±2.3 µL in the AT group, respectively (p<0.05), and the motor neuron cell number of the anterior horn in lesion periphery was 8.3±5.1 cells/HPF in the control and 13.4±6.3 cells/HPF in the AT group, respectively (p<0.05).

Conclusion

Anthocyanin seemed to reduce lesion volume and neuronal loss by its antioxidant effect and these resulted in improved functional recovery.     

Ambulation following spinal cord injury and its correlates

Objectives:

To assess walking ability of spinal cord injury (SCI) patients and observe its correlation with functional and neurological outcomes.

Patients and Methods:

The present prospective, observational study was conducted in a tertiary research hospital in India with 66 patients (46 males) between January 2012 and December 2013. Mean age was 32.62 ± 11.85 years (range 16-65 years), mean duration of injury was 85.3 ± 97.6 days (range 14-365 days) and mean length of stay in the rehabilitation unit was 38.08 ± 21.66 days (range 14-97 days) in the study. Walking Index for spinal cord injury (WISCI II) was used to assess ambulation of the SCI patients. Functional recovery was assessed using Barthel Index (BI) and Spinal Cord Independence Measures (SCIM). Neurological recovery was assessed using ASIA impairment scale (AIS). We tried to correlate ambulatory ability of the patients with functional and neurological recovery.

Results:

Ambulatory ability of the patients improved significantly using WISCI II (P < 0.001) when admission and discharge scores were compared (1.4 ± 3.5 vs 7.6 ± 6.03). Similarly, functional (BI: 31.7 ± 20.5 vs 58.4 ± 23.7 and SCIM: 29.9 ± 15.1 vs 56.2 ± 20.6) and neurological recovery were found to be very significant (P < 0.001) when admission vs discharge scores were compared. Improvement in WISCI II scores was significantly correlated with improvement in neurological (using AIS scores) and functional status (using BI and SCIM scores) (P < 0.001).

Conclusions:

Significant improvement was seen in WISCI II, BI, and SCIM scores after in-patient rehabilitation. Improvement in WISCI II scores also significantly correlated with functional and neurological recovery.     

移植人羊膜间充质干细胞促进脊髓损伤大鼠神经功能恢复

目的研究移植人羊膜间充质干细胞（hAMSCs）是否促进脊髓损伤大鼠神经功能恢复,探索其可能作用机制。 方法60只雌性SD大鼠按照随机数字表法分为磷酸盐缓冲液（PBS）治疗组（30只）和hAMSCs治疗组（30只）。脊髓损伤采用脊髓撞击损伤模型,hAMSCs或PBS立刻移植到离脊髓损伤中心2 mm的头尾两端。免疫荧光检测细胞分化,血管再生和轴突再生。酶联免疫吸附剂测定试剂盒检测脑源性神经营养因子（BDNF）和血管内皮生长因子（VEGF）含量,BBB运动功能评分检测行为学。 结果在脊髓损伤后14 d、21 d和28 d,hAMSCs治疗组BBB评分分别为（8.75±0.701）、（10.375±0.532）和（12.125±0.350）,高于PBS组（6.0±0.463）、（7.25±0.412）和（9.125±0.440）,差异具有统计学意义（P<0.05）。在第7天和第14天,hAMSCs治疗组BDNF表达水平分别为（75.138±4.367）pg/mg和（66.483±4.099）pg/mg,高于PBS组（43.901±3.607）pg/mg和（41.108±3.848）pg/mg,差异具有统计学意义（P<0.05）。在第7天,第14天和第28天,hAMSCs治疗组VEGF表达水平分别为（23.328±2.463）pg/mg,（22.301±2.223）pg/mg和（14.855±1.282）pg/mg,高于PBS组（9.978±1.572）pg/mg,（9.271±1.496）pg/mg和（7.113±1.123）pg/mg,差异具有统计学意义（P<0.05）。hAMSCs治疗组血管数目（17.5±2.102）高于PBS组（6.25±1.750）,差异具有统计学意义（P<0.05）。hAMSCs治疗组小鼠抗5羟色胺阳性神经纤维面积（3486±203.643）和GAP43阳性神经纤维面积（4568.25±253.881）高于PBS组（2070.25±156.344）和（2455.725±314.475）,差异具有统计学意义（P<0.05）。 结论移植hAMSCs能促进脊髓损伤大鼠神经功能恢复,其作用机制可能是通过增加神经营养因子表达,促进血管再生和轴突再生。因此hAMSCs移植是治疗脊髓损伤的理想方法。     

Differential effect of aging on axon sprouting and regenerative growth in spinal cord injury

The demographics of acute spinal cord injury (SCI) are changing with an increased incidence in older age. However, the influence of aging on the regenerative growth potential of central nervous system (CNS) axons following SCI is not known. We investigated axonal sprouting along with the efficiency of the infusion of the stromal cell-derived growth factor-1 (SDF-1/CXCL12) and regenerative growth along with the anti-scarring treatment (AST) in young (2–3 months) and geriatric (22–28 months) female rats following SCI. AST included local injection of iron chelator (2,2′-dipyridine-5,5′-dicarboxylic acid) and 8-bromo-cyclic adenosine monophosphate solution into the lesion core. Axon outgrowth was investigated by immunohistological methods at 5 weeks after a partial dorsal hemisection at thoracic level T8. We found that aging significantly reduces spontaneous axon sprouting of corticospinal (CST), serotonergic (5-HT) raphespinal and catecholaminergic (TH) coerulospinal tracts in distinct regions of the spinal cord rostral to the lesion. However, impairment of axon sprouting could be markedly attenuated in geriatric animals by local infusion of SDF-1. Unexpectedly and in contrast to rostral sprouting, aging does not diminish the regenerative growth capacity of 5-HT-, TH- and calcitonin gene-related peptide (CGRP)-immunoreactive axons at 5 weeks after SCI. Moreover, 5-HT and TH axons maintain the ability to react upon AST with significantly enhanced regeneration in aged animals. These data are the first to demonstrate, that old age compromises axonal plasticity, but not regenerative growth, after SCI in a fiber tract-specific manner. Furthermore, AST and SDF-1 infusions remain efficient, which implicates that therapy in elderly patients is still feasible.     

Effect of Pulsed Radiofrequency Neuromodulation on Clinical Improvements in the Patients of Chronic Intractable Shoulder Pain

Objective

The aim of this study was to evaluate effect of pulsed radiofrequency (PRF) neuromodulation of suprascpaular nerve (SSN) in patients with chronic shoulder pain due to adhesive capsulitis and/or rotator cuff tear.

Methods

The study included 11 patients suffering from chronic shoulder pain for at least 6 months who were diagnosed with adhesive capsulitis (n=4), rotator cuff tear (n=5), or adhesive capsulitis+rotator cuff tear (n=2) using shoulder magnetic resonance imaging or extremity ultrasonography. After a favorable response to a diagnostic suprascapular nerve block twice a week (pain improvement >50%), PRF neuromodulation was performed. Shoulder pain and quality of life were assessed using a Visual Analogue Scale (VAS) and the Oxford Shoulder Score (OSS) before the diagnostic block and every month after PRF neuromodulation over a 9-month period.

Results

The mean VAS score of 11 patients before PRF was 6.4±1.49, and the scores at 6-month and 9 month follow-up were 1.0±0.73 and 1.5±1.23, respectively. A significant pain reduction (p<0.001) was observed. The mean OSS score of 11 patients before PRF was 22.7±8.1, and the scores at 6-month and 9 month follow-up were 41.5±6.65 and 41.0±6.67, respectively. A significant OSS improvement (p<0.001) was observed.

Conclusion

PRF neuromodulation of the suprascapular nerve is an effective treatment for chronic shoulder pain, and the effect was sustained over a relatively long period in patients with medically intractable shoulder pain.     

大鼠脊髓损伤后轴突病理变化与胶质瘢痕的关系

目的 观察脊髓损伤(SCI)后轴突变化及其与胶质瘢痕的关系.方法 应用Allen's法建立大鼠脊髓损伤模型,通过行为学评分、免疫荧光及神经束路示踪等观察SCI后轴突的病理变化,及其与胶质瘢痕的关系,并测量胶质瘢痕的厚度.结果 SCI后损伤处的轴突呈断裂、扭曲状,SCI后1 周损伤轴突呈再生趋势,2周时再生明显,与此相应动物运动功能逐渐恢复,4周时胶质瘢痕形成,再生的轴突被瘢痕阻挡.头尾侧胶质瘢痕厚度(107.00±20.12)μm大于两侧边厚度(69.92±24.37)μm.结论 SCI后轴突仍具有再生能力,但被胶质瘢痕所阻挡,瘢痕厚度的测量为将来去除胶质瘢痕提供了实验依据.     

Neuroprotective Effects of Sacral Epidural Neuromodulation Following Spinal Cord Injury : An Experimental Study in Rats

Objective

The purpose of this study is to evaluate neuroprotective effect of sacral neuromodulation in rat spinal cord injury (SCI) model in the histological and functional aspects.

Methods

Twenty-one female Sprague Dawley rats were randomly divided into 3 groups : the normal control group (CTL, n=7), the SCI with sham stimulation group (SCI, n=7), and the SCI with electrical stimulation (SCI+ES, n=7). Spinal cord was injured by dropping an impactor from 25 mm height. Sacral nerve electrical stimulation was performed by the following protocol : pulse duration, 0.1 ms; frequency, 20 Hz; stimulation time, 30 minutes; and stimulation duration, 4 weeks. Both locomotor function and histological examination were evaluated as scheduled.

Results

The number of anterior horn cell was 12.3±5.7 cells/high power field (HPF) in the CTL group, 7.8±4.9 cells/HPF in the SCI group, and 6.9±5.5 cells/HPF in the SCI+ES group, respectively. Both the SCI and the SCI+ES groups showed severe loss of anterior horn cells and myelin fibers compared with the CTL group. Cavitation and demyelinization of the nerve fibers has no significant difference between the SCI group and the SCI+ES group. Cavitation of dorsal column was more evident in only two rats of SCI group than the SCI+ES group. The locomotor function of all rats improved over time but there was no significant difference at any point in time between the SCI and the SCI+ES group.

Conclusion

In a rat thoracic spinal cord contusion model, we observed that sacral neuromodulation did not prevent SCI-induced myelin loss and apoptosis.     

Effects of short-term training on heart rate dynamics in individuals with spinal cord injury

Increased risk of cardiovascular diseases and autonomic dysregulation are common health concerns in individuals with spinal cord injury (SCI). Two therapies that may help improve cardiovascular control are body-weight supported treadmill training (BWSTT) and head-up tilt training (HUTT). The purpose of this study was to examine the effects of short-term BWSTT and HUTT on cardiac autonomic function. Seven participants (6 male, 37.1 ± 7.7 years) with SCI (C5-T10, ASIA A-C; 5.0 ± 4.4 years post-injury) completed the study protocol. In this randomized cross-over design, participants were required to complete 4 weeks of thrice-weekly BWSTT and HUTT (i.e. 12 sessions each), separated by a 4 week detraining period. Cardiac autonomic function was assessed at rest, before and after, each 4 week training period using linear and non-linear measures (sample entropy and detrended fluctuation analysis (α₁)) of heart rate dynamics. Participants completed equivalent amounts of time performing BWSTT and HUTT (453.7 ± 27.3 min vs. 471.6 ± 19.7 min, p = 0.24). There were no significant differences in linear heart rate variability following BWSTT or HUTT (p > 0.05). In contrast, there was a significant change in sample entropy following BWSTT (1.05 ± 0.14 to 1.42 ± 0.12, p < 0.05). Due to the bi-directional pattern of α₁ values, distance scores were calculated (│1 − α₁│) and demonstrated a significant reduction following BWSTT (0.54 ± 0.06 to 0.26 ± 0.05, p = 0.001). In conclusion, 4 weeks of BWSTT but not HUTT training are sufficient to increase sample entropy and reduce the fractal scaling distance score in participants with SCI.     

Efficacy of olfactory ensheathing cells to support regeneration after spinal cord injury is influenced by method of culture preparation

Olfactory ensheathing cells (OEC) have been shown to stimulate regeneration, myelination and functional recovery in different spinal cord injury models. However, recent reports from several laboratories have challenged this treatment strategy. The discrepancy in results could be attributed to many factors including variations in culture protocols. The present study investigates whether the differences in culture preparation could influence neuroprotective and growth-promoting effects of OEC after transplantation into the injured spinal cord. Primary OEC cultures were purified using method of differential cell adhesion (a-OEC) or separated with immunomagnetic beads (b-OEC). After cervical C4 hemisection in adult rats, short-term (3 weeks) or long-term (7 weeks) cultured OEC were transplanted into the lateral funiculus at 1 mm rostral and caudal to the transection site. At 3–8 weeks after transplantation, labeled OEC were mainly found in the injection sites and in the trauma zone. Short-term cultured a-OEC supported regrowth of rubrospinal, raphaespinal and CGRP-positive fibers, and attenuated retrograde degeneration in the red nucleus. Short-term cultured b-OEC failed to promote axonal regrowth but increased the density of rubrospinal axons within the dorsolateral funiculus and provided significant neuroprotection for axotomized rubrospinal neurons. In addition, short-term cultured OEC attenuated sprouting of rubrospinal terminals. In contrast, long-term cultured OEC neither enhanced axonal growth nor prevented retrograde cell death. The results suggest that the age of OEC in culture and the method of cell purification could affect the efficacy of OEC to support neuronal survival and regeneration after spinal cord injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.     

Lithium attenuates blood–brain barrier damage and brain edema following intracerebral hemorrhage via an endothelial Wnt/β‐catenin signaling‐dependent mechanism in mice

BackgroundVasogenic cerebral edema resulting from blood–brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). Although augmentation of endothelial Wnt/β‐catenin signaling substantially alleviates BBB breakdown in animals, no agents based on this mechanism are clinically available. Lithium is a medication used to treat bipolar mood disorders and can upregulate Wnt/β‐catenin signaling.MethodsWe evaluated the protective effect of lithium on the BBB in a mouse model of collagenase IV‐induced ICH. Furthermore, we assessed the effect and dependency of lithium on Wnt/β‐catenin signaling in mice with endothelial deletion of the Wnt7 coactivator Gpr124.ResultsLithium treatment (3 mmol/kg) significantly decreased the hematoma volume (11.15 ± 3.89 mm³ vs. 19.97 ± 3.20 mm³ in vehicle controls, p = 0.0016) and improved the neurological outcomes of mice following ICH. Importantly, lithium significantly increased the BBB integrity, as evidenced by reductions in the levels of brain edema (p = 0.0312), Evans blue leakage (p = 0.0261), and blood IgG extravasation (p = 0.0009) into brain tissue around the hematoma. Mechanistically, lithium upregulated the activity of endothelial Wnt/β‐catenin signaling in mice and increased the levels of tight junction proteins (occludin, claudin‐5 and ZO‐1). Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, suggesting that its protective effect on BBB function was mainly dependent on Gpr124‐mediated endothelial Wnt/β‐catenin signaling.ConclusionOur findings indicate that lithium may serve as a therapeutic candidate for treating BBB breakdown and brain edema following ICH.     

Review of transplantation of neural stem/progenitor cells for spinal cord injury

Spinal cord injury (SCI) is a debilitating condition often resulting in paralysis, yet currently there is no effective treatment. Stem cell transplantation is a promising therapeutic strategy for promoting tissue repair after SCI. Stem cells offer a renewable source of cells with inherent plasticity for tissue regeneration. Neural stem/progenitor cells (NSPCs) are multipotent cells that self-renew and are committed to the neural lineage, and thus, they are especially suited to SCI repair. NSPCs may differentiate into neural cells after transplantation into the injured spinal cord, replacing lost or damaged cells, providing trophic support, restoring connectivity, and facilitating regeneration. Here, we review experimental studies and considerations for clinical translation of NSPC transplantation for SCI.     

Evaluation of the Combination of Methylprednisolone and Tranilast after Spinal Cord Injury in Rat Models

ObjectiveThe aim of our study was to evaluate the neuroprotective functions of the combination therapy using methylprednisolone (MP) and tranilast (TR) after spinal cord injury (SCI) in adult rats.MethodsSpinal cord compression injury model was achieved using Yasargil aneurysm clip. Rats were divided into control group, MP group, TR group, and combination therapy group using TR and MP. Rat models were assessed for locomotor functional recovery using Basso, Beattie, and Bresnahan (BBB) score, spinal cord water content and myeloperoxidase (MPO) activity 24 hours post SCI, haematoxylin and eosin staining and glial fibrillary acid protein (GFAP) staining at 7 and 14 days post SCI.ResultsThe spinal cord water content and MPO activity in the combination therapy group was significantly lower than the control group and the individual therapy groups p<0.05. The combination therapy group had significantly higher BBB scores than control group and individual therapy groups (p<0.05). At one week after SCI, GFAP expression in the combination group was significantly lower than the control group (p<0.05) but there was no significant difference compared to the individual therapy groups (p>0.05). At 2 weeks after SCI there was a slight decrease in GFAP expression compared to the first week but the difference was not statistically significant (p>0.05), GFAP expression between the groups was not statistically significant p>0.05.ConclusionCombining MP and TR is therapeutically more effective in improving functional recovery, inhibiting inflammation and glial scar formation after acute SCI.     

骨髓基质细胞移植治疗脊髓全横断损伤超微结构观察

目的观察骨髓基质细胞（MSCs）移植治疗脊髓全横断损伤（SCI）超微结构,探讨内源性细胞与再生轴突关系。方法通过全骨髓法培养、纯化MSCs,SCI9d后移植MSCs,通过免疫荧光组化观察细胞移植后损伤区轴突再生情况,免疫荧光双标、免疫电镜观察再生轴突与内源性细胞关系。结果移植8W后实验组脊髓损伤区可见大量神经微丝蛋白200（NF200）阳性纤维,对照组脊髓损伤区未见明显的NF200阳性纤维。免疫荧光双标结果显示损伤区NF200阳性纤维和2,3＇-环核苷酸磷酸而酯酶（CNP）阳性细胞之间存在密切的空间关系,免疫电镜显示CNP阳性细胞通过伸长丝状伪足形成再生轴突支架,内源性施万细胞参与再生轴突髓鞘形成。结论MSCs移植可促进损伤区轴突再生,宿主自身CNP阳性细胞和施万细胞参与损伤轴突的再生和髓鞘形成。     

Apigenin inhibits fibrous scar formation after acute spinal cord injury through TGFβ/SMADs signaling pathway

AimTo investigate the effect of apigenin on fibrous scar formation after mouse spinal cord injury (SCI).MethodsThe pneumatic impactor strike method was used to establish an SCI model. Mice were intraperitoneally injected with 5 mg/kg or 20 mg/kg apigenin daily for 28 days after SCI. The Basso Mouse Scale (BMS) score, hematoxylin–eosin staining, and immunohistochemical staining were used to assess the effect of apigenin on scar formation and motor function recovery. Western blotting and qRT‐PCR were used to detect the expression of fibrosis‐related parameters in spinal cord tissue homogenates. NIH‐3 T3 cells and mouse primary spinal cord fibroblasts, α‐Smooth muscle actin (α‐SMA), collagen 1, and fibronectin were used to evaluate apigenin''s effect in vitro. Western blotting and immunofluorescence techniques were used to study the effect of apigenin on TGFβ/SMADs signaling.ResultsApigenin inhibited fibrous scar formation in the mouse spinal cord and promoted the recovery of motor function. It reduced the expression of fibroblast‐related parameters and increased the content of nerve growth factor in vivo, decreasing myofibroblast activation and collagen fiber formation by inhibiting TGFβ‐induced SMAD2/3 phosphorylation and nuclear translocation in vitro.ConclusionApigenin inhibits fibrous scar formation after SCI by decreasing fibrosis‐related factor expression through TGFβ/SMADs signaling.     

骨髓间质细胞源性神经球移植对大鼠脊髓损伤的修复作用

目的探讨骨髓间质细胞源性神经球(BMSCs-NS)移植在大鼠脊髓损伤修复中的作用。方法体外原代培养大鼠BMSCs-NS。Wistar大鼠45只,玻璃器压迫法制备胸髓(T9-10)完全损伤模型,随机分成三组:BMSCs-NS移植组(A组,n=15)、BMSCs移植组(B组,n=15)和培养液组(C组,n=15)。BBB评分评估三组大鼠后肢运动功能在不同时间段的恢复及双下肢运动诱发电位(MEPs)情况。结果 BBB平均分值为:A组,7.8±1.42;B组,5.7±0.90;C组,1.4±0.29。移植修复21d后BBB绝对分值,A组较B组和C组明显改善(B组:p<0.001;C组:p<0.0001)。移植5w后B组少数大鼠可以诱发出MEPs,但潜伏期较长(20ms)且波幅较低。2w时A组未能诱发出MEPs,但5w时的MEPs接近正常。结论细胞移植治疗能够恢复大鼠后肢的承重运动功能并能成功诱导出理想的MEPs。