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Background/Aims: Hypertrophic scars (HS) formation results from reduced apoptosis and increased proliferation of fibroblasts. Therefore, apoptosis of fibroblasts is a key target for the development of novel therapeutic strategies for HS. Previous reports demonstrated that FK506 could attenuate scar formation in vivo and FK506 could also induce endoplasmic reticulum stress (ER stress). However, the effects of FK506 on ER stress-mediated apoptosis in fibroblasts remain unclear. Methods: Rat skin fibroblasts were used in the study. Cell viability was examined using cell counting Kit-8. Apoptosis was detected by Annexin V/Propidium Iodide Double Staining. Gene silencing was performed using Small Interfering RNAs (siRNAs) or via lentiviral infection. The expression of apoptosis-related proteins was determined via Western blot. Interaction between proteins was explored by co-immunoprecipitation. Results: FK506 significantly reduced cell viability and induced apoptosis in fibroblasts. Interestingly, ER stress was also activated after FK506 treatment. We further demonstrated that FK506-induced apoptosis was mediated by ER stress via activating CHOP, evidenced by decreased apoptosis after inhibition of ER stress using TUDCA or silencing expression of CHOP. Furthermore, Co-immunoprecipitation results indicated that treatment of FK506 induced disassociation of FKBP12.6 from RyR2 and its translocation from ER membrane to cytosol, consequently promoting ER stress-mediated apoptosis. Conclusion: FK506-induced fibroblasts apoptosis was mediated by ER stress via CHOP signaling pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: Epidural fibrosis, a common complication after laminectomy, has been demonstrated to be closely associated with poor surgical outcomes. Previous studies showed that taurine had remarkable anti-fibrotic effects on lung and liver fibrosis. We performed this study to investigate the effects of taurine in rat models of epidural fibrosis after laminectomy and to explore the potential molecular mechanism. Methods: Laminectomy was performed on each rat to establish epidural fibrosis model. After taurine treatment, Masson's trichrome and immunohistochemistry staining were used to examine epidural fibrosis. Cell viability was determined using the Cell Counting Kit-8 assay. Annexin V/Propidium Iodide double staining was performed to detect fibroblasts apoptosis. Microarray was adopted to identify significantly changed mRNAs. mRNA expression was measured by qRT-PCR. Lentivirus infection was performed to establish stable knockdown and overexpression cell lines. The expression of fibrosis-related proteins was determined via Western blot. Results: Taurine treatment markedly reduced laminectomy-induced epidural fibrosis in rat models. However, this effect of taurine was independent on TGF-β/Smad pathway, evidenced by no change in the expression of TGF-β and its receptors. Besides, taurine had almost no effect on cell apoptosis. Interestingly, taurine treatment significantly decreased expression of EGR1 (Early growth response protein 1), an enhancer of fibrosis, both in vivo and in vitro. Furthermore, overexpression of EGR1 increased activation of fibroblasts, while EGR1 knockdown achieved an opposite effect, indicating that EGR1 plays a key role in the inhibitory effect of taurine on TGF-β-induced fibrosis. Conclusions: Reduced epidural fibrosis in vivo and decreased activation of fibroblasts in vitro after taurine treatment was mediated by EGR1. Taurine promises to be a potential prevention for epidural fibrosis after laminectomy. [ABSTRACT FROM AUTHOR]

Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to investigate the effects of topical application mitomycin-C (MMC) on wound healing after laminectomy. 60 adult male SD rats were equally and randomly divided into five groups. Laminectomy was performed at the level of L1 in all rats. After hemostasis was achieved, cotton pads soaked with saline and MMC (0.1mg/ml, 0.3mg/ml, 0.5mg/ml and 0.7mg/ml) were directly subjected to the exposed dura for 5min in each group. Two weeks after laminectomy all the rats were killed. The vertebral column including the back scar tissue and muscles was obtained to make paraffin sections. The hematoxylin–eosin staining and Masson staining were performed with the obtained paraffin sections. The number of the fibroblast and the capillary density were counted by the hematoxylin–eosin staining slice. The extent of epidural fibrosis and the expression of vascular endothelial growth factor (VEGF) were evaluated by the immunohistochemical slice through a computer image analysis system. Our data showed that the number of fibroblast, capillary density and fibrotic tissue in the 0.5 and 0.7mg/ml MMC groups was significantly lower than the control, 0.1 and 0.3mg/ml MMC groups; while the expression of VEGF in control and 0.1mg/ml MMC groups was notably higher than 0.3, 0.5 and 0.7mg/ml MMC groups. Topical application of MMC above the concentration of 0.3mg/ml could affect all steps of the wound healing process via inhibiting the angiogenesis and fibroblast proliferation, thus delayed the wound healing after laminectomy. [Copyright &y& Elsevier]

Abstract: There is increasing evidence that topical application of mitomycin-C can be beneficial in reducing epidural scar adhesion. However, the ideal concentration of mitomycin-C is unknown. The purpose of this study was to verify its efficacy for preventing epidural adhesion and the immediate electrophysiological responses caused by it in a laminectomy model. Seventy rats underwent laminectomy at L-1 and L-2. Cotton pads soaked with saline and various concentrations of mitomycin-C (0.1mg/ml, 0.3mg/ml, 0.5mg/ml and 0.7mg/ml) were applied to the exposed dura for 5min. Spine somatosensory evoked potentials (SSEP) were monitored in preoperative and the immediate electrophysiological responses of mitomycin-C used. Four weeks postlaminectomy the rats were killed. The area of epidural scar tissue and degree of epidural adhesion were determined by 7.0T Micro MR imaging. Macroscopic evaluations were performed according to the Rydell standard. The results showed that severe epidural adhesion was formed in the saline group and no dural adherence or incomplete adhesions were found in the mitomycin-C group. The Rydell classification and the degree of epidural adhesion and the area of the scar in 0.5mg/ml group and 0.7mg/ml mitomycin-C group revealed a significant decrease compared with the control group and 0.1mg/ml group and 0.3mg/ml mitomycin-C group. The spine sensory evoked potentials did not alter obviously in both preoperative and the immediate electrophysiological responses of mitomycin-C used. In conclusion, locally applied mitomycin-C in a concentration of 0.5mg/ml and 0.7mg/ml mitomycin-C may be the optimal concentration in preventing postlaminectomy epidural adhesion. [Copyright &y& Elsevier]

Abstract: Astrocytes play an important role in protecting neurons during ischemia and reperfusion in the central nervous system. Although many studies have shown that oxygen–glucose deprivation (OGD) can induce astrocyte apoptosis, the role of PERK/eIF2α/ATF4 integrated stress response (ISR) in astrocyte apoptosis mediated by oxygen–glucose–serum deprivation (OGSD)/restoration remains uncertain. Astrocytes were subjected to a combination of oxygen, glucose, and serum deprivation for 8h followed by restoration. Hoechst 33342 staining was performed to quantify apoptotic astrocytes and cell viability was assessed with Cell Counting Kit-8 (CCK8). Immunocytochemical analysis and Western blotting for some related molecules, including pancreatic ER stress kinase (PERK), p-PERK, eukaryotic initiation factor 2 alpha (eIF2α), p-eIF2α, activating transcription factor 4 (ATF4), caspase-12, were examined. Caspase activation and apoptosis were detected in neonatal rat astrocytes from spinal cord subjected to OGSD/restoration. We also observed an increase in cytoplasmic staining of p-eIF2α in astrocytes (8h OGSD/15min restoration) compared with that of non-treated cells. In addition, we found the sequential activation of PERK, eIF2α, and ATF4 during OGSD/restoration by Western blotting. These results indicate that both the PERK/eIF2α/ATF4 ISR and activation of caspase-12 may be involved in apoptosis of spinal cord astrocytes induced by OGSD/restoration. [Copyright &y& Elsevier]

Abstract: Background context: Recovery from spine surgery is usually accompanied with the development of epidural scar adhesions from the abnormal proliferation of fibroblast and extracellular matrix–related metabolism. Polyethylene glycol (PEG) could alleviate the postsurgical adhesion formation with still leaving a gap between the sheet and the dura. However, PEG film could not function as a three-dimensional barrier to prevent adhesion completely. Mitomycin C (MMC) could also reduce the scar formation after surgery, but cytotoxicity and the administrative pathway prevent its application. Purpose: Our purpose was to design and attest the role of new delivery system MMC-PEG controlled-release film in preventing the epidural scar adhesions after laminectomy in the rat model. Study design/setting: A total laminectomy of L1 in the rat model was used to assess epidural fibrosis between and around the spinal nerves using a histochemistry assessment along with flow cytometry analysis. Patient sample: The sample comprises 64 adult male Sprague-Dawley rats. Outcome measures: The outcome measures are macroscopic evaluation, histological analysis, and flow cytometry analysis. Methods: Lumbar laminectomies at L1 and L2 with a L1–L2 disc injury were performed on 64 adult male Sprague-Dawley rats. The rats were then randomized into four groups. In Group I, 25 mg PEG film was applied on the dura mater in the laminectomy area before the layers were sutured. In Group II, a cotton pad soaked with 0.05% MMC solution was kept on the laminectomy area for 5 minutes. In Group III, 25 mg PEG film containing 0.01% MMC was implanted on the laminectomy area. In Group IV, the laminectomy area was flushed with saline before wound closure. The rats were sacrificed 4 weeks after the operation. Macroscopic evaluation and histological analysis of epidural scar adhesion with the hematoxylin and eosin stain and Masson stain were used followed by the quantification of hydroxyproline (Hyp) and flow cytometry analysis of the apoptosis of fibroblasts in the scar tissues. Results: The recovery of all rats was uneventful after the operations. In the laminectomy sites of rats treated with MMC or MMC-PEG, the dura mater was clean without any evident adhesion or membrane. Collagen tissue hyperplasia significantly decreased in the MMC- or MMC-PEG–treated models. Accordingly, Hyp concentration was significantly reduced in these two groups compared with saline-control group. In addition, the apoptosis of fibroblasts, however, was significantly elevated in the MMC or MMC-PEG group compared with the saline-control group. Conclusions: These results demonstrate that the treatment of postlaminectomy wounds with MMC-PEG film reduces the severity of adhesion by decreasing the concentration of Hyp and increasing the apoptosis of fibroblasts. [Copyright &y& Elsevier]