Basso, Beattie And Bresnahan Research Articles

Combination therapy with astaxanthin and epidermal neural crest stem cells improves motor impairments and activates mitochondrial biogenesis in a rat model of spinal cord injury

Spinal cord injury (SCI), a multifactorial disease, can lead to irreversible motor and sensory disabilities. Cell therapy in combination with pharmacological agents can be a promising approach to attenuate SCI damages. Epidermal neural crest stem cells (EPI-NCSCs) extracted from bulge hair follicle in adults are attractive candidates due to the possibility of autologous transplantation. This study evaluated the effect of EPI-NCSCs combined with astaxanthin (Ast), a potent antioxidant, on damages induced by SCI.Male rats were treated with Ast (0.2 mM) and EPI-NCSCs (106/10 μl PBS) alone and combined together after SCI contusion. Motor function was assessed by Basso, Beattie and Bresnahan (BBB) test on days 1, 3, 7, 14, 21, 28, 35 and 42 post-injury. Motor neurons number and myelin level were evaluated on days 14 and 42 using Nissl and Luxol Fast Blue staining. The gene expression of mitochondrial biogenesis involved factors (PGC1α, NRF1 and TFAM) was measured by qPCR.All treatments improved motor function, with the highest BBB score in Ast + Cell compared to Ast and Cell. Decreased motor neurons number and myelin level following SCI, were increased by Ast, Cell and Ast + Cell, but combination therapy significantly had a better effect. We observed reduction in PGC1α, NRF1, and TFAM expression in spinal tissue after SCI, and treatment with Cell and Ast + Cell significantly restored NRF1 and TFAM mRNA levels.These results suggested that Ast in combination with EPI-NCSCs has better effects on behavioral dysfunction, motor neuron loss and demyelination after SCI. These protective effects may be attributed to mitochondrial biogenesis activation.

Diffusion tensor imaging in the evaluation of the long-term efficacy of HBO2 therapy in rats after traumatic spinal cord injury

Objective: This study explored the feasibility of diffusion tensor imaging (DTI) in the evaluation of the long-term efficacy of hyperbaric oxygen (HBO2) therapy in rats after traumatic spinal cord injury (TSCI) with different degrees of injury. Method: Adult Sprague-Dawley rats (total n = 60) were randomly separated into three groups of mild, moderate and severe TSCI (20 rats per group). Each group was then randomly divided into TSCI and TSCI+HBO2 subgroups (10 rats per subgroup). Basso Beattie and Bresnahan (BBB) scores and DTI parameters including fractional anisotropy (FA), mean apparent diffusivity (MD), radial diffusivity (RD) and axial diffusivity (AD) were collected at pre-TSCI and at 0, six and 24 hours, and three, seven, 14, 21, 28 and 56 days post-TSCI. Two-way repeated measures analysis of variance was used for comparison between the TSCI and TSCI+HBO2 subgroups over time in the mild, moderate and severe TSCI groups. Pearson correlation analysis was applied to analyze the correlations between BBB scores and DTI parameters. Results: BBB scores, FA, MD and RD values showed significant differences between the TSCI and TSCI+HBO2 subgroups over time in the mild, moderate and severe TSCI groups (all p<0.01). FA, MD and RD values were positively correlated with BBB scores in all TSCI and TSCI+HBO2 subgroups (all p<0.05). Conclusions: DTI parameters, especially MD, could quantifiably assess the long-term efficacy of HBO2 therapy and reflect the functional recovery in rats after TSCI with different degrees of injury.

Effect of exosomes derived from mir-126-modified mesenchymal stem cells on the repair process of spinal cord injury in rats.

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.

Characterization of transection spinal cord injuries by monitoring somatosensory evoked potentials and motor behavior

Standardization of spinal cord injury (SCI) models is crucial for reproducible injury in research settings and their objective assessments. Basso, Beattie and Bresnahan (BBB) scoring, the traditional behavioral evaluation method, is subjective and susceptible to human error. On the other hand, neuro-electrophysiological monitoring, such as somatosensory evoked potential (SSEP), is an objective assessment method that can be performed continuously for longitudinal studies. We implemented both SSEP and BBB assessments on transection SCI model. Five experimental groups are designed as follows: left hemi-transection at T8, right hemi-transection at T10, double hemi-transection at left T8 and right T10, complete transection at T8 and control group which receives only laminectomy with intact dura and no injury on spinal cord parenchyma. On days 4, 7, 14 and 21 post-injury, first BBB scores in awake and then SSEP signals in anesthetized rats were obtained. Our results show SSEP signals and BBB scores are both closely associated with transection model and injury progression. However, the two assessment modalities demonstrate different sensitivity in measuring injury progression when it comes to late-stage double hemi-transection, complete transection and hemi-transection injury. Furthermore, SSEP amplitudes are found to be distinct in different injury groups and the progress of their attenuation is increasingly rapid with more severe transection injuries. It is evident from our findings that SSEP and BBB methods provide distinctive and valuable information and could be complementary of each other. We propose incorporating both SSEP monitoring and conventional BBB scoring in SCI research to more effectively standardize injury progression.

Evaluation of the Effect of Nandrolone Decanoate on Experimental Spinal Cord Injury in Rats

Background: Acute spinal cord injury, a common cause of neurological dysfunction in humans and animals, impairs motor, sensory and autonomic functions and may result in permanent disability. Nandrolone decanoate (ND) is a steroid widely studied for its predominantly anabolic effect and low androgenic potential. Several researchers have described the positive interference of ND in neurological tissue, such as increased synthesis and release of neurotrophic substances, but to date no studies have evaluated the action of this steroid in acute spinal cord injury. The aim of this study was therefore to evaluate the effect of ND in rats subjected to acute spinal cord injury. Materials, Methods & Results: Thirty-two young adult Wistar rats (Rattus norvegicus), weighing between 240 and 260 g, were divided into three groups. The first group (GNAN) (n=13) was subjected to acute spinal cord injury and treated with ND; the control group (GCON) (n=13) was subjected to spinal cord injury without treatment; and the third group (GLAM) (n=6) underwent laminectomy without prior spinal cord injury, in order to control changes caused by the procedure. A 20 g metal device was released from a height of 25 cm to produce the spinal cord injury. After exposing the spinal canal, a 2-mm diameter metal rod was placed directly in contact with the spinal cord, and when the weight was released, the rod was struck, causing the spinal cord injury. An intramuscular injection of 2 mg/kg of ND was administered the immediate postoperative period. The animals were assessed to ascertain the recovery of their motor function on five occasions, namely at 24 h, 48 h, 72 h, 7 and 14 days after undergoing spinal cord injury. This assessment was performed using the Basso, Beattie and Bresnahan (BBB) model. The animals were euthanized 14 days post-op and fragments of the spinal cord and urinary bladder were collected for histological evaluation. Discussion: The animals subjected to spinal cord injury presented paraplegia, failing to score on the BBB scale in the first three assessments. Starting 7 days after surgery, the GNAN (0-13) and GCON (0-5) groups gradually began showing locomotor improvements, with scale variations. On day 14 after spinal cord injury, 22% of the animals in GNAN and 11% in GCON had failed to recover their locomotor function, scoring zero on the BBB scale. After spinal cord injury, all the animals showed urine retention. The urinary function returned on average on day 5 post surgery, with no significant difference between the groups. The locomotor assessment of the animals subjected to acute spinal cord injury revealed that the injury varied in intensity in GNAN and GCON, with signs of pelvic limb paraplegia and asymmetric non-ambulatory paraparesis. Time was a determining factor in the clinical evolution of the animals, with no evidence of the influence of ND. The histological findings revealed variations in the intensity of the injury, with a tendency for lower intensity in the cranial and epicentral segments of the lesion in the animals subjected to ND treatment, albeit without statistically significant evidence (P ≥ 0.05). The spinal cord assessments of the GLAM group indicated that the surgical procedure did not cause histological alterations, since the normal architecture of the neural tissue was preserved. The histopathological evaluations of the urinary bladder revealed an inflammatory response characterized by lymphohistiocytosis and neutrocytosis in the animals of GNAN and GCON, without interference of ND in the change (P ≥ 0.05). The method to elicit spinal cord injury reproduced functional, sensory and motor incapacity heterogeneously in rats. In the dose evaluated here, ND did not significantly influence the return of locomotor function and the intensity of spinal cord histopathological alterations.

MiR-325-3p promotes locomotor function recovery in rats with spinal cord injury via inhibiting the expression of neutrophil elastase.

This study aims to investigate the potential function of miR-325-3p in vascular integrity and inflammatory response following spinal cord injury (SCI). The protein levels of ANG-1, ANG-2, and caspase-3 in HUVECs incubated with 0, 100, 200, 400, and 800 ng/ml NE for 24 h were determined. The regulatory effect of overexpressed miR-325-3p on the protein levels of ANG-1 and ANG-2 was determined by Western blot. The SCI model in SD rats was established by spinal injury at T10. Subsequently, the relative levels of miR-325-3p, ANG-1, and ANG-2 were determined in SCI rats and controls. Furthermore, SCI rats were administrated with miR-325-3p mimics or negative control and the relative levels of miR-325-3p, ANG-1, and ANG-2 were examined as well. At day 14, the protein levels of iNOS and GFAP in SCI rats and those overexpressing miR-325-3p were detected. BBB (Basso, Beattie, and Bresnahan) locomotor rating scale was applied for evaluating the locomotor function recovery at day 1, 3, 7, 14, 21, and 28 following SCI. NE treatment in HUVECs downregulated ANG-1 and upregulated ANG-2 and caspase-3 in a dose-dependent manner. The overexpression of miR-325-3p upregulated NE-induced decreased the level of ANG-1 and downregulated NE-induced increased level of ANG-2. After the establishment of the SCI model in rats, the miR-325-3p level gradually decreased in SCI rats relative to controls in a time-dependent manner. ANG-1 level in SCI rats decreased to the lowest on the first day following SCI, and gradually increased at day 3, 5, and 7. ANG-2 level was firstly upregulated and achieved the peak on day 3, and then decreased at day 5 and 7. Moreover, SCI rats overexpressing miR-325-3p showed a higher level of ANG-1 and lower level of ANG-2 than those of SCI rats. Overexpression of miR-325-3p downregulated the protein levels of iNOS and GFAP in SCI rats. BBB scale showed elevated locomotor function recovery in SCI rats overexpressing miR-325-3p compared with SCI rats. MiR-325-3p protects the integrity of the vascular wall, reduces infiltration of inflammation, and improves locomotor function recovery at post-SCI.

MiRNA-19a exerts an anti-apoptotic effect in spinal cord injured rats via the PTEN pathway.

Contusion spinal cord injury is involved in a number of cellular, biochemical and molecular changes. We studied the overall expression pattern of miRNAs on day 1 and 3 after spinal cord injury and the involved pathways. A spinal cord injury model was developed by contusion injury in rats. Microarray analysis and qRT-PCR were done for expression of miRs. The Basso, Beattie and Bresnahan (BBB) locomotor score was determined after spinal injury. Lesions at the injured site were analyzed by cresyl staining. Western blot analysis was carried out to analyze protein levels. Immunohistochemical staining was done to evaluate immunoreactivity. TUNEL assay was performed to determine the number of apoptotic cells. The microarray analysis data suggested that about eight miRs were upregulated whereas four were downregulated in rats subjected to spinal cord injury on day 1. On comparing sham operated rats from the day 3 group two miRs were overexpressed and four were downregulated. miR-19a was the most deregulated. miR-19a antagomir was used as an inhibitor, which aggravated the functional deficit, decreased the protection of spinal cord tissue and elevated the number of apoptotic cells. The treatment of miR-19a antagomir increased the expression of FasL along with PTEN, but it failed to affect the levels of PDCD4. The results suggested that miR-19a plays a potential role in halting the neuronal cell death spinal cord injury and that the protective role of miR-19a may be due to its regulatory effect on pro-apoptotic genes.

Therapeutic effect of methylprednisolone combined with high frequency electrotherapy on acute spinal cord injury in rats.

Acute spinal cord injury (SCI) has a high rate of disability and mortality. Although secondary SCI results in local tissue hypoxia and the release of inflammatory mediators, it is both controllable and reversible. Therefore, timely rehabilitation treatment is beneficial for the partial recovery of patients with SCI. The present study aimed to investigate the use of methylprednisolone combined with high-frequency electrotherapy as a method of rehabilitation treatment in rats with SCI. The rat SCI model was prepared using the modified Allen's method with the animals randomly divided into the following 4 groups (n=10 for each group): SCI; methylprednisolone (300 mg/kg); high-frequency electrotherapy; and combination treatment with electrotherapy combined with methylprednisolone (300 mg/kg). The Basso, Beattie and Bresnahan (BBB) score, somatosensory evoked potential (SEP) and motor evoked potential (MEP) were used to assess spinal function. Brain-derived neurotrophic factor (BDNF) and NF-κB expression levels were detected using reverse transcription-quantitative PCR and western blotting. Tumor necrosis factor (TNF)-α and IL-2 expression levels were determined by ELISA, and caspase 3 activity was also assessed. In all treatment groups, BDNF mRNA and protein expression levels were significantly increased, whilst those of NF-κB were reduced. Additionally, an elevated BBB score, improved SEPs and MEPs, inhibited caspase 3 activity and downregulated TNF-α and IL-2 expression levels were observed, compared with the SCI group (P<0.05). However, the combination group exhibited more significant effects on SCI. In conclusion, methylprednisolone combined with high frequency electrotherapy may improve the symptoms of SCI by increasing the expression level of BDNF, reducing that of NF-κB, and suppressing the secretion of inflammatory factors.

Effect of electroacupuncture on urodynamics and expression of Wnt-1, β-catenin, and Ngn1 in the spinal cord in rats with bladder detrusor hyperreflexia due to supersacral spinal cord transection

To investigate the effect of electroacupuncture (EA) at "Dazhui" (GV14) and "Ciliao" (BL32) on rats with bladder detrusor hyperreflexia (DH) after supersacral spinal cord transection, as well as the mechanism of EA in improving the urinary function by regulating the expression of Wnt-1, β-catenin and Neurogenin 1(Ngn1). A total of 48 female Sprague-Dawley rats were randomly divided into sham-operation group, model control group, EA group, and EA control group, with 12 rats in each group. T10 spinal cord transection (SCT) was performed by surgery. The Basso, Beattie and Bresnahan (BBB) score was used to evaluate the motor function of SCT rat, and the Crede technique was used to assist urination. After the urine volume became stable, the urodynamic test was used to determine whether a rat model of DH was successfully established. The rats in the EA group were given EA at GV14 and BL32, and those in the EA control group were given EA (10 Hz/50 Hz, 20 min) at the acupuncture points at 1 cm next to GV14 and BL32 at both sides alternatively. EA was performed once a day for one week. Urodynamic parameters were used to evaluate urinary function. Western blot and immunohistochemistry were used to measure the expression of Wnt-1 and β-catenin in the spinal cord, and immunofluorescence assay was used to measure the expression of Ngn1 in the spinal cord. The BBB score of the model control group significantly decreased compared with that of the sham-operation group(P<0.01), and the EA group was significantly higher than the model control group and the EA control group. Compared with the sham-operation group, the model control group had significant increases in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant reductions in maximum bladder capacity and compliance (P<0.01). Compared with the model control group, the EA group had significant reductions in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant increases in maximum bladder capacity and compliance (P<0.01, P<0.05). Compared with the EA group, the EA control group had significant increases in bladder base pressure, maximum pressure, and leak point pressure (P<0.01) and significant reductions in maximum bladder capacity and compliance (P<0.01, P<0.05). Compared with the sham-operation group, the model control group had significant increases in the protein expression of Wnt-1 and β-catenin (P<0.05, P<0.01) and a signi-ficant reduction in the protein expression of Ngn1 in the spinal cord (P<0.01). Compared with the model control group, the EA group had significant increases in the protein expression of Wnt-1, β-catenin and Ngn1 in the spinal cord (P<0.01). Compared with the EA group, the EA control group had significant reductions in the protein expression of Wnt-1, β-catenin, and Ngn1 in the spinal cord (P<0.01). EA at GV14 and BL32 can significantly improve urinary function in rats with bladder DH due to SCT, partially by activating the Wnt/β-catenin signaling pathway and promoting the protein expression of Wnt-1, β-catenin and Ngn1.

Trehalose protects against spinal cord injury through regulating heat shock proteins 27 and 70 and caspase-3 genes expression

Background Heat shock proteins (HSPs) are a class of highly conserved proteins responsible for various functions critical to cell survival. Pharmacological induction of HSPs has been implicated in the regulation of neuronal loss and functional deficits in peripheral and central nervous system injuries. Accordingly, the present study was conducted to investigate the effect of trehalose on spinal expression of HSP27, HSP70 and caspase-3 genes following traumatic spinal cord injury (SCI) in rats. Methods Male rats weighing 250-300 g underwent laminectomy and were divided into four groups including sham, SCI (received SCI), vehicle (received SCI and phosphate buffer saline intrathecally) and trehalose (received 10 mM trehalose intrathecally following SCI). On days 1, 3 and 7 after injury, HSP27, HSP70 and caspase-3 genes transcripts were quantified in spinal cord tissues via a real-time PCR technique. In addition, locomotor function was assessed using the Basso, Beattie and Bresnahan (BBB) rating scale. Results SCI induced the expression of HSP27, HSP70 and caspase-3 genes and BBB score at all time points. Trehalose treatment upregulated HSP27, HSP70 genes expression at 1 day after SCI. Interestingly, a significant reduction in the expression of HSP27 and HSP70 genes was observed on days 3 and 7 following trauma compared with the vehicle group (p < 0.01). Caspase-3 gene showed a decrease in expression in the trehalose-treated group at all times. In addition, neurological function revealed an improvement after treatment with trehalose. Conclusion This study suggests that the neuroprotective effect of trehalose is mediated via regulation of HSP27 and HSP70, which are involved in cytoprotection and functional recovery following SCI.

Adenovirus vector‑mediated invivo gene transfer of nuclear factor erythroid‑2p45‑related factor 2 promotes functional recovery following spinal cord contusion.

The aim of the present study was to investigate whether nuclear factor erythroid 2p45‑related factor2(Nrf2) overexpression by gene transfer may protect neurons/glial cells and the association between neurons/glial cells and axons in spinal cord injury (SCI). In the present study, Nrf2 recombinant adenovirus (Ad) vectors were constructed. The protein levels of Nrf2 in the nucleus and of the Nrf2‑regulated gene products heme oxygenase‑1 (HO‑1) and NAD (P)H‑quinone oxidoreductase‑1 (NQO1), were detected using western blot analysis in PC12 cells following 48 h of transfection. Furthermore, the expression of Nrf2 was localized using an immunofluorescence experiment, and the expression of Nrf2, HO‑1 and NQO1 were detected using an immunohistochemical experiment in the grey matter of spinal cord in rats. Post‑injury motor behavior was assessed via the Basso, Beattie and Bresnahan (BBB) locomotor scale method. In PC12 cells, subsequent to Ad‑Nrf2 transfection, nuclear Nrf2, HO‑1 and NQO1 levels were significantly increased compared with the control (P<0.01). There was statistically significant changes in the PC12‑Ad‑Nrf2 group [Nrf2 (1.146±0.095), HO‑1 (1.816±0.095) and NQO1 (1.421±0.138)] compared with the PC12‑control group [Nrf2 (0.717±0.055), HO‑1 (1.264±0.081) and NQO1 (0.921±0.088)] and PC12‑Ad‑green fluorescent protein group [Nrf2 (0.714±0.111), HO‑1 (1.238±0.053) and NQO1 (0.987±0.045); P<0.01]. The BBB scores of the rats indicated that they had improved functional recovery following the local injection of Ad‑Nrf2. On the third day following the operation, BBB scores in the adenovirus groups (0.167±0.408) were significantly decreased compared with the SCI group (1±0.894; P<0.05). In the injured section of the spinal cord in the rats, the number of positive cells expressing Nrf2, HO‑1 and NQO1 were raised compared with the control and SCI groups, indicating that the adenovirus vector‑mediated gene transfer of Nrf2 promotes functional recovery following spinal cord contusion in rats.

EXPERIMENTAL STUDY OF THE NEUROTOXIC EFFECTS OF PHOTODYNAMIC THERAPY ON THE SPINAL CORD

ABSTRACT Objective To evaluate the effects of photodynamic therapy (PDT) on the dura mater using the photosensitizers aluminum chloride phthalocyanine and methylene blue in in vivo assays. Methods Fifty-six male Wistar rats were divided into two groups; one submitted to PDT and the other submitted to the photosensitizers without their photoactivation (control). The photosensitizers were applied to the dura mater after laminectomy at the T10 level. The methods used for assessment were the Basso, Beattie and Bresnahan (BBB) functional evaluation scale and study of the dura mater by light microscopy. Results No changes in motor activity were observed in the animals submitted to PDT compared to control. Histological and pathological evaluation did not show any differences between the group exposed to activated photosensitizers and the control group with regard to the inflammatory process and tissue necrosis. Conclusion The joint use of PDT with the photosensitizing pharmaceuticals aluminum chloride phthalocyanine and methylene blue did not induce any clinical neurotoxic effects or histological changes in the dura mater of the animals studied. Level de evidence V; Expert Opnion.

Curcumin promotes functional recovery and inhibits neuronal apoptosis after spinal cord injury through the modulation of autophagy

Objective: The study was aimed to investigate whether the neuroprotective role of curcumin is associated with regulation of autophagy. Methods: Rat spinal cord injury (SCI) models were established according to Allen’s weight-drop trauma method. Curcumin was administered 30 min after the contusion and continued weekly. At 3, 7, 14, 21, and 28 days after SCI, functional recovery was evaluated using the Basso, Beattie and Bresnahan (BBB) scoring and the oblique plate test, following which, spinal cord tissues were obtained. Histological changes and apoptosis were then measured with H&E staining and TUNEL assay. Glia activation, inflammatory infiltration, inflammatory factor release, and myelination were observed through immunohistochemical (IHC) staining and ELISA. Autophagy and Akt activation were detected by western blotting. After autophagy was inhibited by injection of chloroquine, TUNEL, inflammatory factor release, myelin basic protein (MBP) IHC staining and functional recovery evaluation were performed again. Results: Curcumin treatment promoted functional recovery after SCI and reduced neuron apoptosis, improved spinal cord integrity, recovery, and re-myelination, and suppressed the inflammatory response. Autophagy was enhanced and Akt/mTOR pathway was inhibited by curcumin. Autophagy inhibition partially eliminated the protective effect of curcumin on SCI. Conclusion: Curcumin may exert its therapeutic effect on SCI through the enhancement of autophagy, in which, inhibition of the Akt/mTOR signaling pathway may be also involved.

The Effect of Prophylactic Hypothermia on Neurophysiological and Functional Measures in the Setting of Iatrogenic Spinal Cord Impact Injury

Iatrogenic spinal cord injury (iSCI) during spinal corrective surgery can result in devastating complications, such as paraplegia or paraparesis. Perioperatively, iSCI often occurs as a direct injury during spinal cord instrumentation placement. Currently, treatment of iSCI remains limited to posttraumatic hypothermia, which has demonstrated some value in recent clinical trials. Here we report the outcomes of preinjury hypothermia initiated preprocedurally and maintained for a considerable time after iSCI. Twenty-six female Sprague-Dawley rats were assigned at random to either a normothermic group (36 °C) or a hypothermic group (32 °C) and then underwent a laminectomy procedure at the T8 level. Each group was further divided at random to receive a 200-kdyn force contusive spinal cord injury or a sham impact. Hypothermic rats were then rewarmed after 2 hours of hypothermic treatment. Behavioral scores, temperature profiles, weights, and somatosensory evoked potentials were obtained at baseline and at specified time points after the procedure. The median survival was 42 days for the iSCI hypothermic group and 11 days for the iSCI normothermic group (hazard ratio, 3.82; 95% confidence interval, 1.52-9.57). The probability of survival was significantly higher in the iSCI hypothermic group compared with the iSCI normothermic group (χ2= 4.18; P= 0.040). The hypothermic group exhibited a higher Basso, Beattie and Bresnahan (BBB) locomotor rating scale score (17 vs. 14; P < 0.01), lower normalized latencies (1.06 ± 0.16 seconds vs. 1.34 ± 0.17 seconds; P= 0.04), and higher peak-to-peak amplitudes (0.32 ± 0.10 μV vs. 0.12 ± 0.09 μV; P= 0.005). The use of prophylactic hypothermia before iSCI was significantly associated with an increased survival rate, higher BBB scores, and improved neurophysiological measures.

Co-Transplantation of Human Umbilical Cord Mesenchymal Stem Cells and Human Neural Stem Cells Improves the Outcome in Rats with Spinal Cord Injury

Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) are promising graft materials for cell therapies in spinal cord injury (SCI) models. Previous studies have demonstrated that MSCs can regulate the microenvironment of NSCs and promote their survival rate. Furthermore, several studies indicate that MSCs can reduce stem cell transplantation-linked tumor formation. To our knowledge, no previous studies have determined whether co-transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and human neural stem cells (hNSCs) could improve the outcome in rats with SCI. Therefore, we investigated whether the transplantation of hUC-MSCs combined with hNSCs through an intramedullary injection can improve the outcome of rats with SCI, and explored the underlying mechanisms. In this study, a moderate spinal cord contusion model was established in adult female Wistar rats using an NYU impactor. In total, 108 spinal cord-injured rats were randomly selected and divided into the following five groups: 1) hUC-MSCs group, 2) hNSCs group, 3) hUC-MSCs+hNSCs group, 4) PBS (control) group, and 5) a Sham group. Basso, Beattie and Bresnahan (BBB) behavioral test scores were used to evaluate the motor function of all animals before and after the SCI weekly through the 8th week. Two weeks after transplantation, some rats were sacrificed, immunofluorescence and immunohistochemistry were performed to evaluate the survival and differentiation of the transplanted stem cells, and brain-derived neurotrophic factor (BDNF) was detected by ELISA in the injured spinal cords. At the end of the experiment, we evaluated the remaining myelin sheath and anterior horn neurons in the injured spinal cords using Luxol Fast Blue (LFB) staining. Our results demonstrated that the surviving stem cells in the hUC-MSCs+hNSCs group were significantly increased compared with those in the hUC-MSCs alone and the hNSCs alone groups 2 weeks post-transplantation. Furthermore, the results of the BBB scores and the remaining myelin sheath evaluated via LFB staining in the injured spinal cords demonstrated that the most significantly improved outcome occurred in the hUC-MSCs+hNSCs group. The hUC-MSCs alone and the hNSCs alone groups also had a better outcome compared with that of the PBS-treated group. In conclusion, the present study demonstrates that local intramedullary subacute transplantation of hUC-MSCs, hNSCs, or hUC-MSCs+hNSCs significantly improves the outcome in an in vivo moderate contusion SCI model, and that co-transplantation of hUC-MSCs and hNSCs displayed the best outcome in our experiment.

Reduction of Inflammation and Enhancement of Motility after Pancreatic Islet Derived Stem Cell Transplantation Following Spinal Cord Injury.

ObjectiveSpinal cord injury (SCI) is a very serious health problem, usually caused by a trauma and accompanied by elevated levels of inflammation indicators. Stem cell-based therapy is promising some valuable strategies for its functional recovery. Nestin-positive progenitor and/or stem cells (SC) isolated from pancreatic islets (PI) show mesenchymal stem cell (MSC) characteristics. For this reason, we aimed to analyze the effects of rat pancreatic islet derived stem cell (rPI-SC) delivery on functional recovery, as well as the levels of inflammation factors following SCI. MethodsrPI-SCs were isolated, cultured and their MSC characteristics were determined through flow cytometry and immunofluorescence analysis. The experimental rat population was divided into three groups : 1) laminectomy & trauma, 2) laminectomy & trauma & phosphate-buffered saline (PBS), and 3) laminectomy+trauma+SCs. Green fluorescent protein (GFP) labelled rPI-SCs were transplanted into the injured rat spinal cord. Their motilities were evaluated with Basso, Beattie and Bresnahan (BBB) Score. After 4-weeks, spinal cord sections were analyzed for GFP labeled SCs and stained for vimentin, S100β, brain derived neurotrophic factor (BDNF), 2’,3’-cyclic-nucleotide 3'-phosphodiesterase (CNPase), vascular endothelial growth factor (VEGF) and proinflammatory (interleukin [IL]-6, transforming growth factor [TGF]-β, macrophage inflammatory protein [MIP]-2, myeloperoxidase [MPO]) and anti-inflammatory (IL-1 receptor antagonis) factors. ResultsrPI-SCs were revealed to display MSC characteristics and express neural and glial cell markers including BDNF, glial fibrillary acidic protein (GFAP), fibronectin, microtubule associated protein-2a,b (MAP2a,b), β3-tubulin and nestin as well as antiinflammatory prostaglandin E2 receptor, EP3. The BBB scores showed significant motor recovery in group 3. GFP-labelled cells were localized on the injury site. In addition, decreased proinflammatory factor levels and increased intensity of anti-inflammatory factors were determined. ConclusionTransplantation of PI-SCs might be an effective strategy to improve functional recovery following spinal cord trauma.

Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury

To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P<0.001), but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group (P>0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P<0.05). In contrast to the model group, the injured severity of the spinal cord AH region was relatively milder, and the expression levels of NR2B mRNA and protein were considerably down-regulated in both EA and MP groups (P<0.05). However, there were no significant differences between the EA and MP groups in the expression levels of NR2B mRNA and protein (P>0.05). EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

MicroRNA-30c abrogation protects against spinal cord ischemia reperfusion injury through modulating SIRT1

Spinal cord ischemia/reperfusion (I/R) injury is a severe complication in many surgeries. Although microRNAs (miRNAs) are involved in I/R-caused spinal cord injury (SCI), the mechanism that underlies miR-30c interacted with SCI remains elusive. In this study, I/R surgery or oxygen-glucose deprivation (OGD) were performed to establish SCI model in vivo or in vitro, respectively. Basso, Beattie and Bresnahan (BBB) score, spinal cord infarct, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining, flow cytometry and enzyme linked immunosorbent assays (ELISA) were used to investigate SCI. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the abundances of miR-30c and sirtuin 1 (SIRT1) either in spinal cord or PC12 cells. Luciferase assay and RNA immunoprecipitation (RIP) were performed to probe the interaction between miR-30c and SIRT1. Western blot and immunofluorescence assays were used to analyze SIRT1 protein expression. Our results showed that I/R increased miR-30c expression and induced SCI, revealed by decreasing BBB score, enhancing apoptosis, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression. However, miR-30c knockdown attenuated I/R-induced SCI in vivo. Moreover, depletion of miR-30c protected PC12 cells against OGD-caused apoptosis and inflammatory response. In addition, SIRT1 was limited by miR-30c, silencing of which reversed anti-miR-30c-mediated inhibitory effect on apoptosis and secretion of inflammatory cytokines in PC12 cells after OGD treatment. Collectively, abrogation of miR-30c inhibited spinal cord ischemia reperfusion injury through targeting SIRT1, providing a promising biomarker of prognosis and therapeutic for SCI.

Effect of electroacupuncture with different frequencies on hindlimb locomotor function and expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in spinal cord injury rats

To investigate the effect of different frequencies of electroacupuncture (EA) on motor function and expression of autophagy-related proteins microtubule-associated protein light chain 3 (LC3), etc. in spinal cord injury (SCI) rats, so as to reveal its underlying mechanisms and provide a reference for clinical application. A total of 100 male SD rats were randomly divided into sham control, model, 2 Hz-EA, 100 Hz-EA and 2 Hz/100 Hz-EA groups(n=20 in each group). EA (2 Hz, 100 Hz or 2 Hz/100 Hz, 0.4-0.5-0.6 mA increased by 0.1 mA every10 min) was applied to "HuatuoJiaji" (EX-HN1, T9 and T11) for 30 min, once a day for 7 days. The rat's hindlimb motor function was assessed by using Basso, Beattie and Bresnahan(BBB) locomotor rating scale, inclined plane test and plantar imprinting test, separately. The histological changes and neuronal apoptosis of the spinal cord tissue were observed by Fluoro-Jade B (FJB) and Nissl staining, respectively. The expression of LC3, Beclin 1 and cleaved Caspase-3 proteins in the spinal cord was detected by Western blot. After modeling, the BBB scores and the angles of inclined plane on day 1, 3 and 7 were significantly decreased (P<0.001), and the number of FJB positive cells, and the expression levels of Beclin 1 and cleaved Caspase-3 proteins were considerably increased in the model group compared with the sham control group (P<0.001, P<0.05). After EA intervention, the BBB score and the angles of inclined plate on day 3 in the 2 Hz/100 Hz-EA group (rather than in the 2 Hz- and 100 Hz-EA groups), the BBB score and the angles of inclined plate on day 7 in both 2 Hz/100 Hz and 100 Hz-EA groups(rather than in the 2 Hz-EA group), and the expression levels of Beclin 1 and LC3-Ⅱ/Ⅰ in both 2 Hz/100 Hz and 100 Hz-EA groups (rather than in the 2 Hz-EA group) on day 7 were obviously increased (P<0.001, P<0.01, P<0.05), while the number of FJB-positive neurons in the 3 EA groups, and the expression of cleaved Caspase-3 protein in both 2 Hz/100 Hz and 100 Hz-EA groups and the LC3-Ⅱ/Ⅰ in the 2 Hz-EA group were obviously decreased relevant to the model group (P<0.001, P<0.05). The expression of Beclin 1 in the 100 Hz-EA group was obviously decreased relevant to the 2 Hz/100 Hz-EA group (P<0.05) .Nissl staining displayed appearance of cavities and fuzzy shape of Nissl bodies with light coloration in the injured spinal cord of model group, which was milder in both 2 Hz/100 Hz-EA and 100 Hz-EA groups. Plantar imprinting tests showed dragging gait prints in the model group due to disability in movement, and relatively distinct foot imprints in both 2 Hz/100 Hz and 100 Hz-EA groups. Both 100 Hz and 2 Hz/100 Hz-EA can effectively promote the recovery of hindlimb locomotor function of SCI rats, which may be related to its function in promoting autophagy of damaged nerve cells and in reducing neuronal apoptosis.

Cyclic adenosine phosphate improves functional recovery after spinal cord injury via activating unfolded protein response.

The current study aimed to evaluate the role and underlying mechanism of cyclic adenosine phosphate (cAMP) on the functional recovery of spinal cord injury (SCI). Basso, Beattie and Bresnahan (BBB) scoring and inclined plane test indicated that cAMP treatment improved the functional recovery of SCI rats. Real time PCR and western blot analysis showed the mRNA and protein levels of IRE1, PERK, and ATF6 were increased in the SCI rats than those of sham control. However, higher levels of IRE1, PERK, and ATF6 were indicated after cAMP treatment. Meanwhile, more apoptotic cells were observed in the SCI rats, as evidenced by TUNEL staining and increased expression of GRP78, CHOP, and caspase12. In contrast, the expression of GRP78, CHOP, and caspase12 was decreased in SCI rats after cAMP treatment. In summary, we showed novel data that cAMP reduced cell apoptosis and functional recover after SCI mainly via activating UPR.