Guodong Shi

Increased miR‐195 aggravates neuropathic pain by inhibiting autophagy following peripheral nerve injury

Following peripheral nerve injury (PNI) microglia proliferates and adopts inflammation that contributes to development and maintenance of neuropathic pain. miRNAs and autophagy are two important factors in the regulation of inflammation. However, little is known about whether miRNAs regulate neuroinflammation and neuropathic pain by controlling autophagy. In the study, we demonstrated that miR‐195 levels were markedly increased in rats subjected to L5 spinal nerve ligation (SNL). Upregulated miR‐195 was also found in spinal microglia of rats with SNL. The overexpression of miR‐195 contributed to lipopolysaccharide‐induced expression of proinflammatory cytokines IL‐1β, TNF‐α, and iNOS in cultured microglia. Upregulated miR‐195 also resulted in increased mechanical and cold hypersensitivity after PNI, whereas miR‐195 inhibition reduced mechanical and cold sensitivity. We further demonstrated that PNI significantly inhibited microglial autophagy activation, whereas miR‐195 inhibitor treatment increased autophagy activation and suppressed neuroinflammation in vivo and in vitro. More important, autophagy inhibition impaired miR‐195 inhibitor‐induced downregulation of neuroinflammation and neuropathic pain. Additionally, ATG14 was identified as the functional target of miR‐195. Conclusions: These data demonstrated that miR‐195/autophagy signaling represents a novel pathway regulating neuroinflammation and neuropathic pain, thus offering a new target for therapy of neuropathic pain.

Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury

It is increasingly clear that microRNAs (miRNAs) play an important role in controlling cell survival. However, the functional significance of miRNAs in ischemic brain injury remains poorly understood. In the present study, we assayed the expression levels of miR-29b after ischemic brain injury, and defined the target genes and biological functions of miR-29b. We found that the miR-29b levels were significantly increased in rat brain after transient middle cerebral artery occlusion and neurons after oxygen–glucose deprivation. Moreover, ectopic expression of miR-29b promoted neuronal cell death, whereas its repression decreased cell death. Furthermore, we verified that miR-29b directly targeted and inhibited Bcl2L2 gene expression, and then increased neuronal cell death. Importantly, Bcl2L2 overexpression rescued neuronal cell death induced by miR-29b. These results suggest an important role of miR-29b in regulating neuronal cell death, thus offering a new target for the development of therapeutic agents against ischemic brain injury.

RUNX2 Polymorphisms Associated with OPLL and OLF in the Han Population

BackgroundRunt-related transcription factor 2 (RUNX2), BMP-2, COL6A1, and VDR are four genes that may be related to ossification of the spinal ligament. However, their pathogenetic relevance remains unclear. Most cases of ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF) have been reported in Asian populations, but the polymorphic loci in these genes may vary among people of different races.PurposesWe identified (1) polymorphic loci in four genes (RUNX2, BMP-2, COL6A1, and VDR) in OPLL and OLF in Chinese Han patients and (2) identified loci related to these diseases.MethodsWe analyzed 19 single nucleotide polymorphisms (SNPs) in four candidate genes in 200 Han individuals (82 patients and 118 control subjects) by the Sequenom system. The genotype distribution and allele frequency of each SNP in the control and patient groups were compared. We then determined the relationships between the loci and the occurrence of OPLL and OLF.ResultsGenotyping showed RS1321075 and RS12333172 in RUNX2 differed between the patients and the control subjects. Both loci were located on chromosome 6 and exhibited linkage disequilibrium. One of the two blocks was a haplotype, thus suggesting a link between this block and increased incidence of OPLL and OLF.ConclusionAlthough the detailed mechanism of the SNP is unclear, our data suggest RUNX2 could be responsible for ectopic bone formation in the spinal ligament in the Chinese Han population. However, we found no obvious connection between polymorphic loci of COLA1, BMP-2, and VDR and the diseases.Clinical RelevanceMolecular genetic studies have identified several candidate genes that may be responsible for increased susceptibility to the diseases. Information regarding SNPs among the certain candidate genes may improve understanding of the disease and assist in developing new diagnostic gene tools during early episodes of the disease.

Lysyl oxidase polymorphisms and susceptibility to osteosarcoma.

Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Lysyl oxidase (LOX) catalyzes the cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. In the current study, we performed genomic sequencing on all seven exons -including the intron-exon splice sites, and the putative promoter region of LOX gene - followed by luciferase reporter assay to analyze the function of newly identified polymorphisms. Associations between LOX polymorphisms and osteosarcoma were then evaluated. Our sequencing data revealed three polymorphisms (−22G/C, 225C/G, and 473G/A) in the exons and promoter region of LOX. The −22G/C polymorphism lies in the downstream core promoter element (DPE) region and caused a decrease in promoter activity of LOX. The prevalence of the −22C allele and 473A allele were significantly increased in osteosarcoma patients compared to controls (odds ratio [OR]u200a=u200a3.88, 95% confidence interval [CI] u200a=u200a1.94−7.78, pu200a=u200a4.18×10−5, and ORu200a=u200a1.38, 95%CIu200a=u200a1.07−1.78, pu200a=u200a0.013; p 0.0167 was considered significant after Bonferroni correction). Analyzing haplotype showed that the frequency of CCG haplotype (−22, 225, 473) was significantly higher in osteosarcoma cases than in healthy controls after Bonferroni correction (pu200a=u200a4.46×10−4). These results indicate that the −22G/C polymorphism may affect the expression of LOX, and that −22G/C and 473G/A polymorphisms may be new risk factors for osteosarcoma. These findings reveal a potential new pathway by which genetic polymorphisms may affect human diseases.

Study design: in vitro and in vivo assessment of bone morphogenic protein 2 combined with platelet-rich plasma on treatment of disc degeneration

ObjectiveOur aim was to investigate the biological effects of bone morphogenic protein 2 (BMP2) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocyte-like cells in platelet-rich plasma (PRP) gel in vitro. In addition, the effectiveness of BMP2-transduced BMSCs in combined with PRP gel to repair the degenerated intervertebral disc in a rabbit model was also evaluated.Summary of background dataPrevious studies have shown that tissue engineering provides many promising advantages to treating disc degeneration and may reverse the pathological process of disc degeneration.MethodsThe expressions of types I, II and X collagen, aggrecan and Sox9 of the BMP2-transduced BMSCs in monolayer or PRP gel were examined by reverse transcriptase polymerase chain reaction (RT-PCR). Sixty New Zealand white rabbits were divided into five groups: 12 normal controls; 12 puncture operated with only disc degeneration being induced; 12 PRP transplantation animals; 12 BMSC and PRP-transplantation animals; 12 BMP2-transduced BMSCs and PRP-transplantation animals. The effect of BMP2-transduced BMSCs on degenerated discs were evaluated by magnetic resonance image (MRI) scan, histology, immunohistochemistry and Western blot analysis.ResultsBMP2 could facilitate chondrogenic differentiation of BMSCs in monolayer or PRP gel. The discs treated with BMP2-transduced BMSCs exhibited relatively well-preserved nucleus pulposus (NP) structure. Significantly higher T2-weighted signal intensity and a greater amount of extracellular matrix were observed in the BMP2-transduced BMSC group compared with other groups. In addition, the presences of BMP2-transduced BMSCs were identified at week 12 postoperatively in vivo.ConclusionsBMP2-transduced BMSCs can maintain the chondrocyte-like phenotype in PRP gel in vitro, and the combined use of these two agents can significantly promote repair of the degenerated discs in vivo.

Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification.

Aims: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. Methods: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. Results: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. Conclusion: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

Comparing effects of cervical anterior approach and laminoplasty in surgical management of cervical ossification of posterior longitudinal ligament by a prospective nonrandomized controlled study

INTRODUCTIONnThe laminoplasty has been the mostxa0widelyxa0used surgical method for OPLL. In recent years, increasing attention has been drawn to the anterior operative approaches for surgical treatment of cervical OPLL. However, which method is the optimum selection for therapy of cervical OPLL is still obscure. Therefore, we performed this prospective nonrandomized clinical study in patients with multilevel cervical myelopathy due to OPLL and compare the therapeutic efficiency of laminoplasty and anteriorxa0approach (cervical discectomy and/or cervical corpectomy) in the management of multilevel cervical OPLL.nnnHYPOTHESISnThere is no difference in clinical effects between anterior cervical spine surgery and laminoplasty in the treatment of multilevel cervical OPLL.nnnMATERIAL AND METHODSnA total of 150 consecutive patients with multilevels of cervical OPLL underwent anterior approaches (ACDF, ACCF and HDF) from July 2010 to June 2014, which were enrolled in this study. During the same period, one hundred and two patients receiving the laminoplasty were enrolled in the study. The clinical effects, alignment and range of motion (ROM) of cervical spine in patients of the anterior group and posterior group were assessed, respectively. The effects of high signals in T2 weighed MRI scans and percentage of spinal canal stenosis in these patients were also evaluated. Finally, postoperative complications regarding each group were analyzed.nnnRESULTSnAlthough significant differences in types of OPLL and preoperative sagittal alignment of cervical spine occurred in the two groups (P<0.05), clinical effects of the two groups were similar (P>0.05). The cervical curvature in laminoplasty group showed significant decrease at final follow-up (P<0.05). For ROM of cervical spine, no significant alteration was observed in both groups. The high T2 weighed signals and rate of spinal canal stenosis can influence clinical effects of both anterior group and laminoplasty group. In addition, significantly higher complication rate was observed in laminoplasty group compared with anterior group (P<0.05).nnnDISCUSSIONnBoth anterior and laminoplasty approaches can be considered effective and safe procedures in the treatment of the multilevel OPLL. However, the anterior approach with relatively lower incidence of postoperative complications is a better choice for cases with poor cervical curvature and serious spinal canal stenosis.nnnTYPE OF STUDY AND LEVEL OF PROOFnLevel 3 nonrandomized, controlled clinical trials.

Surgical Incision and Approach in Thoracolumbar Extreme Lateral Interbody Fusion Surgery: An Anatomic Study of the Diaphragmatic Attachments.

Study Design. Cadaveric study. Objective. To provide anatomical basis for deciding the surgical approach and skin incision in thoracolumbar extreme lateral interbody fusion (XLIF) by delineating the attachment points of diaphragm. Summary of Background Data. Although the general anatomy of the thoracic diaphragm is well described, the specific attachment points of diaphragm concerned with the XLIF approach is yet to be elaborated. Methods. Dissections were performed on 21 cases of formalin fixed specimens (12 males, 9 females, a total of 42 sets of data). Special attention was paid to the attachment points of diaphragm on both sides at the midaxillary line (MAL point) and the vertebral level parallel to the MAL point (VL-MAL). The attachment points of diaphragm on the front and back edge of the spinal column (FES point and BES point) were also described. Results. The MAL point of diaphragm muscle lied between the inferior edge of the 10th rib and the superior edge of the 12th rib (20 out of 21 on left, 21 out of 21 on right). VL-MAL lied between L1 and L2 vertebrae level (20 out of 21 on left, 18 out of 21 on right). The attachments on both sides of the vertebral column mainly located between the upper edge of T12 vertebrae and L1-L2 disc (38 out of 42). Conclusion. A transthoracic approach should be considered when the target level was above T12 vertebrae, whereas a retroperitoneal approach should be chosen when target level was below L1-L2 disc. If the target level is located between T12 and L1-L2 disc, whether via transthoracic, retropleural, or retroperitoneal approach should be determined according to the conditions of patients and the skill and experience of the surgeon. Incision should be made above the 10th rib for the transthoracic approach and below the 12th rib for the retroperitoneal approach. Level of Evidence: 4

miR-32-5p-mediated Dusp5 downregulation contributes to neuropathic pain

Previous studies have demonstrated that microRNAs (miRNAs) play important roles in the pathogenesis of neuropathic pain. In the present study, we found that miR-32-5p was significantly upregulated in rats after spinal nerve ligation (SNL), specifically in the spinal microglia of rats with SNL. Functional assays showed that knockdown of miR-32-5p greatly suppressed mechanical allodynia and heat hyperalgesia, and decreased inflammatory cytokine (IL-1β, TNF-α and IL-6) protein expression in rats after SNL. Similarly, miR-32-5p knockdown alleviated cytokine production in lipopolysaccharide (LPS)-treated spinal microglial cells, whereas its overexpression had the opposite effect. Mechanistic investigations revealed Dual-specificity phosphatase 5 (Dusp5) as a direct target of miR-32-5p, which is involved in the miR-32-5p-mediated effects on neuropathic pain and neuroinflammation. We demonstrated for the first time that miR-32-5p promotes neuroinflammation and neuropathic pain development through regulation of Dusp5. Our findings highlight a novel contribution of miR-32-5p to the process of neuropathic pain, and suggest possibilities for the development of novel therapeutic options for neuropathic pain.

Anterior Controllable Antedisplacement Fusion (ACAF) for Severe Cervical Ossification of the Posterior Longitudinal Ligament: Comparison with Anterior Cervical Corpectomy with Fusion (ACCF)

OBJECTIVEnAnterior cervical corpectomy and fusion (ACCF), in which a ventral constriction is resected, can decompress myelopathy and is considered the optimal treatment for ossification of the posterior longitudinal ligament (OPLL) up to now. However, its disadvantages are incomplete decompression, high surgery- and implant-related complication rates, and extremely surgical technique demanding. Our object was to introduce anterior controllable antedisplacement fusion (ACAF), a new surgical technique to treat OPLL, and compare it with ACCF.nnnMETHODSnACAF was performed on 34 patients with spinal stenosis with myelopathy due to severe (occupying rate ≥50%) OPLL. Pre- and postoperatively, we measured decompression width and spinal canal area on cross-sectional computed tomography and morphology and anteroposterior diameter of the spinal cord at the most severely affected segment on cross-sectional magnetic resonance imaging and cross-sectional computed tomography. Japanese Orthopedic Association scoring was used to evaluate neurologic status. The ACAF group and a control group of 36 patients with ACCF were compared.nnnRESULTSnPostoperatively, decompression width (17.9 ± 1.0 vs. 15.1 ± 0.8 mm; P < 0.01), spinal canal area (150.4 ± 31.6 vs. 127.0 ± 27.0 mm2; P < 0.01), and anteroposterior spinal cord diameter (5.4 ± 0.6 vs. 5.0 ± 1.1 mm; P < 0.05) were significantly greater in the ACAF group. At 6 months,xa0mean Japanese Orthopedic Association score was significantly better in the ACAF group (15.4 ± 0.9 vs. 14.5 ± 2.5 points; Pxa0= 0.04).nnnCONCLUSIONSnACAF, providing adequate decompression of the spinal cord and good outcomes, is a well choice in the treatment of spinal stenosis due to severe OPLL.