RESUMO
Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17+ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17+ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.
Assuntos
Animais , Ratos , Interleucina-17 , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Linfócitos T Reguladores , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
BACKGROUND: Change of microenvironment after acute spinal cord injury is the main factor causing secondary injury, so it is of great significance to investigate the changes of microenvironment after acute spinal cord injury for clinical diagnosis and treatment. OBJECTIVE: To investigate the expression levels and clinical significance of interleukin-6, brain derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3 and neurotrophin-4 in peripheral blood within 48 hours after acute spinal cord injury. METHODS: Twenty-nine patients with acute spinal cord injury admitted at the Department of Spinal Osteopathia, the First Affiliated Hospital of Guangxi Medical University from October 2016 to June 2018 were enrolled, and were divided into two groups according to American Spinal Injury Association impairment scale: complete spinal cord injury (n=11) and incomplete spinal cord injury (n=18). Thirteen patients with avascular necrosis of the femoral head were selected as controls. The expression levels of interleukin-6, brain derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3 and neurotrophin-4 in peripheral blood of 42 patients were determined by ELISA and compared. RESULTS AND CONCLUSION: The ELISA results showed that the expression levels of interleukin-6, brain derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3 and neurotrophin-4 in peripheral blood in the spinal cord injury group were significantly higher than those in the control group (P ＜ 0.05). The expression levels of all above cytokines in the complete spinal cord injury group were significantly higher than those in the incomplete spinal cord injury group (P ＜ 0.05). In summary, increased expression of interleukin-6, brain derived neurotrophic factor, basic fibroblast growth factor, neurotrophin-3, neurotrophin-4 after acute spinal cord injury indicates that it may participate in the important pathophysiological process after acute spinal cord injury.
RESUMO
BACKGROUND: miRNA-136-5 p plays a crucial regulatory role in pathological changes, inflammatory response and regeneration after spinal cord injury. OBJECTIVE: To investigate the effect of miRNA-136-5 p on the expression of cytokines in serum and NF-κB protein in spinal cord in rats with spinal cord injury and to explore the molecular mechanism. METHODS: Thirty-six male Sprague-Dawley rats, of SPF grade were provided by Laboratory Animal Center of Guangxi Medical University. The lentiviral vector system was prepared and transfected into spinal cord injured rats. Thirty-six rat models of spinal cord injury were established by modified Allen's method. Basso Beattie Bresnahan scores were performed. Rats were randomly divided into normal control, modeling (LV-ctrl plus spinal cord injury), overexpression (spinal cord injury plus LV-miRNA-136-5 p), and inhibition (spinal cord injury plus LV-sponge) groups (n=9/group). Seven days before surgery and the day of surgery, the overexpression and inhibition groups were continuously injected with the lentivirus suspension into the injured area, and the normal control and modeling groups were injected with the same amount of normal saline. Three rats were sacrificed at 1, 3 and 7 days, and blood and spinal cord tissues were taken. The levels of interleukin-1β, interleukion-6 and interferon-α in rat serum were determined by ELISA. The expression of NF-κB protein was detected by western blot assay and double immunofluorescence. RESULTS AND CONCLUSION: (1) There was no significant difference in preoperative Basso Beattie Bresnahan scores (P＞ 0.05). In the modeling group, the rats showed prone walking, vary degrees of urinary retention, and spinal shock, with complete loss of function of both hind limbs and muscle strength of 0. (2) Compared with the normal control group, the levels of inflammatory factors in the other groups were increased significantly (P ＜ 0.05). The expression levels of inflammatory factors were highest in the overexpression group, followed by modeling group, and lowest in the inhibition group. (3) Results of western blot assay and double immunofluorescence showed that the expression level of NF-κB protein in the modeling, overexpression and inhibition groups was significantly higher than that in the normal control group (P ＜ 0.05), and the level was highest in the overexpression group. (4) In summary, miRNA-136-5 p can affect inflammatory factors and NF-κB in rats with acute spinal cord injury.
RESUMO
BACKGROUND:Our previous studies have found that polygonatum sibiricum polysaccharide (PSP) promotes osteogenic differentiation of bone marrow mesenchymal stem cel s (BMSCs) by Wnt/β-catenin signaling pathway, but the molecular mechanism is unclear.OBJECTIVE:To investigate the effect of PSP promoting the osteogenic differentiation via Wnt signaling pathways in BMSCs after LRP5 silencing. METHODS:LRP5 interference vectors were constructed and then transfected into C57BL/6 mouse BMSCs cultured in vitro. The transfection efficiency of cel s was calculated under fluorescence inverted microscope and the expression of LRP5 protein was detected by western blot assay. The osteogenic potential of BMSCs after LRP5-siRNA transfection was analyzed by alkaline phosphatase staining, alizarin red staining and western blot assay. Effect of PSP on the osteogenic differentiation of LIRP5-silenced mouse BMSCs was detected by real-time PCR and dual luciferase assay. RESULTS AND CONCLUSION:Compared with the control group, the mineralization ability, the mRNA expressions of Runx2 and Osterix, and the protein expression of LRP5 were significantly decreased in the LRP5-siRNA group (P<0.05). PSP could promote LRP5-siRNA transfected mouse BMSCs differentiating into osteoblasts and significantly upregulated the expressions ofβ-catenin and Osterixin, and also induced the high expression of luciferase reporter gene (TOPFlash) containing wild type TCF binding sites (P<0.05). To conclude, LRP5 plays an important role in the process of mouse BMSCs differentiating into osteoblasts. PSP can promote the osteogenic differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway independent on LRP5.
RESUMO
BACKGROUND: miRNA plays a critical regulatory role in the development and plasticity of spinal cord, and pathological changes after spinal cord injury. OBJECTIVE: To study the effect of miR-136-5p on the A20 expression in mouse astrocytes stimulated by interleukin-17 (IL-17). METHODS: C57BL/6 mouse astrocytes were cultured in vitro, identified by immunofluorescence staining, and then stimulated by 100 μg/L IL-17 for 0, 3, 6, 12 and 24 hours, respectively. The relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected by RT-PCR to determine the optimal stimulation time of IL-17. The mouse astrocytes were respectively stimulated by 10, 20, 50, 100 and 200 μg/L IL-7 for 6 hours, and similarly, the relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected to determine the optimal concentration of IL-17. At 6 hours after IL-17 (50 μg/L) stimulation, the mRNA expression levels of miR-136-5p and A20 in mouse astrocytes were detected by RT- PCR, and the protein expression level of A20 was detected by western blot assay. In addition, the lentiviral expression vector (miR-136-5p-inhibition) was constructed and transfected into the mouse astrocytes that were also stimulated by IL-7 to detect the expression levels of miR-136-5p, A20 mRNA and A20 protein. RESULTS AND CONCLUSION: Compared with the blank control group, the expression level of miR-136-5p in the miR-136-5p-inhibition group was significantly decreased after 6-hour IL-17 stimulation (P 0.05). To conclude, miR-136-5p makes certain effect on the expression of A20 protein in astrocytes after IL-17 stimulation.
RESUMO
Objective To investigate PSP on bone microstructures,Ca,P,OPG and RANKL of osteoporotic rat model.Methods Thirty female rats randomly divided into five groups:Sham,OVX,H-,M-,L-PSP.Sham and OVX were irrigated stomachsaline;PSP solution was gavaged to other groups.After 8-week,bone microstructures of tibial metaphyseal,Ca,P,OPG and RANKL were measured.Results Body weight,Ca,P,RANKL,Tb.Sp of OVX were significantly increased compared to Sham,OPG,BV/TV,Tb.Th,Tb.N decreased.Body weight of H-,M-PSP,Ca and Tb.Sp of PSP,P and RANKL in H-PSP were decreased compared to OVX,OPG in H-,M-PSP,BV/TV,Tb.Th,Tb.N of PSP group increased.The differences were statistically significant (P ＜ 0.05).Conclusion PSP prevents osteoporosis by improving the microstructure of trabecular bone,reducing bone turnover,increasing OPG and reducing RANKL expression.
RESUMO
BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) hold the potential of differentiating into osteoclasts. Polygonatum sibiricum polysaccharide (PSP) may inhibit the differentiation of BM-MNCs into osteoclasts and it is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE: To investigate the effect of PSP on the differentiation of mouse BM-MNCs into osteoclasts induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and bone resorption in vivo. METHODS: Mouse bone marrow-derived macrophages cultured in vitro, the effect of macrophage colony stimulating factor and PSP (5, 10, 20, 40, 80,160, 320, 640, 1280, 2560 mg/L) on the proliferation of mouse BM-MNCs was detected by cell counting kit-8 assay to determine the PSP concentration range; the mouse BMMs were cultured and induced in DMEM medium containing macrophage colony stimulating factor, RANKL and 5, 10, 20, 40, 80,160, 320, 640 mg/L PSP, respectively; those cultured without PSP served as control group. The morphological changes of cells were observed under an inverted microscope.; the number of osteoclasts was detected by tartrate-resistant acid phosphatase staining; the mRNA expression levels of osteoclast-related genes including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 were evaluated by quantitative real-time PCR. A mouse model of calvarial osteolysis induced by lipopolysaccharide was established to receive PSP intervention, and then micro CT scanning, three-dimensional reconstruction and relevants software were used for quantitative analysis of bone volume/volume percentage, trabecular number, trabecular bone spacing and thickness. The number of osteoclasts was identified by tartrate-resistant acid phosphatase staining and quantitative analysis of bone resorption area was conducted. RESULTS AND CONCLUSION: Compared with the control group, the concentration of PSP below 640 mg/L showed no significant effect on the proliferation of BMMs (P > 0.05). Different concentrations of PSP (40-640 mg/L) significantly reduced the number of osteoclasts, osteoclast differentiation and maturation, and the mRNA expression levels of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 TRAP, MMP-9, CtsK and NFATc1 (P < 0.05). Compared with lipopolysaccharide, PSP could effectively alleviate the lipopolysaccharide-induced calvarial osteolysis, and the bone volume/volume percentage, trabecular number, and trabecular bone spacing were significantly decreased (P < 0.05); additionally, the number of osteoclasts and the area of bone resorption were decreased significantly (P < 0.01). To conclude, PSP can inhibit the differentiation and maturation of mouse BMMs to osteoclasts and alleviate lipopolysaccharide-induced calvarial osteolysis.
RESUMO
BACKGROUND:Bone marrow mesenchymal stem cels (BMSCs) have the ability of multi-directional differentiation. Polygonatum sibiricum polysaccharide can promote osteogenetic differentiation of mouse BMSCs by activating Wnt/β-catenin signaling pathway, which is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE:To investigate the effects of Polygonatum sibiricum polysaccharide on Wnt/β-catenin signaling pathway in the osteogenic differentiation of mouse BMSCs. METHODS:The mouse BMSCs were cultured and induced in osteoblast medium containing final concentrations (5, 10, 25, 50mg/L) of Polygonatum sibiricum polysaccharide. The mouse BMSCs treated without Polygonatum sibiricum polysaccharide was set as the negative control group. The morphological changes of cels were observed under an inverted microscope. Alkaline phosphatase (ALP) activity assay was performed by PNPP method. The mineralization nodules were observed and stained with alizarin red S and the number and area fraction were recorded under an inverted microscope. The mRNA expressions of osteogenesis-related genes ALP, Runx2, and osteocalcin were evaluated by quantitative real-time PCR (qRT-PCR). qRT-PCR and western blot were used to determine the expression level of β-catenin. The downstream β-catenin/TCF transcriptional activity was evaluated with the Dual-Luciferase Reporter Assay System. RESULTS AND CONCLUSION: Compared with the control group, polygonatum sibiricum polysaccharide significantly enhanced the alkaline phosphatase activity, the mineralization ability of cels, and the mRNA expression of ALP, Runx2 and osteocalcin in the differentiated BMSCs in a dose dependent manner (P <0.05). After induction, the mRNA expression of β-catenin was the highest on the 3rd day. Polygonatum sibiricum polysaccharide significantly increased the expression of β-catenin (P < 0.05) in the process of promoting the differentiation of BMSCs into osteoblasts, and also promoted the high-level expression of luciferase reporter gene (TOPFlash) which contains wild type TCF binding sites (P < 0.05). These results demonstrate that Polygonatum sibiricum polysaccharide can promote the osteoblast differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway.
RESUMO
BACKGROUND:Posterior lamina resection often causes loss of spinal stability, so screw rod internal fixation technology is needed to maintain the stability of lumbar spine. Finite element analysis can be used to simulate the stress distribution of the spine and internal fixation system after spinal surgery. OBJECTIVE: To build three-dimensional finite element model of spinal L1 to L3, analyze the spinal stability and stress distribution after the total laminectomy and insertion of bilateral pedicle screw using finite element method. METHODS: L1-L3 CT data could be colected from an adult healthy male volunteer. Mimics14.01, 3-matic(V6.0) and Ansys 15.0 could be used to set up the intact lumbar spine finite element model of L1-L3 (group A), the L1-L3 finite element model after L2 total laminectomy (group B), and the finite element model of L2 total laminectomy and insertion of bilateral pedicle screw (group C). We used software to simulate flexion, extension, lateral bending and axial rotation, and three kinds of models received finite element analysis. RESULTS AND CONCLUSION: (1) Based on the maximum of Von Mises under different motion states, the maximum stress was significantly lower in group A than in group B (P< 0.05). The maximum stress was significantly lower in group B than in group C (P < 0.05). (2) Based on the total deformation under different motion states, the total deformation was significantly lower in group A than in group B (P < 0.05). The total deformation was significantly lower in group C than in groups A and B (P < 0.05). (3) After the total laminectomy, vertebral body stress increased, especialy in the lamina, pedicle and joints. The range of motion of the vertebral body increased, which influenced the stability of the vertebral body. Internal fixation could decrease range of motion. Stress concentrated on the screw. Stress on the vertebral plate and pedicle decreased. The stability of vertebral body increased. Excessive stress concentrated on screw system wil increase the risk of screw breakage.
RESUMO
BACKGROUND:Posterior pedicle screw fixation is an important method to treat various diseases of the spine and to stabilize the spine. Computer navigation system can completely, intuitively and truly reveal the morphology of various tissues and their positions so that the performer can obtain three-dimensional images in time and avoid the risk area of the operation to the utmost, and can directly introduce accurate placement of the screw in the vertebral body. OBJECTIVE:To evaluate the accuracy and safety of computer navigation technique-assisted posterior spinal pedicle screw placement.METHODS:307 patients with spine diseases, who were treated in the First Affiliated Hospital of Guangxi Medical University from July 2008 to January 2014, were enrol ed in this study. They received computer navigation technique-assisted posterior spinal pedicle screw placement and laminectomy for decompression. C-arm fluoroscopy was applied to assess the precision of pedicle screw position during the operation. The mean implantation time per screw and the exposure time to radiation were recorded. 3-day postoperative radiographs and CT examination, which al owed measurements of screw position relative to pedicle position according to Andrew classification, were performed routinely. RESULTS AND CONCLUSION:Of the 1 820 screws inserted by computer-assisted navigation, 1 778 were grade I (accuracy 97.69%). A total of 92 screws were implanted in the cervical vertebrae, including 90 grade-I screws (accuracy 97.82%). 502 screws were implanted in the thoracic vertebrae, including 492 grade-I screws (accuracy 98%). 1 226 screws were implanted in the lumbar vertebrae, including 1 196 grade-I screws (accuracy 97.2%). The mean implantation time per screw was (7.0±1.5) minutes. 215 patients were fol owed up for (12±6) months. No complications such as fixator displacement or breakage or neurovascular injury occurred. Above findings suggested that computer navigation system-assisted spinal pedicle screw implantation provides real-time, multi-perspective, three-dimensional visualization of spinal anatomy, ensures the accuracy and safety of spinal pedicle screw implantation, and apparently reduces exposure time to radiation.
RESUMO
BACKGROUND:Cytokines secreted from neurons and glial cel s in early stage of spinal cord injury probably are essential factors for inducing secondary immunologic injury. OBJECTIVE:To investigate the effects of interleukin-1β, interleukin-6 and interleukin-17 on inflammatory reaction after acute spinal cord injury. METHODS:A total of 75 adult male Sprague-Dawley rats were randomly divided into control group and the spinal cord injury groups:1, 6, 24 and 72 hours. A rat model of incomplete spinal cord injury was established by the modified Al en weight drop method. The control group just underwent laminectomy. Injured spinal cord and spleen tissues were col ected at corresponding time points after model induction. Immunohistochemical staining was used to detect the distribution and expressions of interleukin-6 and interleukin-17 in spinal cord tissue. Western blot assay was utilized to detect the changes in p-STAT3 expression in injured spinal cord. RT-PCR was applied to measure the mRNA expression of interleukin-1β, interleukin-6 and interleukin-17 in the spleen. RESULTS AND CONCLUSION:The expression levels of p-STAT3, interleukin-1β, interleukin-6, and interleukin-17 were significantly higher in the spinal cord injury groups than those in the control group (P<0.05). The expression of inflammatory cytokines increased immediately after injury. Interleukin-1βand interleukin-6 levels peaked at 6 hours, and then decreased. p-STAT3 and interleukin-17 levels peaked at 24 hours, and then decreased. The expression was stil higher at 72 hours than that in the control group. Results suggested that the expression of p-STAT3-mediated pro-inflammatory cytokines interleukin-1βand interleukin-6 in early stage increased. Inflammatory cascade would enlarge in the injured area, which probably induced secondary spinal cord injury and increased interleukin-17 levels. These possibly played a key role in secondary inflammatory reaction.
RESUMO
Objective Laminectomy is destructive to bone structure in spine , which affect spinal stability .This article was to investigate the effect on spinal stability after laminectomy in different segments of vertebral plate in the treatment of lumbar intraspinal tumors. Methods Retrospective analysis were made on the data of 143 patients with lumbar intraspinal tumors from January 2009 to June 2012 in 6 hospitals.All the patients underwent laminectomy with no use of inner regular apparatus during the operation .JOA evalu-ation was applied to observe short-term efficacy , while ASIA scale for long-term efficacy .Comparison was made on lumbar spinal stability before operation and in the last visiting . Results From the observation of short-term efficacy, JOA evaluation score rised from (1.12 ± 0.65)to (1.97 ±0.71).Form the observation of long-term efficacy, ASIA scale classification was as follows:4 cases of Grade I, 6 cases of Grade II, 14 cases of Grade III, 53 cases of Grade IV and 66 cases of Grade V.In the following 12-30 months′visiting, all patients were covered.In the last postoperative visiting, patients suffering spinal instability after laminectomy were as follows:2 of 45(one seg-ment), 9 of 47(two segments), 5 of 27 (three segments) and 2 of 14 (four segments).From the observation on the postoperative spinal sta-bility and the segments in laminectomy , spinal stability of one-segment group was significantly higher than that of multi-segment group ( P=0.047).No significant difference exist between the groups of less than 2 segments and more than 3 segments as well as the groups of less than 3 segments and more than 4 segments. Conclusion A single seg-ment laminectomy on lumbar intraspinal tumors showed good postopera-tive spinal stability .But laminectomy in two or more segments implied greater risk of postoperative spinal instability .
RESUMO
Objective To investigate advantages and disadvantages of unilateral hemilaminectomy for the microsurgical removal of cervical intraspinal tumors. Methods Twenty-three patients with cervical intraspinal tumors were treated microsurgically with unilateral hemilaminectomy. In condition that the vertabral lamina was kept intact in bony structure, an opening (key-hole) was made in the unilateral lamina by using a drill or a small-sized bone rongeur. Then cervical intraspinal tumors were removed via the “key-hole”. Results Tumors were completely removed in all the 23 patients and no symptoms relating nerve injuries were observed. Postoperative follow-up for 3~16 months (mean, 8 months) revealed no recurrence and spinal deformity. Conclusions Unilateral hemilaminectomy for the treatment of cervical intraspinal tumors is a mini-invasive procedure which benefits the spinal stability. The pitfall of the technique lies in limited surgical field.
RESUMO
Objective To discuss the microsurgical treatment for tethered cord syndrome (TCS) caused by intraspinal lipomas. Methods Thirty-two patients with intraspinal lipoma TCS were treated microsurgically. On surgery the tethering lesions of lipoma were excised and anomalies restricting the spinal cord were removed. Results Following the operation, cerebrospinal fluid subcutaneous leakage had occurred in 3 patients, who were cured by expectant treatment. The patients were followed for 3~12 months (mean, 6 months). Five patients were cured, 21 got improved, and 6 had no change. Conclusions Application of microsurgical technique for intraspinal lipoma TCS has advantages of fewer complications and better improvement of nerve functions.