Abstract： Objective To study the spinal cord protection effect of cerebrospinal fluid drainage （CSFD）for patients undergoing thoracoabdominal aortic aneurysm surgery. Methods We randomly allocated 30 patients undergoing thoracoabdominal aortic aneurysm surgery in Beijing Anzhen Hospital from December 2008 to August 2009 into a CSFD group with 15 patients（12 males, 3 females; average age of 45.0 years） and a control group with 15 patients（11 males, 4 females; average age at 45.8 years）by computer. All the patients underwent replacement of ascending aorta and aortic arch, implantation of descending aorta stent, or thoracoabdominal aorta replacement. Some patients underwent Bentall operation or replacement of half aortic arch. Patients in the CSFD group also underwent CSFD. Serum S100B, glial fibrillary acidic protein and neuron-specific enolase were measured at set intraoperative and postoperative times. All the patients were scored preoperatively, 72 hours postoperatively, and before discharge according to the National Institutes of Health Stroke Scale and International Standards for Neurological Classification of Spinal Cord Injury. Results Central nervous system injury occurred in four patients in the control group： one died of both brain damage and spinal cord damage; one patient had spinal cord injury and became better after treatment by early CSFD; two patients had brain damage（one patient died, another patient had concomitant acute renal failure and acute respiratory failure, recovered and was discharged after treatment）. In the CSFD group, only one patient died of acute respiratory failure and subsequent multiple organ system failure, and all other patients recovered very well. There was no late death during three months follow-up in both groups. The average serum S100B, glial fibrillary acidic protein，and neuron-specific enolase concentrations of the CSFD group patients were significantly lower than those of the control group （F=7.153，P=0.012；F=3.263，P=0.082；F=4.927，P=0.035）. Conclusion Selected CSFD is a safe, effective and feasible procedure to protect the spinal cord from ischemic damage during the perioperative period of thoracoabdominal aortic aneurysm surgery.
Spinal cord injury is one of severe complications after thoracic aortic surgery. The degree and time of spinal cord ischemia during surgery, reconstruction of the blood supply of spinal cord ,biochemistry factors ,ischemiareperfusion injury, etc. are considered as factors influence on the complication of spinal cord after surgery. At present, to improve the surgical technique, to increase the blood supply of spinal cord, such as the mechanical dynamic blood perfusion, arterial shunt and cerebrospinal fluid shunt, to degrade the metabolic rate of spinal cord using hypothermia, and to prevent the ischemia-reperfusion injury using drugs are the methods for spinal cord protection during the aortic surgery. The feature of blood circulation of spinal cord, mechanisms of spinal cord injury and the latest progress of spinal cord protection is reviewed in this article.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To investigate the effect of curcumin on calcitionin gene related peptide (CGRP) expression after spinal cord injury (SCI) in rats. Methods A total of 200 rats, weighing 250-300 g, were randomly divided into 4 groups (n=50): sham-operation group, normal saline (NS) group, low-dose curcumin group (30 mg/kg), and high-dose curcumin group (100 mg/kg). In sham-operation group, only vertebral lamina excision was performed without SCI; the SCI model was established in the other 3 groups. At immediate after modeling, 30 mg/kg and 100 mg/kg curcumin were injected intraperitoneally in 2 curcumin groups, equivalent NS was given in NS group (30 mg/kg), but no treatment in sham-operation group. At 1, 3, 7, 14, and 21 days after operation, the motor neural function was evaluated by the inclined plane test and Basso-Beattie-Bresnahan (BBB) scores; immunohistochemical staining and Western blot assay were used to observe CGRP expression. Results BBB score and inclined plane test score of NS group, low-dose curcumin group, and high-dose curcumin group were significantly lower than those of sham-operation group at each time point (P lt; 0.05). BBB score of low-dose curcumin group and high-dose curcumin group was significantly higher than that of NS group at 3, 7, 14, and 21 days after SCI (P lt; 0.05), and the score of high-dose group was significantly higher than that of low-dose curcumin group at 7, 14, and 21 days after SCI (P lt; 0.05). Inclined plane test score of low-dose curcumin group and high-dose curcumin group was significantly higher than that of NS group at 7, 14, and 21 days after SCI (P lt; 0.05), and the score of high-dose curcumin group was significantly higher than that of low-dose curcumin group at 7, 14, and 21 days after SCI (P lt; 0.05). Immunohistochemical staining results showed that the CGRP positive cells of sham-operation group was significantly more than those of the other 3 groups, and the CGRP positive cells of high-dose curcumin group were significantly more than those of low-dose curcumin group at each time point (P lt; 0.05); the CGRP positive cells of low- and high-dose curcumin groups were significantly more than those of NS group at 3, 7, 14, and 21 days after SCI (P lt; 0.05). Western blot assay results showed that the CGRP protein expressed at each time point after SCI in sham-operation group; the CGRP protein expression gradually decrease with time passing in NS group; but the CGRP protein expression gradually increased with time passing in low- and high-dose curcumin groups, and reached the peak at 14 days, then maintained a high level. Conclusion After SCI in rats, 30 mg/kg curcumin can improve rats’ motor function, and 100 mg/kg curcumin effect is more obvious, especially in promoting the expression of CGRP. That may be the mechanism of protection of the nervous system.
Objective To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats. Methods Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups A and B, the SCI model was established with modified Allen’s method and then the rats of groups A and B were administrated by subarachnoid injection of 6 μL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord. Results At different time points, the BBB score of group C was significantly higher than those of groups A and B (P lt; 0.05), and the BBB score of group A was significantly better than that of group B at 14 and 28 days after operation (P lt; 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P lt; 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P lt; 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P lt; 0.05). Conclusion The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.
Objective To investigate the effects of chondroitinase ABC (ChABC) combined with bone marrow mesenchymal stem cells (BMSCs) in repair spinal cord injury of rats. Methods Primary BMSCs were isolated and cultured from the femur and tibia of neonatal Sprague Dawley (SD) rats. The spinal cord injury model was established in 24 adult SD male rats (weighing, 200-230 g), which were randomly divided into control group (group A), BMSCs transplantation group (group B), ChABC injection group (group C), and ChABC and BMSCs transplantation group (group D), 6 rats in each group. At 7 and 14 days after injury, Basso-Beattie-Bresnahan (BBB) score criteria was used to evaluate the hindlimb motor function; at 14 days after injury, the injured spinal cord tissue was perfused and stained by HE for further calculation of the injury area. Immunofluorescence staining were used for observing the expressions of glial fibrillary acidic protein (GFAP)/chondroitin sulfate proteoglycan (CSPG) and GFAP/growth associated protein 43 (GAP43). Results At 7 days after injury, three joints movement of the hindlimbs were recovered in all groups, and no significant difference in the BBB score was found among 4 groups (P gt; 0.05). At 14 days after injury, no load drag was observed in 3 joints of the hindlimbs in groups A, B, and C, but weight-bearing plantar or occasional dorsalis pedis weight-bearing walking was observed in group D with no plantar walking. The BBB score of group D was significantly higher than that of the other 3 groups (P lt; 0.05). HE staining showed that the cavity formed in the damage zone, and there were a large number of macrophages in the cavity and its surrounding, which was wrapped by scar tissue. The damage area of group D was significantly smaller than that of the other 3 groups (P lt; 0.05). At 14 days after injury, the GFAP/CSPG double immunofluorescence staining showed that the astroglial scar damage zone in group D was significantly reduced, and no cavity formation was found. And the fluorescence intensity in groups C and D was significantly lower than that in group B (P lt; 0.05). The GFAP/GAP43 double immunofluorescence staining showed that GAP43-positive fibers passed through the damage zone in group D and the fluorescence intensity in group D was significantly higher than those in groups B and C (P lt; 0.05). Conclusion Inhibition of astrocytes secreting CSPG by ChABC combined with BMSCs transplantation in early injury may promote the regeneration of nerve fibers, and repair spinal cord injury in rats.
Objective To investigate the expressions of heat shock protein 27 (HSP27), Bcl-2, and Bax proteins of the nerve cells after spinal cord ischemia/reperfusion injury (SCII) in rats and their relationship. Methods Seventy adult male Sprague Dawley rats (weighing, 200-220 g) were randomly divided into the sham operated group (sham group, n=35) and the SCII group (n=35). Only the left renal artery was exposed with no occlusion of the abdominal aorta in the rats of sham group. The left renal artery was exposed with occlusion of the abdominal aorta for 20 minutes in the rats of SCII group. At 4, 8, and 12 hours and at 1, 2, 3, and 5 days, reperfusion treatment was performed in 5 rats respectively, and then the spinal cord tissue was harvested to detect the expressions of HSP27, Bcl-2, and Bax protein of the nerve cells by using immunohistochemistry staining. Results The HSP27 began to express at 4 hours, reached the peak at 3 days, and decreased at 5 days in SCII group; significant differences were found between at 3 and 5 days and at the other time points (P lt; 0.05). The Bcl-2 expression increased at 4 hours, reached the peak at 1 day and maintained a high level at 2 days, and then gradually decreased; significant differences were found between at 1 and 2 days and at the other time points (P lt; 0.05). The Bax expression reached the peak at 12 hours and 3 days, and decreased at 5 days; significant differences were found between at 12 hours and 3 days and at the other time points (P lt; 0.05). A little expression of each protein was observed in sham group at different time points; the expressions of HSP27, Bcl-2, and Bax proteins in SCII group were significantly higher than those in sham group at different time points (P lt; 0.05). Conclusion There may be the time window of self repair after SCII. High expression of HSP27 has an obvious protective effect on the SCII in rat, by promoting the expression of the anti-apoptotic protein Bcl-2 and reducing the expression of the pro-apoptotic protein Bax so as to inhibit spinal cord cell apoptosis.
Objective To investigate the relationship between the expression of apoptosis-related gene Fas and recovery of neurological function after surgical decompression at different time points in acute spinal cord injury (SCI) rat model by cerclage. Methods A total of 100 13-week-old male Sprague Dawley rats (weighing, 255-376 g) were randomly divided into 4 groups (n=25). The rats only received laminectomy in group A as control; the rats were made the acute SCI models by cerclage in groups B, C, and D. The spinal cord decompression was performed in group B at 8 hours and in group C at 72 hours, no spinal cord decompression in group D. At 1, 3, 7, 14, and 21 days, Basso-Beattie-Bresnahan (BBB) score and inclined plane test were used to evaluate the recovery of neurological function; the neuronal apoptosis level of spinal cord was examined by TUNEL staining; HE staining and immunohistochemical staining were applied to analyze the expressions of Fas. Results The BBB score and inclined plane test score in group A were significantly better than those in groups B, C, and D at different time points (P lt; 0.05); group B was significantly better than groups C and D, and group C than group D at 3, 7, 14, and 21 days (P lt; 0.05). In group A, no bleeding, edema, or necrosis was found. The edema, hemorrhage, and neuron death were observed in spinal cord tissue of groups B, C, and D at 1 day after operation, especially in group D. The degree of cell degeneration in group B was lighter than that in groups C and D at 3 and 7 days after operation; few glial cells and fibroblast proliferation were found at damaged zone in group B at 14 and 21 days, but necrosis and cystic cavity in groups C and D. Fas and TUNEL expression was little in group A at different time points. Fas and TUNEL were expressed in groups B, C, and D; the expressions of Fas and TUNEL reached the maximum at 3 days, and then gradually decreased at 7 and 21 days. The number of positive cells was highest in group D, and the number of positive cells in group B was significantly less than that in groups C and D (P lt; 0.05). Conclusion Early decompression of SCI is beneficial to recovering the neurological function. The Fas signal pathway may play an important role in the apoptosis of neuron and glial cells after SCI.
【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nervegrowth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.