Functional Recovery Of Hind Limbs Research Articles

Therapeutic mechanism of basic fibroblast growth factor on spinal cord injury in rats based on the Notch/signal transducer and activator of transcription 3 signaling pathway

To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T 10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T 10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group ( P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group ( P<0.05); the bFGF group showed an increase compared to model group ( P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant ( P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group ( P<0.05). The above indexes in bFGF group significantly improved when compared with model group ( P<0.05). bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.

MiR-219 overexpressing oligodendrocyte progenitor cells for treating compression spinal cord injury.

Oligodendrocyte progenitor cells (OPCs) transplantation has been considered a promising treatment for spinal cord injury, according to previous studies. Recent research shed light on the importance of microRNA 219 (miR-219) in oligodendrocyte development, so here miR-219-overexpressing OPCs (miR-219 OPCs) were transplanted in animal models of spinal cord injury to evaluate the impact of miR-219 on oligodendrocyte differentiation and functional recovery in vivo. Our findings demonstrate that transplanted cells were distributed in the tissue sections and contributed to reducing the size of cavity in the injury site. Interestingly, miR-219 promoted OPC differentiation into mature oligodendrocyte expressing MBP in vivo whereas in absence of miR-219, less number of cells differentiated into mature oligodendrocytes. An eight week evaluation using the Basso Beattie Bresnahan (BBB) locomotor test confirmed improvement in functional recovery of hind limbs. Overall, this study demonstrated that miR-219 promoted differentiation and maturation of OPCs after transplantation and can be used in cell therapy of spinal cord injury.

Effects of Edaravone on Functional Recovery of a Rat Model with Spinal Cord Injury Through Induced Differentiation of Bone Marrow Mesenchymal Stem Cells into Neuron-Like Cells.

Edaravone can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells and replace lost cells by transplanting neuron-like cells to repair spinal cord injury (SCI). In this study, BMSCs were derived from the bone marrow of male Wistar rats (4 weeks old) through density gradient centrifugation (1.073 g/mL), and the cell purity of BMSCs was up to 95%. The combined injection of basic fibroblast growth factor and edaravone was conducted to differentiate BMSCs into neuron-like cells. In this study, 120 male Wistar rats were used to establish the model of semitransverse SCI; on the seventh day, neuron-like cells were labeled by BrdU and then injected into the epicenter of the injury of rats. On the 14th day after cell transplantation, the biotin dextran amine (BDA) fluorescent agent was used to track the repair of nerve damage. At 7, 14, 21, and 30 days after SCI, the Basso, Beattie, and Bresnahan (BBB) locomotor scale method was used to measure the functional recovery of hind limbs in rats. Additionally, hematoxylin and eosin (H&E) staining, Nissl staining, immunohistochemistry, transmission electron microscopy (TEM), Western blotting, and Real-time quantitative reverse transcripion PCR (qRT-PCR) were used to observe the regeneration of nerve cells. In the edaravone+BMSC group, behavioral analysis of locomotor function showed that functional recovery was significantly enhanced after transplantation of the cells, BrdU-positive cells could be observed scattered in the injured area and extended to both the head and tail, and the BDA tracer shows that the edaravone+BMSC group emits more fluorescent signals. Additionally, H&E staining, Nissl staining, and immunohistochemistry revealed that the space of spinal cord tissue was attenuated and the neurons were increased. Western blotting and qRT-PCR showed that the expression levels of neuron-specific enolase (NSE), Nestin, and neurofilament 200 (NF) were increased, while the expression of glial fibrillary acidic protein (GFAP) was decreased. TEM showed that cytoplasmic edema was reduced, mitochondrial vacuoles were attenuated, and nuclear chromatin concentration was declined after transplantation of neuron-like cells. Moreover, with the extension of time of edaravone+BMSC transplantation, the structures of mitochondria and endoplasmic reticulum tended to be normal. In summary, the induced differentiation of BMSC transplantation can significantly promote the functional repair of SCI.

Olfactory ensheathing cells seeded decellularized scaffold promotes axonal regeneration in spinal cord injury rats.

Spinal cord decellularized (DC) scaffolds can promote axonal regeneration and restore hindlimb motor function of spinal cord defect rats. However, scarring caused by damage to the astrocytes at the margin of injury can hinder axon regeneration. Olfactory ensheathing cells (OECs) integrate and migrate with astrocytes at the site of spinal cord injury, providing a bridge for axons to penetrate the scars and grow into lesion cores. The purpose of this study was to evaluate whether DC scaffolds carrying OECs could better promote axon growth. For these studies, DC scaffolds were cocultured with primary extracted and purified OECs. Immunofluorescence and electron microscopy were used for verification of cells adhere and growth on the scaffold. Scaffolds with OECs were transplanted into rat spinal cord defects to evaluate axon regeneration and functional recovery of hind limbs. Basso, Beattie, and Bresnahan (BBB) scoring was used to assess motor function recovery, and glial fibrillary acidic protein (GFAP) and NF200-stained tissue sections were used to evaluate axonal regeneration and astrological scar distribution. Our results indicated that spinal cord DC scaffolds have good histocompatibility and spatial structure, and can promote the proliferation of adherent OECs. In animal experiments, scaffolds carrying OECs have better axon regeneration promoting protein expression than the SCI model, and improve the proliferation and distribution of astrocytes at the site of injury. These results proved that the spinal cord DC scaffold with OECs can promote axon regeneration at the site of injury, providing a new basis for clinical application.

Review of the Current Knowledge on the Role of Stem Cell Transplantation in Neurorehabilitation.

The management involving stem cell (SC) therapy along with physiotherapy offers tremendous chance for patients after spinal cord injury (SCI), traumatic brain injury (TBI), stroke, etc. However, there are still only a limited number of reports assessing the impact of stem cells (SCs) on the rehabilitation process and/or the results of the simultaneous use of SC and rehabilitation. Additionally, since there is still not enough convincing evidence about the effect of SCT on humans, e.g., in stroke, there have been no studies conducted concerning rehabilitation program formation and expected outcomes. It has been shown that bone marrow-derived mesenchymal stem cell (BMSCs) transplantation in rats combined with hyperbaric oxygen therapy (HBO) can promote the functional recovery of hind limbs after SCI. An anti-inflammatory effect has been shown. One case study showed that, after the simultaneous use of SCT and rehabilitation, an SCI patient progressed from ASIA Grade A to ASIA Grade C. Such promising data in the case of complete tetraplegia could be a breakthrough in the treatment of neurologic disorders in humans. Although SCT appears as a promising method for the treatment of neurological conditions, e.g., complete tetraplegia, much work should be done towards the development of rehabilitation protocols.

Reconditioning effect of oligodendrocyte-like cell transplantation in acute spinal cord injury in rats

To observe the reconditioning effect of oligodendrocyte-like cell transplantation in acute spinal cord injury in rats, bone marrow mesenchymal stem cells were differentiated into oligodendrocyte-like cells induced by cytokines including insulin-like growth factor-1(IGF-1). Rats with acute spinal cord injury were treated with the oligodendrocyte-like cells for 8 weeks by local injection. The therapeutic effect was compared with oligodendrocytes transplantation and saline. Regeneration of axon myelin sheath and functional recovery of hind limbs were significantly improved after transplantation with oligodendrocyte-like cells than that with saline. But there was no significant difference between rats treated with oligodendrocyte-like cells and oligodendrocytes. Bone marrow mesenchymal stem cells differentiating into oligodendrocyte-like cells can be induced by cytokines including IGF-1, with similar specific surface markers of oligodendrocytes. These cells can survive in injury sites of the spinal cord for a long duration after transplantation to improve recovery of neural function and reduce cavity area at injury site after acute spinal injury in rats. The possible mechanism may be regeneration of axon myelin sheath, which is helpful for reconstruction of neural reflex function. Key words: Oligodendrocyte-like cells, differentiation, spinal cord injury, transplantation.

Cholecalciferol (vitamin D₃) improves myelination and recovery after nerve injury.

Previously, we demonstrated i) that ergocalciferol (vitamin D2) increases axon diameter and potentiates nerve regeneration in a rat model of transected peripheral nerve and ii) that cholecalciferol (vitamin D3) improves breathing and hyper-reflexia in a rat model of paraplegia. However, before bringing this molecule to the clinic, it was of prime importance i) to assess which form – ergocalciferol versus cholecalciferol – and which dose were the most efficient and ii) to identify the molecular pathways activated by this pleiotropic molecule. The rat left peroneal nerve was cut out on a length of 10 mm and autografted in an inverted position. Animals were treated with either cholecalciferol or ergocalciferol, at the dose of 100 or 500 IU/kg/day, or excipient (Vehicle), and compared to unlesioned rats (Control). Functional recovery of hindlimb was measured weekly, during 12 weeks, using the peroneal functional index. Ventilatory, motor and sensitive responses of the regenerated axons were recorded and histological analysis was performed. In parallel, to identify the genes regulated by vitamin D in dorsal root ganglia and/or Schwann cells, we performed an in vitro transcriptome study. We observed that cholecalciferol is more efficient than ergocalciferol and, when delivered at a high dose (500 IU/kg/day), cholecalciferol induces a significant locomotor and electrophysiological recovery. We also demonstrated that cholecalciferol increases i) the number of preserved or newly formed axons in the proximal end, ii) the mean axon diameter in the distal end, and iii) neurite myelination in both distal and proximal ends. Finally, we found a modified expression of several genes involved in axogenesis and myelination, after 24 hours of vitamin supplementation. Our study is the first to demonstrate that vitamin D acts on myelination via the activation of several myelin-associated genes. It paves the way for future randomised controlled clinical trials for peripheral nerve or spinal cord repair.

Immediate physical therapy in dogs with rupture of the cranial cruciate ligament submitted to extracapsular surgical stabilization

The study evaluated the influence of immediate physical therapy on the functional recovery of hind limbs of dogs with experimental cranial cruciate ligament rupture which underwent surgical extracapsular stabilization as well as to verify its interference in joint stability. Eight dogs were randomly divided into two groups: GI (control) (n=4) and GII (physical therapy) (n=4). The dogs in GII underwent the following therapeutic treatments in the postoperative period: cryotherapy, passive joint movement, massage, passive straightening, neuromuscular electrical stimulation, hydrotherapy (bath and aquatic mat) and therapeutic exercises. We performed evaluations of the thigh circumference, goniometry, X-ray, and knee stability (drawer test). Results did not demonstrate a significant difference between the groups nor between different post-operative times. Regarding gait analysis, we found that the 4 dogs in GI remained in degree 3 of lameness 45 and 90 days postoperatively. However, in GII, one dog remained in degree 3 45 and 90 days after surgery; one dog changed from degree 3 to 4 90 days after surgery and the other 2 dogs changed from degree 3 to 5 90 days after surgery. It is possible to conclude that dogs with CCL rupture that undergo immediate physical therapy demonstrate better results in regards to functional gait recovery. The therapeutic modalities used in the immediate post-operative period did not cause instability of the operated knee. Further studies are needed with a larger number of dogs to indicate the immediate physical therapy in dogs with CCL ligament rupture which underwent extracapsular stabilization.

Lentivirus-siNgR199 Promotes Axonal Regeneration and Functional Recovery in Rats

ABSTRACTBackground: The limited regenerative capability of central neurons and inhibitory factors are the main causes of axonal regeneration failure after spinal cord injury (SCI). Nogo receptors (NgR) are a family of receptors shared by three factors that inhibit axon outgrowth. In a previous study, siNgR199, an effective lentiviral siRNA vector of NgR1, was constructed and transfected into cortical neurons in vitro, and it effectively promoted axon outgrowth. The present study focused on the therapeutic effect of delivery of a recombinant lentivirus containing siNgR199 in vivo in rats. Methods: Rat models of traumatic SCI were constructed according to the method developed by Allen. The animals were randomly divided into three groups: group 1, injected with physiological saline; group 2, injected with empty lentivirus vehicle; and group 3, injected with lentivirus carrying siNgR199. The Basso, Beattie, and Bresnahan (BBB) scale was used for assessing hindlimb locomotor function after SCI. The neural tracer biotinylated dextran amine (BDA) and immunohistochemical methods were employed to study axon outgrowth. Results: After injection for 8 weeks, BBB locomotion scores showed that the motor function of the hindlimb recovered better in animals in group 3 (injected with lentivirus carrying siNgR199) than those in groups 1 and 2, which were injected with saline and empty lentivirus vehicle, respectively. In group 3, regenerative nerve fibers were observed at and across the injury site, while very few axonal sprouts were observed in groups 1 and 2. Conclusions: Injection with lentivirus carrying siNgR199 into the sensorimotor cortex improved axonal regeneration and functional recovery of hindlimb after SCI in rats.

Vitamin D2Potentiates Axon Regeneration

To date, the use of autograft tissue remains the "gold standard" technique for repairing transected peripheral nerves. However, the recovery is suboptimal, and neuroactive molecules are required. In the current study, we focused our attention on vitamin D, an FDA-approved molecule whose neuroprotective and neurotrophic actions are increasingly recognized. We assessed the therapeutic potential of ergocalciferol--the plant-derived form of vitamin D, named vitamin D2--in a rat model of peripheral nerve injury and repair. The left peroneal nerve was cut out on a length of 10 mm and immediately autografted in an inverted position. After surgery, animals were treated with ergocalciferol (100 IU/kg/day) and compared to untreated animals. Functional recovery of hindlimb was measured weekly, during 10 weeks post-surgery, using a walking track apparatus and a numerical camcorder. At the end of this period, motor and sensitive responses of the regenerated axons were calculated and histological analysis was performed. We observed that vitamin D2 significantly (i) increased axogenesis and axon diameter; (ii) improved the responses of sensory neurons to metabolites such as KCl and lactic acid; and (iii) induced a fast-to-slow fiber type transition of the Tibialis anterior muscle. In addition, functional recovery was not impaired by vitamin D supplementation. Altogether, these data indicate that vitamin D potentiates axon regeneration. Pharmacological studies with various concentrations of the two forms of vitamin D (ergocalciferol vs. cholecalciferol) are now required before recommending this molecule as a potential supplemental therapeutic approach following nerve injury.

Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins alpha-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.

Repetitive transcranial magnetic stimulation improves open field locomotor recovery after low but not high thoracic spinal cord compression-injury in adult rats.

Electromagnetic fields are able to promote axonal regeneration in vitro and in vivo. Repetitive transcranial magnetic stimulation (rTMS) is used routinely in neuropsychiatric conditions and as an atraumatic method to activate descending motor pathways. After spinal cord injury, these pathways are disconnected from the spinal locomotor generator, resulting in most of the functional deficit. We have applied daily 10 Hz rTMS for 8 weeks immediately after an incomplete high (T4-5; n = 5) or low (T10-11; n = 6) thoracic closed spinal cord compression-injury in adult rats, using 6 high- and 6 low-lesioned non-stimulated animals as controls. Functional recovery of hindlimbs was assessed using the BBB locomotor rating scale. In the control group, the BBB score was significantly better from the 7th week post-injury in animals lesioned at T4-5 compared to those lesioned at T10-11. rTMS significantly improved locomotor recovery in T10-11-injured rats, but not in rats with a high thoracic injury. In rTMS-treated rats, there was significant positive correlation between final BBB score and grey matter density of serotonergic fibres in the spinal segment just caudal to the lesion. We propose that low thoracic lesions produce a greater functional deficit because they interfere with the locomotor centre and that rTMS is beneficial in such lesions because it activates this central pattern generator, presumably via descending serotonin pathways. The benefits of rTMS shown here suggest strongly that this non-invasive intervention strategy merits consideration for clinical trials in human paraplegics with low spinal cord lesions.