Objective Bone marrow mesenchymal stem cells (BMSCs) play an important role in repairing nerve injury, meanwhile external temperature has significant effect on BMSCs transplantation, prol iferation, and differentiation. To investigate the effect of BMSCs transplantation and mild hypothermia on repair of rat spinal cord injury (SCI). Methods Forty-five female adult SD rats (weighing 200-250 g) were made the models of hemitransection SCI and divided randomly into 3 groups according to different treatments: group A (SCI group), group B (BMSCs transplantation group), and group C [BMSCs transplantation combined with mild hypothermia (33-35 ) group]. At 1, 2, 4, 6, and 8 weeks after injury, the fuction of hind l imb was evaluated with Basso Beattie and Bresnahan (BBB) score and incl ined plane test. At 4 weeks after injury, histopathology and BrdU immunohistochemistry staining were performed. At 8 weeks after injury, horseradishperoxidase (HRP) retrograde nerve trace and transmission electron microscope (TEM) testing were performed to observe the regeneration of axon. Results After 4 weeks, the function of hind l imb obviously recovered in groups B and C, there were significant differences in BBB score between groups B, C and group A (P lt; 0.05), between group B and group C (P lt; 0.05). There was no significant difference (P gt; 0.05) in tilt angle among 3 groups after 1 and 2 weeks, and there were significant differences (P lt; 0.05) among 3 groups after 4 weeks. HE staining showed that significant cavity could be seen in group A, l ittle in group B, and no cavity in group C. BrdU immunohistochemistry staining showed that the number of positive cells was 0, 90.54 ± 6.23, and 121.22 ± 7.54 in groups A, B, and C, respectively; showing significant differences (P lt; 0.01) among 3 groups. HRP retrograde neural tracing observation showed that the number of HRP positive nerve fibers was 10.35 ± 1.72, 43.25 ± 2.65, and 84.37 ± 4.59 in groups A, B, and C, respectively, showing significant differences (P lt; 0.01) among 3 groups. TEM observation showed that a great amount of unmyel inated nerve fibers and myel inated nerve fibers were found in central transverse plane in group C. Conclusion The BMSCs transplantation play an impontant role in promotion of recovering the function of hind l imb after SCI, and mild hypothermia has synergism effects.
Objective To explore the factors to affect severity of hyperextension injury of the cervical spinal cord (HEICSC). Methods Forty-five patients with HEICSC, 35 males and 10 females, aged 27-67 years old (mean 48.2 years old), were retrospectively analyzed. The disease course was 30 minutes to 16 days. According to modified Frankel grading, there were 6 cases of grade A, 8 cases of grade B, 16 cases of grade C and 15 cases of grade D. Spinal cord injuries (SCI) segments were determined according to SCI plane and high signal change (HSC) in spinal cord on MR images. The whole or large part of HSC segments were supposed to be main injured spinal cord segments (MISCSs) and the staccato or patchy HSC ones were supposed to be common injured spinal cord segments (CISCSs). When the external force acting on head or face suffered was larger, the force produced during high-speed movement or forehead and/or face had severe contused and/or) lacerated wound, the force was defined severe traumatic strength, whereas the reverse was true for sl ight traumatic strength. According to signal magnitude of the cervical discs on T2-weighted MR images, degeneration of cervical discs and cervical vertebras were classified into 5 grades: grade 0-4. Cervical spinal stenosis were graded to 5 grades according to the width of anterior or posterior cerebrospinal fluid layer to spinal cord on T2-weighted MR images and compressed degree of spinal cord on T1-weighted MR images. The influence of traumatic strength, cervical spinal degeneration or cervical spinal stenosis on SCI were explored. Results Among the 45 cases, 12 cases were caused by sl ight traumatic strength, 33 cases were caused by severe one. The cervical spinal cord was injuried more sl ightly and the patients were older in the sl ight traumatic strength cases than in the severe ones (P lt; 0.05). The number of MISCSs were 45 in 40 cases and the 25 segments were located at C3, 4 level. The number of CISCSs were 39 in 21 cases. All the cervical vertebraes of the 45 patients had degenerated. The most were in grade 3 in 22 patients and the severest degenerative segments were mostly located in C5,6 discs in 35 ones. The number of the MISCSs in different degenerative grades of discs was 0 in grade 0, 9 in grade 1, 20 in grade 2, 14 in grade 3, and 2 in grade 4. The ratios of the segment number of injuried spinal cord to the segment number of spinal stenosis in every grade of stenosis were 1/62 in grade 0, 2/11 in grade 1, 27/52 in grade 2, 33/33 in grade 3, 21/22 in grade 4. Conclusion Three main factors including the magnitude of traumatic strength, the degree of instabil ity of cervical vertebrae and the degree of cervical stenosis contribute to development and progress of HEICSC.
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.
Spinal cord injuries (SCI) seriously impair the quality of life, functional status, and social independence of the patients. Since the last century, a series of basic research on spinal cord injury has made us a deep understanding of its mechanisms and pathophysiology. But so far, how to repair damaged nerve functions after SCI is still a neurological problem. There are still controversies surrounding some treatment strategies for SCI, including the use of magnetic resonance imaging, type and timing of anticoagulant prevention, the timing of surgical intervention, the use of corticosteroids such as methylprednisolone sodium, as well as the type and timing of rehabilitation. For patients with SCI, early surgical intervention and neuroprotective therapy may be the best treatment. At the same time, rehabilitation and psychological intervention are equally important.
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 investigate the effect of M2 microglia (M2-MG) transplantation on spinal cord injury (SCI) repair in mice. Methods Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1. Then the primary MG were co-cultured with interleukin 4 for 48 hours (experimental group) to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1 (Arg-1) and Iba1. The normal MG were harvested as control (control group). The dorsal root ganglion (DRG) of 5 C57BL/6 mice born 1 week old were co-cultured with M2-MG for 5 days to observe the axon length, the DRG alone was used as control. Forty-two 6-week-old female C57BL/6 mice were randomly divided into sham group (n=6), SCI group (n=18), and SCI+M2-MG group (n=18). In sham group, only the laminae of T10 level were removed; SCI group and SCI+M2-MG group underwent SCI modeling, and SCI+M2-MG group was simultaneously injected with M2-MG. The survival of mice in each group was observed after operation. At immediate (0), 3, 7, 14, 21, and 28 days after operation, the motor function of mice was evaluated by Basso Mouse Scale (BMS) score, and the gait was evaluated by footprint experiment at 28 days. The spinal cord tissue was taken after operation for immunofluorescence staining, in which glial fibrillary acidic protein (GFAP) staining at 7, 14, and 28 days was used to observe the injured area of the spinal cord, neuronal nuclei antigen staining at 28 days was used to observe the survival of neurons, and GFAP/C3 double staining at 7 and 14 days was used to observe the changes in the number of A1 astrocytes. Results The purity of MG in vitro reached 90%, and the most of the cells were polarized into M2 phenotype identified by Arg-1 immunofluorescence staining. M2-MG promoted the axon growth when co-cultured with DRGs in vitro (P<0.05). All groups of mice survived until the experiment was completed. The hind limb motor function of SCI group and SCI+M2-MG group gradually recovered over time. Among them, the SCI+M2-MG group had significantly higher BMS scores than the SCI group at 21 and 28 days (P<0.05), and the dragging gait significantly improved at 28 days, but it did not reach the level of the sham group. Immunofluorescence staining showed that compared with the SCI group, the SCI+M2-MG group had a smaller injury area at 7, 14, and 28 days, an increase in neuronal survival at 28 days, and a decrease in the number of A1 astrocytes at 7 and 14 days, with significant differences (P<0.05). ConclusionM2-MG transplantation improves the motor function of the hind limbs of SCI mice by promoting neuron survival and axon regeneration. This neuroprotective effect is related to the inhibition of A1 astrocytes polarization.
Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsallaminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion TherAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.
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.
ObjectiveTo fabricate the bionic scaffolds of rat spinal cord by combining three dimensional (3D) printer and 3D software, so as to lay the foundation of theory and technology for the manufacture of scaffolds by using biomaterials.
MethodsThree female Sprague Dawley rats were scanned by 7.0T MRI to obtain the shape and position data of the cross section and gray matter of T8 to T10 spinal cord. Combined with data of position and shape of nerve conduction beam, the relevant data were obtained via Getdata software. Then the 3D graphics were made and converted to stereolithography (STL) format by using SolidWorks software. Photosensitive resin was used as the materials of spinal cord scaffolds. The bionic scaffolds were fabricated by 3D printer.
ResultsMRI showed that the section shape of T8 to T10 segments of the spinal cord were approximately oval with a relatively long sagittal diameter of (2.20±0.52) mm and short transverse diameter of (2.05±0.24) mm, and the data of nerve conduction bundle were featured in the STL format. The spinal cord bionic scaffolds of the target segments made by 3D printer were similar to the spinal cord of rat in the morphology and size, and the position of pores simulated normal nerve conduction of rat spinal cord.
ConclusionSpinal cord scaffolds produced by 3D printer which have similar shape and size of normal rat spinal cord are more bionic, and the procedure is simple. This technology combined with biomaterials is also promising in spinal cord repairing after spinal cord injury.
