A Novel Stem Cell Line Derived from Mesenchymal Stem Cells and Its Culture Supernatant Ameliorates Spinal Cord Injury in Rats

Spinal cord injury models: neurophysiology.

Background/Aims: Shikonin, a compound extracted from Zicao, has been demonstrated to hold anti-bacterial, anti-inflammatory, and anti-tumor activities in various diseases and it has been shown to protect human organs from injuries. However, the effect of shikonin on the recovery of spinal cord injury (SCI) remains unknown. This study was designed to estimate the potential therapeutic effect and underlying mechanism of shikonin on SCI in vivo. Methods: In the study, we used HE staining, ELISA assay, transfection assay, TUNEL assay, real time PCR and Western blot to detect the effects of shikonin on spinal cord injury in rats. Results: we showed that shikonin could promote the recovery of motor function and tissue repair after SCI treatment in rats SCI model. Moreover, we demonstrated that shikonin inhibited the spinal cord edema in SCI model of rats. According to further investigation, shikonin induced the reduction of inflammatory response through decreasing the expression levels of HMGB1, TLR4 and NF-κB after SCI injury. In addition, we also found that shikonin could suppress the apoptosis and expression of caspase-3 protein in SCI model of rats. Conclusion: Our results demonstrated that shikonin induced the recovery of tissue repair and motor function via inactivation of HMGB1/TLR4/NF-κB signaling pathway in SCI model of rats. Meanwhile, shikonin regulated the inflammation response in SCI by suppressing the HMGB1/TLR4/NF-κB signaling pathway. The described mechanism sheds novel light on molecular signaling pathway in spinal cord injury and secondary injury including inflammatory response.

To investigate the effect of the micro ribonucleic acid (miR)-126-modified mesenchymal stem cell (MSC)-derived exosomes (MSC-exos) on the repair process of spinal cord injury (SCI) in rats. MiR-126-modified MSCs were cultured, the exosomes were extracted, and a rat model of SCI was established. The Quantitative Polymerase Chain Reaction (qPCR) was carried out to detect the expression of miR-126 in the injured spinal cord, and the Western blotting (WB) was adopted to detect the expressions of the exosome-related molecules. Subsequently, the motor function recovery of rats was examined via the Basso, Beattie and Bresnahan (BBB) locomotor rating scale. The effects of miR-126 exosomes on the injury volume and NeuN retention following SCI were evaluated via immunohistochemistry. MSCs were able to package miR-126 into exosomes secreted. After SCI, the recovery of the hind limb function of rats was remarkably improved by miR-126-modified MSC-exos relative to the control group. Treatment with miR-126-modified MSC-exos remarkably decreased the injury volume, retained the neuronal cells, and triggered the axon regeneration following SCI. Besides, the expression of Ras homolog gene family member A (RhoA), identified as the downstream gene of miR-126, was downregulated in the group with miR-126-modified MSC-exos. Moreover, miR-126-modified MSC-exos activated the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. MiR-126-modified MSC-exos protect neurons of rats with SCI, stimulates axon regeneration, and improves the recovery of limb motor function after SCI.

Study DesignThis study investigated the effects of transplantation of alginate encapsulated neural stem cells (NSCs) on spinal cord injury in Sprague-Dawley male rats. The neurological functions were assessed for 6 weeks after transplantation along with a histological study and measurement of caspase-3 levels.PurposeThe aim of this study was to discover whether NSCs cultured in alginate transplantation improve recovery from spinal cord injury.Overview of LiteratureSpinal cord injury is one of the leading causes of disability and it has no effective treatment. Spinal cord injury can also cause sensory impairment. With an impetus on using stem cells therapy in various central nervous system settings, there is an interest in using stem cells for addressing spinal cord injury. Neural stem cell is one type of stem cells that is able to differentiate to all three neural lineages and it shows promise in spinal injury treatment. Furthermore, a number of studies have shown that culturing NSCs in three-dimensional (3D) scaffolds like alginate could enhance neural differentiation.MethodsThe NSCs were isolated from 14-day-old rat embryos. The isolated NSCs were cultured in growth media containing basic fibroblast growth factor and endothelial growth factor. The cells were characterized by differentiating to three neural lineages and they were cultured in an alginate scaffold. After 7 days the cells were encapsulated and transplanted in a rat model of spinal cord injury.ResultsOur data showed that culturing in an alginate 3D scaffold and transplantation of the NSCs could improve neurological outcome in a rat model of spinal cord injury. The inflammation scores and lesion sizes and also the activity of caspase-3 (for apoptosis evaluation) were less in encapsulated neural stem cell transplantation cases.ConclusionsTransplantation of NSCs that were cultured in an alginate scaffold led to a better clinical and histological outcome for recovery from spinal cord injury in a rat model.

Cell-based therapy using mesenchymal stem cells (MSCs) is a novel treatment strategy for spinal cord injury (SCI). MSCs can be isolated from various tissues, and their characteristics vary based on the source. However, reports demonstrating the effect of transplanted rat cranial bone-derived MSCs (rcMSCs) on rat SCI models are lacking. In this study, we determined the effect of transplanting rcMSCs in rat SCI models. MSCs were established from collected bone marrow and cranial bones. SCI rats were established using the weight-drop method and transplanted intravenously with MSCs at 24 h post SCI. The recovery of motor function and hindlimb electrophysiology was evaluated 4 weeks post transplantation. Electrophysiological recovery was evaluated by recording the transcranial electrical stimulation motor-evoked potentials. Tissue repair after SCI was assessed by calculating the cavity ratio. The expression of genes involved in the inflammatory response and cell death in the spinal cord tissue was assessed by real-time polymerase chain reaction. The transplantation of rcMSCs improved motor function and electrophysiology recovery, and reduced cavity ratio. The expression of proinflammatory cytokines was suppressed in the spinal cord tissues of the rats that received rcMSCs. These results demonstrate the efficacy of rcMSCs as cell-based therapy for SCI.

Simvastatin mobilizes bone marrow stromal cells migrating to injured areas and promotes functional recovery after spinal cord injury in the rat

Transcranial electrically stimulated motor-evoked potentials (tcMEPs) are widely used to evaluate motor function in humans and animals. However, the relationship between tcMEPs and the recovery of paralysis remains unclear. We previously reported that transplantation of mesenchymal stem cells to a spinal cord injury (SCI) rat model resulted in various degrees of recovery from paraplegia. As a continuation of this work, in the present study, we aimed to establish the longitudinal electrophysiological changes in this SCI rat model after mesenchymal stem cell transplantation. SCI rats were established using the weight-drop method. The model rats were transvenously transplanted with two types of mesenchymal stem cells (MSCs), one derived from rat cranial bones and the other from the bone marrow of the femur and tibia bone, 24 h after SCI. A phosphate-buffered saline (PBS) group that received only PBS was also created for comparison. The degree of paralysis was evaluated over 28 days using the Basso-Beattie-Bresnahan (BBB) scale and inclined plane task score. Extended tcMEPs were recorded using a previously reported bone-thinning technique, and the longitudinal electrophysiological changes in tcMEPs were investigated. In addition, the relationship between the time course of recovery from paralysis and reappearance of tcMEPs was revealed. The appearance of the tcMEP waveform was earlier in MSC-transplanted rats than in PBS-administered rats (earliest date was 7 days after SCI). The MEP waveforms also appeared at approximately the same level on the BBB scale (average score, 11 points). Ultimately, this study can help enhance our understanding of the relationship between neural regeneration and tcMEP recording. Further application of tcMEP in regenerative medicine research is expected.

Transcranial electrically stimulated motor-evoked potentials (tcMEPs) are widely used to evaluate motor function in humans and animals. However, the relationship between tcMEPs and the recovery of paralysis remains unclear. We previously reported that transplantation of mesenchymal stem cells to a spinal cord injury (SCI) rat model resulted in various degrees of recovery from paraplegia. As a continuation of this work, in the present study, we aimed to establish the longitudinal electrophysiological changes in this SCI rat model after mesenchymal stem cell transplantation. SCI rats were established using the weight-drop method. The model rats were transvenously transplanted with two types of mesenchymal stem cells (MSCs), one derived from rat cranial bones and the other from the bone marrow of the femur and tibia bone, 24 h after SCI. A phosphate-buffered saline (PBS) group that received only PBS was also created for comparison. The degree of paralysis was evaluated over 28 days using the Basso–Beattie–Bresnahan (BBB) scale and inclined plane task score. Extended tcMEPs were recorded using a previously reported bone-thinning technique, and the longitudinal electrophysiological changes in tcMEPs were investigated. In addition, the relationship between the time course of recovery from paralysis and reappearance of tcMEPs was revealed. The appearance of the tcMEP waveform was earlier in MSC-transplanted rats than in PBS-administered rats (earliest date was 7 days after SCI). The MEP waveforms also appeared at approximately the same level on the BBB scale (average score, 11 points). Ultimately, this study can help enhance our understanding of the relationship between neural regeneration and tcMEP recording. Further application of tcMEP in regenerative medicine research is expected.

Bone marrow mesenchymal stem cells (BMSCs) on the repair of spinal cord injury (SCI) in rats as well as the role of transforming growth factor-β (TGF-β)/Smads signaling pathway in the repair were investigated. Rat BMSCs and astrocyte-spinal cords (ASCs) were isolated and cultured in vitro, and the cell purity was detected by flow cytometry. ASCs were co-cultured with TGF-β1, BMSCs and BMSCs + TGF-β1, respectively, and grouped accordingly, and ASCs cultured conventionally were included into control group. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-diphenyltetrazoliumbromide (MTT) assay was conducted to detect the proliferation ability of ASCs in each group. Western blotting (WB) was utilized to examine the expression of TGF-β/Smads signaling pathway-related proteins [TGF-β1, Smad2 and phosphorylated (p)-Smad2] in ASCs and ASCs co-cultured with BMSCs. A rat model of SCI was established, and BMSCs were injected locally. Then (BBB) score was used to evaluate spinal cord repair, and WB was adopted to detect the expression of TGF-β1, Smad2 and p-Smad2 at the injured site. BMSCs and ASCs isolated in vitro grew well. According to MTT assay results, TGF-β1 significantly promoted the proliferation of ASCs (P<0.05), and co-culture of ASCs and BMSCs remarkably reduced the proliferation of ASCs (P<0.05). The detection of protein expression at the SCI site via WB demonstrated that the expression of TGF-β1, Smad2 and p-Smad2 in SCI group were obviously upregulated compared with those in Sham group at 1 week (P<0.05), and the injection of BMSCs could markedly downregulate the expression (P<0.05). After 3 week, there were no significant differences in the expression of TGF-β1, Smad2 and p-Smad2 among groups (P>0.05). The transplantation of BMSCs can improve the spinal function of SCI rats probably by inhibiting the TGF-β/Smads signaling pathway and reducing the proliferation of ASCs.

Objective To investigate the effect of argatroban in repair of spinal cord injury in rats. Methods A total of 54 female Wistar rats were selected and divided into three groups according to the random number table: sham group, injury group and Argatroban group, with 18 rats in each group. The sham group only took the T10lamina; the injury group used the spinal cord injury device to make the rat spinal cord injury model; the Argatroban group received Argatroban treatment after spinal cord injury. The recovery of hindlimb motor function was evaluated by BBB score and clined plate test before injury and 7, 14, 21, 28, 35 and 42 days after injury. The sensory evoked potentials (SEP) and motor evoked potentials (MEP) were detected 42 days after operation. HE staining was used to compare the size of the cavity in the local region 42 days after injury. Results At day 7 after injury, the BBB score was (3.7±0.5)points and the inclined plane test was (28.0±2.6)° in the Argatroban group, which were better than those in the injury group [(3.3±0.5)points, (24.3±1.9)°] (P<0.05). At day 42 after injury, the BBB score was (13.0±0.8)points and inclined plane test was (50.7±2.7)° in the Argatroban group, which were significantly better than those in the injury group [(9.7±1.3) points, (40.5±2.7)°] (P<0.05). But all the above values in the Argatroban group were significantly lower than those in the sham group [(21.0±0.0)points, (60.0±0.0)°](P<0.05). At day 42 after operation, the SEP latency [(25.0±0.9)ms] in the Argatroban group was significantly shorter than that in the injury group [(31.5±1.9) ms]; the amplitude [(2.1±0.1)μV] in the Argatroban group was lower than that in the injury group [(0.5±0.1)μV] (P<0.05). The MEP latency [(11.5±1.0)ms] in the Argatroban group was significantly shorter than that in the injury group [(17.5±1.1)ms], and the amplitude [(4.8±0.8)μV] in the Argatroban group was lower than that in the injury group [(2.8±0.7)μV] (P<0.05). And the SEP or MEP latency and amplitude in the Argatroban group showed significant differences compared to the sham group [(7.5±1.0)ms, (7.5±1.0)μV](P<0.05). HE staining showed that the central area of the lesion in the Argatroban group [(0.35±0.04)mm2] was significantly smaller than that in the injury group [(0.71±0.05)mm2]. Conclusion After spinal cord injury, argatroban can protect the spinal cord tissue effectively in the injured area and promote recovery of sensory and motor function in the hind limbs of rats. Key words: Spinal cord injuries; Argatroban; Inflammation; Thrombin

Insufficient local levels of neurotrophic factor after spinal cord injury (SCI) are the leading cause of secondary injury and limited axonal regeneration. Neuritin belongs to a family of neurotrophic factors that promote neurite outgrowth, maintain neuronal survival, and provide a favorable microenvironment for the regeneration and repair of nerve cells after injury. However, it is not known whether the exogenously applied neuritin protein has a positive effect on nerve repair after SCI. This was investigated in the present study using purified human recombinant neuritin expressed in and purified from Pichia pastoris, which was tested in a rat SCI model. A recombinant neuritin concentration of 60 μg/ml induced the recovery of hind limb motor function and stimulated nerve regeneration in rats with SCI. Continuous administration of neuritin at this dose at an early stage after SCI inhibited poly ADP ribose polymerase (PARP) protein degradation and decreased neuronal apoptosis. In addition, during the critical postinjury period of axonal regeneration, exogenous neuritin treatment increased the expression of neurofilament 200 and growth-associated protein 43 in the damaged tissue, which was associated with the restoration of hind limb movement. These results suggest that neuritin creates an environment that promotes nerve cell survival and neurite regeneration after SCI, which contribute to nerve regeneration and the recovery of motor function.

Exosomes Derived from miR-126-modified MSCs Promote Angiogenesis and Neurogenesis and Attenuate Apoptosis after Spinal Cord Injury in Rats

Spinal cord injury (SCI) is one of the most common devastating injuries, which causes permanent disabilities such as paralysis and loss of movement or sensation. The precise pathogenic mechanisms of the disease remain unclear, and, as of yet, there is no effective cure. Mesenchymal stem cells (MSCs) show promise as an effective therapy in the experimental models of SCI. MSCs secrete various factors that can modulate a hostile environment, which is called the paracrine effect. Among these paracrine molecules, exosome is considered to be the most valuable therapeutic factor. Thus, exosomes from MSCs (MSCs-exosomes) can be a potential candidate of therapeutic effects of stem cells. The present study was designed to investigate the effect of whether systemic administration of exosomes generated from MSCs can promote the function recovery on the rat model of SCI in vivo. In the present study, we observed that systemic administration of MSCs-exosomes significantly attenuated lesion size and improved functional recovery post-SCI. Additionally, MSCs-exosomes treatment attenuated cellular apoptosis and inflammation in the injured spinal cord. Expression levels of proapoptotic protein (Bcl-2-associated X protein) and proinflammatory cytokines (tumor necrosis factor alpha and interleukin [IL]-1β) were significantly decreased after MSCs-exosomes treatment, whereas expression levels of antiapoptotic (B-cell lymphoma 2) and anti-inflammatory (IL-10) proteins were upregulated. Further, administration of MSCs-exosomes significantly promoted angiogenesis. These results show, for the first time, that systemic administration of MSCs-exosomes attenuated cell apoptosis and inflammation, promoted angiogenesis, and promoted functional recovery post-SCI, suggesting that MSCs-exosomes hold promise as a novel therapeutic strategy for treating SCI.

Mangiferin has antioxidant, antiviral, apoptosis regulating, anti‑inflammatory, antitumor and antidiabetic effects, which can also inhibit osteoclast formation and bone resorption. However, whether mangiferin ameliorates the neurological pain of spinal cord injury (SCI) in ratS remains to be elucidated. The present study investigated the therapeutic effects of mangiferin on neurological function, the water content of spinal cord, oxidative stress, the expression of inflammatory cytokines and the protein expression of Bcl‑2/Bax in a SCI rat model. In the present study, the Basso, Beattie and Bresnahan scores, and the water content of the spinal cord were used to analyze the therapeutic effects of mangiferin on neurological pain in the SCI rat. The concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and the serum levels of glutathione peroxidase (GSH‑PX), nuclear factor‑κB p65 unit, tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and caspase‑3/9 were detected using commercial kits. The expression levels of Bcl‑2 and Bax were measured using western blot analysis. The results demonstrated that administrating mangiferin began to ameliorate neurological function and the water content of the spinal cord in the SCI rat. The mangiferin‑treated group were found to have lower oxidative stress activity and lower expression levels of inflammatory cytokines, compared with the SCI rat. In addition, mangiferin significantly reduced the protein expression of Bax and promoted the protein expression of Bcl-2 in the SCI rat model. Finally, mangiferin markedly suppressed the expression of caspase‑3/9, indicating that the protective action of mangiferin may be associated with anti‑apoptosis activation. In conclusion, mangiferin attenuated contusive SCI in the rats through regulating oxidative stress, inflammation and the Bcl‑2 and Bax pathway.

ObjectiveThe limited ability to regenerate axons after spinal cord injury (SCI) is influenced by factors such as astrocyte activation, reactive proliferation, and glial scar formation. The TGF-β/Smad (transforming growth factor-β/mothers against decapentaplegic homolog) pathway, associated with astrocytic scarring, plays a crucial role in recovery post-injury. This study aims to investigate how icariin (ICA) interacts with reactive astrocytes in the treatment of spinal cord injury.MethodsA rat SCI model was constructed, and the recovery of motor function was observed after treatment with ICA.HE staining, LFB staining, immunofluorescence staining, and Western blotting were employed to assess ICA's ability to inhibit astrocyte proliferation in rats following spinal cord injury by modulating YAP, as well as to evaluate the reparative effects of ICA on the injured spinal cord tissue. Primary astrocytes were isolated and cultured. Immunoprecipitation-Western Blot (IP-WB) ubiquitination and cytoplasm-nuclear separation were employed to assess PPM1B ubiquitination and nuclear translocation.ResultsThe CatWalk XT gait analysis, BBB (Basso, Beattie, and Bresnahan) score, electrophysiological measurements, HE staining, and LFB staining collectively demonstrated that ICA promotes motor function and tissue recovery following spinal cord injury in rats. Immunofluorescence staining and Western Blot analyses revealed that ICA inhibits astrocyte proliferation in rats post-spinal cord injury by suppressing YAP activity. Furthermore, the activation of YAP by XMU-MP-1 was shown to compromise the efficacy of ICA in these rats after spinal cord injury. Additional immunofluorescence staining and Western Blot experiments confirmed that ICA inhibits TGFβ1-induced astrocyte activation through the regulation of YAP. The knockdown of PPM1B (protein phosphatase, Mg2+/Mn2+-dependent 1B) in astrocytes was found to inhibit TGFβ signaling. Additionally, YAP was shown to regulate PPM1B ubiquitination and nuclear translocation through immunoprecipitation-Western blot analysis, along with the segregation of cytoplasm and nucleus.ConclusionIcariin promotes functional recovery in rats after spinal cord injury by inhibiting YAP and regulating PPM1B ubiquitination to inhibiting the activation of reactive astrocytes.