In order to observe the role of genetically modified Schwann cell (SC) with pSVP0Mcat in the regeneration of injured spinal cord, the cells were implanted into the spinal cord. Ninety SD rats were used to establish a model of hemi-transection of spinal cord at the level of T8, and were divided into three groups, randomly, that is, pSVP0Mcat modified SC implantation (Group A), SC implantation (Group B) and without cell implantation as control (Group C). After three months the presence of axonal regeneration of the injured spinal cord was examined by means of horseradish peroxidase (HRP) retrograde labelling technique and stereography. The results indicated that HRP labelled cells in Group A and B could be found in the superior region of injured spinal cord and the brain stem such as the red nuclei and oculomotor nuclei. The density of ventral hom neurons of the spinal cord and the number of myelinated axons in 100 microns of the white matter was A gt; B gt; C group. In brief, the pSVP0Mcat modified SC intraspinal implantation could promote regeneration of the injured spinal cord.
ObjectiveTo study the local vascular remodeling, inflammatory response, and their correlations following acute spinal cord injury (SCI) with different grades, and to assess the histological changes in SCI rats.MethodsOne hundred and sixteen adult female Sprague Dawley rats were randomly divided into 4 groups (n=29). The rats in sham group were received laminectomy only. A standard MASCIS spinal cord compactor was applied with drop height of 12.5, 25.0, or 50.0 mm to establish the mild, moderate, or severe SCI model, respectively. Quantitative rat endothelial cell antigen 1 (RECA1) and CD68 positive areas and the correlations were studied by double immunofluorescent (DIF) staining at 12 hours, 24 hours, 3 days, 7 days, and 28 days following SCI. Moreover, qualitative neurofilament-H (NF-H) and glial fibrillary acidic protein (GFAP) positive glial cells were studied by DIF staining at 28 days. ELISA was used to detect the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in spinal cord homogenates at 12 hours, 24 hours, and 3 days, and the correlations between TNF-α, IL-1β, or IL-6 levels and microvascular density (RECA1) were accordingly studied. Moreover, the neural tissue integrity and neuron damage were assessed by HE staining at 12 hours, 24 hours, 3 days, 7 days, and 28 days, and Nissl’s staining at 28 days following SCI, respectively.ResultsDIF staining revealed that the ratio of RECA1 positive area was the highest in moderate group, higher in mild and severe groups, and the lowest in sham group with significant differences between groups (P<0.05). The ratio of CD68 positive area was the highest in severe group, higher in moderate and mild groups, and the lowest in sham group with significant differences between groups (P<0.05), except the comparisons between mild and moderate groups at 24 hours and 28 days after SCI (P>0.05). There was no significant correlation between the RECA1 and CD68 expressions in sham group at different time points (P>0.05). At 12 and 24 hours after SCI, the RECA1 and CD68 expressions in mild and moderate groups showed significant positive correlations (P<0.05), while no significant correlation was found in severe group (P>0.05). No significant correlations between the RECA1 and CD68 expressions was shown in all SCI groups at 3 days and in severe group at 7 days (P>0.05), while the negative correlations were shown in mild and moderate groups at 7 days, and in all SCI groups at 28 days (P<0.05). In mild, moderate, and severe groups, the axons became disrupted, shorter and thicker rods-like, or even merged blocks with increased injury, while the astrocytes decreased in number, unorganized and condensed in appearance. ELISA studies showed that TNF-α, IL-1β, and IL-6 levels in sham group were significantly lower than those in other 3 groups at different time points (P>0.05). The differences in TNF-α, IL-1β, and IL-6 levels between SCI groups at different time points were sinificant (P<0.05), except IL-1β levels between the mild and moderate groups at 12 hours (P>0.05). Three inflammatory factors were all significantly correlated with the microvascular density grades (P<0.05). Histological analysis indicated that the damage to spinal cord tissue structure correlated with the extent of SCI. In severe group, local hemorrhage, edema, and infiltration of inflammatory cells were found the most drastic, the grey/white matter boundary was disappeared concurrently with the formation of cavity and shortage of normal neurons.ConclusionIn the acute stage following mild or moderate SCI, progressively aggravated injury result in higher microvessel density and increased inflammation. However, at the SCI region, the relation between microvessel density and inflammation inverse with time in the different grades of SCI. Accordingly, the destruction of neural structures positively relate to the grades of SCI and severity of inflammation.
Objective To explore the construction of a canine model of vascularized allogeneic spinal cord transplantation (vASCT) and preliminarily evaluate its therapeutic efficacy for spinal cord injury (SCI). Methods Sixteen female Beagle dogs aged 8-12 months were randomly selected, with 8 dogs serving as donors for the harvesting of spinal cord tissue with a vascular pedicle [dorsal intercostal artery (DIA) at the T10 level and accompanying vein]. The remaining 8 dogs underwent a 1.5-cm-length spinal cord defect at the T10 level, followed by transplantation of the donor spinal cord tissue for repair. Polyethylene glycol (PEG) was applied to both ends to spinal cord graft; then, using a random number table method, the dogs were divided into an experimental group (n=4) and a control group (n=4). The experimental group received immunosuppressive intervention with oral tacrolimus [0.1 mg/(kg?d)] postoperatively, while the control group received no treatment. The operation time and ischemia-reperfusion time of two groups were recorded. The recovery of hind limb function was estimated by Olby score within 2 months after operation; the motor evoked potentials (MEP) was measured through neuroelectrophysiological examination, and the spinal cord integrity was observed through MRI. ResultsThere was no significant difference in the operation time and ischemia-reperfusion time between the two groups (P>0.05). All dogs survived until the completion of the experiment. Within 2 months after operation, all dogs in the control group failed to regain the movement function of hind limbs, and Olby scores were all 0. In the experimental group, the movement and weight-bearing, as well as walking abilities of the hind limbs gradually recovered, and the Olby scores also showed a gradually increasing trend. There was a significant difference between the two groups from 3 to 8 weeks after operation (P<0.05). Neuroelectrophysiological examination indicated that the electrical signals of the experimental group passed through the transplanted area, and the latency was shortened compared to that at 1 month after operation (P<0.05), showing continuous improvement, but the amplitude did not show significant improvement (P>0.05). The control group was unable to detect any MEP changes after operation. MRI examination showed that the transplanted spinal cord in the experimental group survived and had good continuity with normal spinal cord tissue, while no relevant change was observed in the control group. ConclusionThe vASCT model of dogs was successfully constructed. This surgical procedure can restore the continuity of the spinal cord. The combination of tacrolimus anti-immunity is a key factor for the success of transplantation.
Objective To observe the structural changes of urinary center and the expression of Bcl-2 after conus medullaris injury in rats brain so as to explore the possible influence factors of degeneration in brain. Methods Thirty-six adult Sprague-Dawley rats were randomly divided into experimental group (n=30) and control group (n=6). In the experimental group, the conus medullaris injury model was established by cutting off the spinal nerve below L4, and no treatment was done in the control group. The modeling operations in the experimental group were successful, and 2 rats died at 3 months and 5 months after modeling operation respectively, which may be caused by renal failure or urinary tract infection. In the experimental group, 6, 6, 6, 5, and 5 rats were killed at 1 day, 1 week, and 1, 3, 6 months after operation respectively, and 1 rat was killed at each time point in the control group. The dorsolateral tissue of the pontine tegmentum was harvested to perform HE staining and Bcl-2 immunohistochemical SP staining. Results HE staining showed that there was no obvious difference between the experimental group and the control group at 1 day after operation, the neurons were densely packed, arranged neatly, and the nucleoli were clear; at 1 week, the space between the neurons in the experimental group were slightly widened; at 1 month, nucleus retraction in some neurons happened in the experimental group; at 3 and 6 months, the nuclei in the experimental group were more and more condensed, and even some cells disappeared. Bcl-2 immunohistochemical SP staining showed that the expression of Bcl-2 in the control group was weakly positive. The positive expression of Bcl-2 was found at 1 day after operation in the experimental group; the positive expression of Bcl-2 at 7 days after operation was significantly higher than that in the control group, and reached the peak; the positive expression of Bcl-2 decreased gradually at 1, 3, and 6 months after modeling operation, but it was still higher than that of the control group. Conclusion The urinary center appears structure degeneration and necrocytosis after conus medullaris injury in rats brain. The elevated expression of Bcl-2 may be associated with brain tissue repair and function remodeling.
Objective To review evidences of the relationship between olfactory ensheathing cells (OECs) transplantation and motor functional restitution of spinal cord injury (SCI). Methods We searched the CBM, CNKI, VIP and PubMed databases for collecting relative studies published from January 1989 to December 2009. Randomized controlled experiments of treating rats SCI with OECs transplantation were included. Quality of included experiments was assessed by Jadad scale, and the available data were abstracted and meta-analyzed with RevMan 4.2 software. Results A total of 12 randomized controlled experiments were identified. Meta-analysis showed that, OECs group was higher than control group in both BBB score (WMD=1.67, 95%CI 0.99 to 2.36; WMD=3.61, 95%CI 1.97 to 5.26; WMD=6.50, 95%CI 5.76 to 7.24; WMD=4.23, 95%CI 1.19 to 7.28; WMD=1.90, 95%CI 1.22 to 2.58; WMD=3.30, 95%CI 2.63 to 3.97) and MEP latency period (WMD= –?3.98, 95%CI –?5.71 to –?2.25), but there was no statistical significance in SEP latency period or amplitude period (WMD= –?7.13, 95%CI –?16.49 to 2.23; WMD=3.00, 95%CI –?1.12 to 7.11; WMD=1.95, 95%CI –?0.89 to4.78). Conclusions This meta-analysis based on current experiments suggests that OECs transplantation is superior in motor function restitution after spinal cord injury, but is similar as control group in SEP latency or amplitude.
Objective To investigate the changes of autophagy after spinal cord injury (SCI) in rats and its relationship with multisite phosphorylation of B-cell lymphoma-2 (Bcl-2) protein. Methods Forty male Sprague-Dawley rats aged 8 weeks were used to prepare SCI models by modified Allen method, and the SCI model were prepared successfully in 36 rats. The 36 SCI models were randomly divided into SCI group, autophagy inhibitor group, and autophagy promoter group, with 12 rats in each group. Another 12 rats were selected as sham operation group with only laminectomy and no spinal cord injury. At the end of modeling, the autophagy inhibitor group and the autophagy promoter group were intrathecally injected with 20 μL of 600 nmol/L 3-methyladenine and 25 nmol/L rapamycin, respectively, once a day for 4 weeks. The sham operation group and the SCI group were injected with only 20 μL of normal saline at the same time point. The motor function of rat in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1 day and 1, 2, 4 weeks after modeling. The rats in each group were sacrificed at 24 hours after the last injection and the spinal cord tissues were taken. ELISA assay was used to detect the levels of inflammatory factors in spinal cord tissues, including myeloperoxidase (MPO), tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β); the morphological changes of spinal cord were observed by HE staining; the autophagy of mitochondria in spinal cord tissues was observed by transmission electron microscopy; the expressions of Beclin1 and microtubule-associated protein light chain 3 (LC3) were detected by immunofluorescence staining; neuronal apoptosis in spinal cord tissues were observed by TUNEL staining; LC3/TUNEL positive cells were calculated by immunofluorescence double staining; the expressions of Bcl-2 associated X protein (Bax), Bcl-2, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) were detected by Western blot. Results Compared with sham operation group, BBB score of SCI group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; peripheral space of nerve cells enlarged, cells swelled, vacuoles appeared, and autophagic bodies appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins, and apoptotic rate of neurons significantly increased; the LC3/TUNEL positive cells significantly increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased; all showing significant differences (P<0.05). Compared with SCI group, BBB score in autophagy inhibitor group decreased at each time point, while the levels of MPO, TNF-α, and IL-1β increased; a few autophagic vesicles appeared in mitochondria; the positive rates of Beclin1 and LC3 proteins decreased and the apoptotic rate of neurons increased significantly; the LC3 positive cells decreased and the TUNEL positive cells increased; the expressions of Bax, p-Bcl-2 (Ser87), and p-Bcl-2 (Ser70) proteins increased, while the expression of Bcl-2 protein decreased. The results of autophagy promoter group were opposite to those of autophagy inhibitor group; all showing significant differences between groups (P<0.05). Conclusion Induction of autophagy after SCI in rats can reduce neuronal apoptosis and protect spinal cord function, which may be related to the inhibition of Bcl-2 protein multisite phosphorylation.
ObjectiveTo explore the biological functions of Kip1 ubiquitylation-promoting complex 2 (KPC2) in the repair process of spinal cord injury (SCI) by studying the expression and cellular localization of KPC2 in rat SCI models. MethodsFifty-six adult Sprague-Dawley rats were randomly divided into 2 groups: in the control group (n=7), simple T9 laminectomy was performed;in the experimental group (n=49), the SCI model was established at T9, 7 rats were used to detect follow indexs at 6 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, and 14 days after SCI. Western blot analysis was used to detect the protein expressions of P27kip1, KPC2, CyclinA and proliferating cell nuclear antigen (PCNA) after SCI. Immunohistochemistry was used to observed the cellular localization of KPC2 after SCI, double-labeling immunofluorescence staining to observe the co-localization of KPC2 with neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and PCNA. in vitro astrocytes proliferation model was used to further validate these results, Western blot to detect KPC2, P27kip1, and PCNA expressions. The interaction of P27kip1, KPC1, and KPC2 in cell proliferation was analyzed by co-immunoprecipitation. ResultsThe Western blot analysis showed a significant down-regulation of P27kip1 and a concomitant up-regulation of KPC2, CyclinA, and PCNA after SCI. Immunohistochemistry staining revealed a wide distribution of KPC2 positive signals in the gray matter and white matter of the spinal cord. The number of KPC2 positive cells in the experimental group was significantly higher than that in the control group (t=10.982, P=0.000). Double-labeling immunofluorescence staining revealed the number of KPC2/NeuN co-expression cells in the gray matter of spinal cord was (0.43±0.53)/visual field in the control group and (0.57±0.53)/visual field in the experimental group, showing no significant difference (t=0.548, P=0.604);in the white matter of spinal cord, the number of KPC2/PCNA co-expression cells was (3.86±0.90)/visual field in the control group and (0.71±0.49)/visual field in the experimental group, showing significant difference (t=7.778, P=0.000). And then, the number of KPC2/PCNA co-expression cells were (0.57±0.53)/visual field in the control group and (5.57±1.13)/visual field in the experimental group, showing significant difference (t=8.101, P=0.000). Concomitantly, there was a similar kinetic in proliferating astrocytes in vitro. The Western blot analysis showed a significant down-regulation of P27kip1 and a concomitant up-regulation of KPC2 and PCNA after serum stimulated. Co-immunoprecipitation demonstrated increased interactions between P27kip1, KPC1, and KPC2 after stimulation. ConclusionThe up-regulated expression of KPC2 after SCI is related to the down-regulation of P27kip1, this event may be involved in the proliferation of astrocytes after SCI.
Objective To discuss the clinical value of whole spine magnetic resonance imaging (WSMRI) in practice of neurosurgical spinal surgery. Method A total of 70 cases examined using WSMRI between January 2015 and December 2016 were collected and analyzed retrospectively. Results All patients got clear images of WSMRI. Eighteen cases got important information, including spinal variation (1 case), multiple lesions (3 cases), combined lesions (6 cases) and large range multi-segmental lesions (8 cases), which were missed by single-segment MRI . Conclusions WSMRI can show all the spine, spinal cord and surrounding tissue in one image at one time. It has high clinical value because of its accurate positioning, comprehensiveness, time saving, and low rate of misdiagnosis and missed diagnosis.
OBJECTIVE: To investigate a animal model of spinal cord injury in different degrees of impact. METHODS: A new weight-drop device was designed with the character of controlled degree of impact and time. After thirty-five rats underwent different degrees of impact, their motor function and pathological changes were observed. RESULTS: In control group, the rats could walk after reviving, and the micro-structure of spinal cord was normal. With 0.5 mm depth of impact, the rats also could walk, and the micro-structure of spinal cord did not change obviously. With 0.8 mm depth of impact, the rats could walk after several days of injury and only slight damage could be found in spinal cord. When the impact depth increased to 1.0 or 1.5 mm, the rats were paralyzed completely and could not walk after four weeks of injury. Severe injury was observed in spinal cord. CONCLUSION: This animal model of spinal cord injury is based on different degrees of impact. It has stable and repetitive characters for the research on spinal cord injury.
ObjectiveTo investigate the regulatory effect of miRNA-21-5p (miR-21) on spinal fibroblasts, and to explore the mechanism of miR-21 related pathological process of spinal cord injury.MethodsSpinal cord fibroblasts were identified by immunofluorescence. Spinal fibroblasts damage model was established by scratch method. Quantitative real-time polymerase chain reaction (RT-PCR) was used to determine the relative expression of miR-21 and fibrosis-related genes in spinal cord fibroblasts after injury. The expression of miR-21 in spinal cord fibroblasts was up-regulated and down-regulated by using miR-21 mimics/inhibitor, and the expression levels of apoptosis and proliferation-related proteins were detected by Western Blot (WB).ResultsThe expression of miR-21 and fibrosis-related genes were increased after spinal cord fibroblast scratch (P<0.05). Up-regulation of the miR-21 can increase the expression of apoptosis-related genes in fibroblasts (P<0.05), and vice versa. The proliferation of fibroblasts was consistent with the expression of miR-21, while the apoptosis of fibroblasts was contrary to the expression of miR-21.ConclusionsmiR-21 enhanced the fibrosis and proliferation, inhibited the apoptosis of spinal cord fibroblasts after mechanical injury. This indicates that miR-21 is closely related with the formation of fibrotic scar after spinal cord injury, which also providesa potential therapeutic target for spinal cord injury.