Pirfenidone inhibits fibroblast proliferation, migration or adhesion and reduces epidural fibrosis in rats via the PI3K/AKT signaling pathway.

OBJECTIVE: This present study aims to assess the effect of pirfenidone (PFD) on inhibiting fibroblast proliferation, migration or adhesion in vitro and reducing laminectomy-induced epidural fibrosis in vivo. METHODS: The effect of PFD on proliferation inhibition was evaluated with flow cytometry, CCK-8, EdU and western-blotting assays. Altered properties in migration and adhesion were confirmed by wound-scratch, transwell, immunofluorescence (IF), cell adhesion and western-blotting assays. Additionally, fifty male healthy Sprague-Dawley rats were subjected to laminectomy and then treated with various concentrations of PFD. After 4 weeks, the degree of epidural fibrosis was evaluated by histological analysis. RESULTS: In vitro, the results of flow cytometry, CCK-8, EdU and western-blotting assays showed that PFD reduced fibroblast proliferation by a dose-dependent manner. And the results of wound-scratch, transwell, IF, cell adhesion and western-blotting assays showed that the migration and adhesion of fibroblasts could be inhibited and the cytoskeleton could also be altered in a dose-dependent manner. And the inhibitory effect of PFD could be partially reversed in the PI3K overexpression experiment, which indicated that the capability of PFD to inhibit fibroblast proliferation, migration and adhesion might be through the PI3K/AKT signaling pathway. In vivo, an obvious reduction in epidural fibrosis was observed in groups topically treated with PFD. CONCLUSIONS: Topical PFD application obviously suppressed laminectomy-induced epidural fibrosis, possibly by inhibiting fibroblast proliferation, migration and adhesion via the PI3K/AKT signaling pathway. PFD may be a safe and effective pharmaceutical to reduce clinical epidural fibrosis.

Cervical Epidural Contrast Spread Patterns in Fluoroscopic Antero-Posterior, Lateral, and Contralateral Oblique View: A Three-Dimensional Analysis.

Objective: To describe and to analyze cervical epidural contrast patterns seen in antero-posterior (AP), contralateral oblique (CLO), and lateral view. To identify factors that might help in predicting contrast distribution pattern and extent. Method: Spread of contrast in the cervical epidural space was prospectively studied in AP, lateral, and three CLO views. Results: CLO view showed contrast spread of variable thickness with its posterior margin overlying the ventral interlaminar line (VILL). In the lateral view, the spread was also of variable thickness, but the posterior margin of the contrast lay on the spinolaminar line in only 10 of 24 patients. Ventral contrast spread was not visualized in any patient. In the AP view, bilateral spread was seen in 14 of 24 subjects, and nerve root spread was seen in 16 of 24 subjects. No association of the pattern of spread or dispersion was seen to patient age, volume injected, or needle location. Conclusions: The CLO view provides a consistent radiological landmark for the posterior margin of contrast in the dorsal epidural space; the lateral view fails to provide such a consistent landmark. The thickness of the spread is variable, both in the CLO and in the lateral view. Thick spread extending into the foramen in the CLO view and over the articular pillars in the lateral view is frequent and should not be misconstrued as subdural or intrathecal spread. In contradistinction to previous studies, true ventral spread was not seen in any patient. When using low volumes, contrast spread is independent of patient age, volume injected, or needle tip location in the AP view.

Taurine Reduced Epidural Fibrosis in Rat Models after Laminectomy via Downregulating EGR1.

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-ß/Smad pathway, evidenced by no change in the expression of TGF-ß 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-ß-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.

Anti-adhesive effect of poloxamer-based thermo-sensitive sol-gel in rabbit laminectomy model.

Poloxamer-based thermo-sensitive sol-gel has been developed to reduce the incidence of postoperative scar formation at the laminectomy site. The purpose of this study was to evaluate the anti-adhesive effect of poloxamer based thermo-sensitive sol-gel compared to hyaluronate based solution after laminectomy, using a rabbit model. A thermo-sensitive anti-adhesive with a property of sol-gel transition was manufactured by a physical mixture of Poloxamer188/407, Chitosan and Gelatin. The viscosity in different temperatures was assessed. 72 adult New Zealand rabbits underwent lumbar laminectomy and were randomly divided into experimental (treated with the newly developed agent), positive (treated with hyaluronate based solution), and negative control groups. Each group was subdivided into 1 and 4-week subgroups. Gross and histological evaluations were performed to assess the extent of epidural adhesion. The experimental group showed significantly higher viscosity compared to the positive control group and showed a significant increase of viscosity as the temperature increased. Gross evaluation showed no statistically significant differences between the 1- and 4-week subgroups. However, histologic evaluation showed significant differences both in 1- and 4-week subgroups. Although the 4-week histologic results of the experimental and the positive control subgroups showed no significant difference, both subgroups revealed higher value compared to the negative control subgroup with regard to the ratio of adhesion less than 50 %. The new poloxamer based thermo-sensitive agent showed superior efficacy over the hyaluronate based agent at 1 week postoperatively. At 4 weeks postoperatively, there were no statistically significant differences between the two agents, although both showed efficacy over the sham group.

Hemostasis vs. epidural fibrosis?: A comparative study on an experimental rat model of laminectomy.

AIM: The aim of this study was to evaluate the histopathological and biochemical impact and effectiveness of two hemostatic agents, Ankaferd blood stopper (ABS) and Microporous Polysaccharide Hemospheres (MPH), on epidural fibrosis in an experimental rat laminectomy model. MATERIAL AND METHODS: Twenty adult Wistar albino rats were divided into MPH-treated (n=6), ABS-treated (n=6) and control (n=8) groups. Laminectomy of the lumbar spine was performed in all animals and treatment groups were exposed to MPH and ABS while closure was applied in control group as per usual. Epidural fibrosis was evaluated in all groups macroscopically, histopathologically, biochemically and with electron microscopy four weeks later. RESULTS: Statistically, it was found that MPH-treated group had significantly less epidural fibrosis compared to ABS-treated and control groups. CONCLUSION: We compared two hemostatic agents for their propensity to cause adhesions in the present study. Our results show that MPH significantly reduces epidural scar formation and dural adhesion in a rat model of laminectomy while ABS increases postoperative fibrosis.

CCN2 and CCN5 exerts opposing effect on fibroblast proliferation and transdifferentiation induced by TGF-ß.

Epidural fibrosis might occur after lumbar discectomy and contributes to failed back syndrome. Transforming growth factor (TGF)-ß has been reported to influence multiple organ fibrosis, in which connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 2 (CCN2) and CCN5 are involved. However, the effect of CCN2 and CCN5 on TGF-ß induced fibrosis has not yet been elucidated. This study reports that CCN2 and CCN5 play opposing roles in cell proliferation and transdifferentiation of human skin fibroblasts or rabbit epidural scar-derived fibroblasts exposed to TGF-ß. We observed that TGF-ß1 induced fibroblasts proliferation and differentiation in a dose-dependent manner (from 0 µg/L to 20 µg/L). Meanwhile, CCN2 expression is up-regulated while CCN5 expression is inhibited by TGF-ß1 exposure. Furthermore, it is demonstrated that CCN2 overexpression leads to promoted proliferation and elevated collagen and α-smooth muscle actin (α-SMA) expression, which are inhibited by CCN5 overexpression. Moreover, it is shown that the cysteine knot (CT) domain, present in CCN2 but absent in CCN5, plays an essential part in fibroblast proliferation and differentiation. Additionally, enhanced TGF-ß and CCN2 expression but decreased CCN5 expression is found in rabbit epidural scar-derived fibroblasts. Overall, the results show the opposing effects of CCN2 and CCN5 on fibroblast proliferation and transdifferentiation induced by TGF-ß.

ERK2 small interfering RNAs prevent epidural fibrosis via the efficient inhibition of collagen expression and inflammation in laminectomy rats.

Laminectomy is a widely accepted treatment for lumbar disorders. Epidural Fibrosis (EF) is a common post-laminectomy or post-discectomy complication, which is thought to cause recurrent pain. RNA interference (RNAi) is a process by which double-stranded RNA triggers the destruction of mRNAs sharing the same sequence. Previously, extra-cellular signal-regulated kinase (ERK) 2 plays crucial roles in suppressing the collagen expression. To investigate the effects of lentiviral ERK2 siRNA on the prevention of post-laminectomy EF formation in a rat model, a controlled double-blinded study was conducted in 75 healthy adult Wistar rats that underwent laminectomy. They were divided randomly into 3 groups according to the treatment method: (1) control group; (2) ERK scrRNA group; (3) ERK siRNA group. All rats were euthanized humanely 4 weeks post-laminectomy. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions were performed. The hydroxyproline content, Rydell score, vimentin cells density, fibroblasts density, inflammatory cells density and inflammatory factors expressions all suggested better results in ERK siRNA group than other two groups. None of the rats expired and no obvious adverse effects were observed. Local delivery of a lentiviral siRNA targeting ERK2 can prevent epidural scar adhesion in post-laminectomy rat via inhibiting collagen expression and inflammation.

Ultrasound assessment of cranial spread during caudal blockade in children: the effect of different volumes of local anaesthetics.

BACKGROUND: Despite the large amount of literature on caudal anaesthesia in children, the issue of volume of local anaesthetics and cranial spread is still not settled. Thus, the aim of the present prospective randomized study was to evaluate the cranial spread of caudally administered local anaesthetics in children by means of real-time ultrasound, with a special focus on the effects of using different volumes of local anaesthetics. METHODS: Seventy-five children, 1 month to 6 yr, undergoing inguinal hernia repair or more distal surgery were randomized to receive a caudal block with 0.7, 1.0, or 1.3 ml kg(-1) ropivacaine. The cranial spread of the local anaesthetic within the spinal canal was assessed by real-time ultrasound scanning; the absolute cranial segmental level and the cranial level relative to the conus medullaris were determined. RESULTS: All the blocks were judged to be clinically successful. A significant correlation was found between the injected volume and the cranial level reached by the local anaesthetic both with regards to the absolute cranial segmental level and the cranial level relative to the conus medullaris. CONCLUSIONS: The main finding of the present study was positive, but numerically small correlation between injected volumes of local anaesthetic and the cranial spread of caudally administered local anaesthetics. Therefore, the prediction of the cranial spread of local anaesthetic, depending on the injected volume of the local anaesthetic, was not possible. EudraCT Number: 2008-007627-40.

Neuraxial techniques in patients with pre-existing back impairment or prior spine interventions: a topical review with special reference to obstetrics.

Many anaesthetists consider neurological disorders of all kinds as a contraindication for regional anaesthesia particularly for neuraxial techniques. This hesitation is partly rooted in fears of medicolegal problems but also in the heterogeneous literature. Therefore, the present topical review is an attempt to describe the feasibility and the risks of neuraxial techniques in patients with spinal injury, anatomical compromise, chronic back pain or previous spinal interventions, ranging from 'minor' types like epidural blood patches to major surgery such as Harrington fusions. Most reviews and case reports were describing experiences in obstetrics as these patients are more likely to insist on neuraxial blocks. In the acute phase of new neurologic injury, general anaesthesia may be the technique of choice to prevent further haemodynamic and respiratory deterioration. After the acute phase, current evidence is mostly reassuring with respect to the risks of neuraxial blocks as they may even be recommendable in some conditions. Ultrasound technology may be of additional help to increase the success rate. A careful pre-operative examination remains mandatory, while patients should be sufficiently informed about technical aspects and possible relapses or progression of their disease. When necessary, patients should have additional technical and clinical examinations as close as possible to surgery to establish the actual pre-operative status. Most patients may benefit more from spinal techniques rather than from less reliable epidural ones. High concentrations and volumes of local anaesthetics should be avoided at all times, especially in patients with nerve compression, large disc herniation or spinal stenosis.

Management of hypertrophic pylorus stenosis with ultrasound guided single shot epidural anaesthesia--a retrospective analysis of 20 cases.

AIM: To retrospectively describe the performance of ultrasound guided thoracic epidural anaesthesia under sedation for anaesthesia management of open pyloromyotomy. BACKGROUND: Anaesthesia management for hypertrophic pylorus stenosis (HPS) is usually performed under general anaesthesia with tracheal intubation. Only a few publications describe avoidance of tracheal intubation in infants by using spinal or caudal anaesthesia. The present retrospective analysis describes the performance of ultrasound guided thoracic epidural anaesthesia under sedation for anaesthetic management of open pyloromyotomy. METHODS: Twenty consecutive infants scheduled for pyloromyotomy according to the Weber-Ramstedt technique were retrospectively analysed. After sedation with nalbuphine and propofol, an ultrasound guided single shot thoracic epidural anaesthesia was performed with 0.75 ml·kg(-1) ropivacaine 0.475%. Insufficient blockade was defined as increase of HR > 15% from initial value and/or any movements at skin incision. In those cases we were prepared for rapid sequence intubation according to the departmental standard. RESULTS: All pyloromyotomies could be performed under single shot thoracic epidural anaesthesia and sedation. One case of moderate oxygen desaturation was treated with intermittent ventilation via face mask. CONCLUSIONS: Thoracic epidural anaesthesia under sedation for pyloromyotomy has been a useful technique in this retrospective series of infants suffering from HPS. In 1/20 infants short term assisted ventilation via face mask was required. Undisturbed surgery was possible in all cases.

Cranial epidural spread of contrast medium and new methylene blue dye in sternally recumbent anaesthetized dogs.

OBJECTIVE: To examine the spread of solution in the epidural space of sternally recumbent dogs. STUDY DESIGN: Prospective experimental trial. Animals Ten healthy adult Beagle dogs weighing 7.6 ± 1.1 kg. METHODS: Dogs were anaesthetized with total intravenous propofol infusion, and placed in sternal recumbency. A volume of 0.2 mL kg(-1) contrast medium (CM) containing 1% new methylene blue (MB) dye was administered into the lumbosacral epidural space. Left to right lateral radiographs using a horizontal beam were taken every 5 minutes for 45 minutes. The perpendicular height (PH) between floor of the epidural canal of the highest vertebra and that of lumbosacral spinal canal was measured on radiographs. The angle of slope from the injection point toward the highest vertebral floor was measured. Immediately after taking the last radiographic image, dogs were euthanized and a laminectomy was performed from the cervical to lumbar vertebrae for visual evaluation of MB spread. The spread of CM and of MB as counted in number of stained vertebra were compared, and each of these data sets were further compared to PH and angle, using linear regression analyses. RESULTS: The PH and angle were (mean ± SD) 3.8 ± 0.8 cm and 14.8 ± 2.8° respectively. The most cranial spread of CM was at 12.7 ± 5.7 (range: C6-L3) vertebrae, and at 14.0 ± 5.4 (range: C6-L2) vertebrae for MB staining. There were no significant correlations between PH and spread of CM (R(2) = 0.08) or MB (R(2) = 0.13), between angle and spread of CM (R(2) = 0.05) or MB (R(2) = 0.02), respectively. CM and MB demonstrated proportional relationship (R(2) = 0.82, p < 0.001). CONCLUSIONS: No significant inhibitory effect of upward slope on cranial epidural spread of the solution was observed. Other factors may have greater effect on epidural spread in sternally recumbent dogs.

The peridural membrane of the spine has characteristics of synovium.

The peridural membrane (PDM) is a well-defined structure between dura mater and the wall of the spinal canal. The spine may be viewed as a multi-segmented joint, with the epidural cavity and neural foramina as joint spaces and PDM as synovial lining. The objective of this investigation was to determine if PDM has histological characteristics of synovium. Samples of the PDM of the thoraco-lumbar spine were taken from 23 human cadavers and analyzed with conventional light microscopy and confocal microscopy. Results were compared to reports on similar analyses of synovium in the literature. Histological distribution of areolar, fibrous, and adipose connective tissue in PDM was similar to synovium. The PDM has an intima and sub-intima. No basement membrane was identified. CD68, a marker for macrophage-like-synoviocytes, and CD55, a marker for fibroblast-like synoviocytes, were seen in the lining and sub-lining of the PDM. Multifunctional hyaluronan receptor CD44 and hyaluronic acid synthetase 2 marker HAS2 were abundantly present throughout the membrane. Marked presence of CD44, CD55, and HAS2 in the well-developed tunica muscularis of blood vessels and in the body of the PDM suggests a role in the maintenance and lubrication of the epidural cavity and neural foramina. Presence of CD68, CD55, and CD44 suggests a scavenging function and a role in the inflammatory response to noxious stimuli. Thus, the human PDM has histological and immunohistochemical characteristics of synovium. This suggests that the PDM may be important for the homeostasis of the flexible spine and the neural structures it contains.

MICAL2 regulates myofibroblasts differentiation in epidural fibrosis via SRF/MRTF-A signaling pathway.

AIM: To determine the role of MICAL2 in myofibroblasts differentiation and epidural fibrosis. BACKGROUND: Epidural fibrosis (EF) may develop following laminectomy and aberrant myofibroblasts differentiation and excessive extracellular matrix (ECM) accumulation play key roles in the formation of EF. Dense epidural fibrosis results to the poor surgical outcomes and failed back surgery syndrome (FBSS), and there is no effective treatment available. Molecule interacting with Casl2 (MICAL2) has been demonstrated to participate in multiple cellular processes by regulating actin cytoskeleton dynamics. However, its role in epidural fibrosis remains totally unverified. MATERIALS AND METHODS: The potential functions and mechanisms of MICAL2 were explored using western blotting, immunofluorescence and lentivirus infection. KEY FINDINGS: In our study, we determined that the MICAL2 expression was elevated in epidural fibrotic tissues and TGF-ß1-stimulated fibroblasts. Moreover, knockdown of MICAL2 using MICAL2-specific short hairpin RNA attenuated TGF-ß1-induced myofibroblasts differentiation and epidural fibrosis both in vitro and vivo, as indicated by decreased scar formation, reduced collagen production and down-regulated expression of α-SMA, collagen-1 and fibronectin. We also demonstrated that MICAL2 knockdown affected the migratory capability of fibroblasts in vitro. By further mechanistic research, we revealed that the MRTF-A nuclear translocation was inhibited in response to the knockdown of MICAL2 in fibroblasts and MICAL2 served as a pro-fibrotic factor in an SRF/MRTF-A-dependent manner. SIGNIFICANCE: In conclusion, our results indicated that MICAL2 mediated myofibroblasts differentiation and promoted epidural fibrogenesis via SRF/MRTF-A signaling pathway, suggesting manipulation of MICAL2 activity as a novel alternative strategy for the prevention of epidural fibrosis.

Intradiscal flow during a transforaminal epidural steroid injection.

OBJECTIVE: To present a case of inadvertent intradiscal flow during a L4/5 transforaminal epidural steroid injection. DESIGN: Case report. SETTING: Outpatient private practice. PATIENTS: Single case. INTERVENTION: Lumbar transforaminal epidural steroid injection. RESULTS: Flow of contrast indicating needle entry into extruded disc material with filling of the L4/5 disc. CONCLUSION; This case demonstrates the possibility of needle entry into disc material in the presence of a large lateral disc extrusion with cephalad migration during a transforaminal epidural steroid injection.

The effect of volume of local anesthetic on the anatomic spread of caudal block in children aged 1-7 years.

OBJECTIVES: To examine the anatomic spread of caudal local anesthetic solution in children aged 1-7 years. AIM: To determine whether incremental increases in the volume of caudal injections of 0.5, 0.75, and 1.0 ml·kg(-1) result in reliable (>90%) and potentially clinically significant increases in the number of vertebral segments reached. BACKGROUND: Caudal block is one of the most frequently performed pediatric regional analgesic techniques. Traditional formulae suggest that changes in the volume of caudal injectate in the range 0.5-1.0 ml·kg(-1) would have clinically useful effects. METHODS: In a single blind design, 45 children aged 1-7 years undergoing caudal block received one of the three predetermined volumes (0.5, 0.75, and 1 ml·kg(-1) ) of local anesthetic solution containing radio-opaque contrast under controlled conditions. Following X-ray examination, the anatomic spread of the block was reported by a radiologist blinded to the volume of solution received. RESULTS: There were 15 children in each group, and they were similar in terms of age, height, and weight. Spread was observed between the 5th lumbar (L5) and 12th thoracic (T12) vertebral levels. A volume of 1 ml·kg(-1) results in a small but significantly greater spread of solution than 0.5 ml·kg(-1) (P < 0.05), but there was no difference between 0.5 and 0.75 ml or between 0.75 and 1.0 ml. No volume reliably reached a level higher than the second lumbar vertebra (L2). CONCLUSIONS: Incrementally increasing the volume of injectate between 0.5 and 1.0 results in a modest increase in the spread of the caudal solution. It is unlikely that volumes of <1 ml will reliably reach a vertebral level that is higher than L2.

Effective delivery of mitomycin-C and meloxicam by double-layer electrospun membranes for the prevention of epidural adhesions.

Epidural adhesion between the spinal dura and the surrounding fibrous tissue often occurs post-laminectomy, resulting in clinical symptoms such as nerve compression and severe pain. In this study, we report a drug-loaded double-layered electrospun nanofiber membrane to prevent the occurrence of epidural adhesion. The nanofibers in both layers are made of a mixture of polycaprolactone (PCL) and chitosan (CS) but at different weight ratios. The bottom layer contacting to the spinal dura is loaded with meloxicam (MX) to prevent inflammation. The top layer that contacts to the fibrous tissue is doped with mitomycin-C (MMC) to inhibit the synthesis of DNA and collagen. The two types of drugs are released from the double-layered membrane within about 12 days. Meanwhile, the membrane can inhibit fibroblasts proliferation in vitro while show no cytotoxicity. In a rabbit laminectomy model, the double-layered membrane can effectively prevent the epidural adhesion formation based on the adhesion scores, histological and biochemical evaluations. The combination release of MX and MMC can signally reduce the inflammation reaction and collagen I/III expression relative to the case with the membranes loaded with only either one type of the drugs. This approach offers new progresses in constructing dual drug delivery system and provides innovative barrier strategy in inhibiting epidural adhesion post-laminectomy.

Effect of Cetuximab on the Development of Epidural Fibrosis Based on CD105 and Osteopontin Immunohistochemical Staining.

STUDY DESIGN: The effect of cetuximab on the development of epidural fibrosis (EF) was assessed using immunohistochemical methods as well as antibodies for CD105 and osteopontin (OPN). OBJECTIVE: The goal of this study was to assess of EGFR inhibition for the postoperative treatment of fibrosis. SUMMARY OF BACKGROUND DATA: EF is one of most common causes of failed back surgery syndrome, which occurs after laminectomy. Numerous causes and mechanisms have been proposed to explain its development after laminectomy. Many agents have been tested to prevent the development of EF. EGFR, a multi-functional transmembrane glycoprotein, causes cell growth, proliferation, and EF by interacting with epidermal growth factor and TGF-ß1. The inhibition of postoperative fibrosis using cetuximab, an epidermal growth factor receptor blocker, is theoretically possible. However, this has not been tested to date. METHODS: Sixteen Wistar-Albino rats were divided into two groups, namely, control and cetuximab groups. L1-2 laminectomy alone was performed in both groups, and topical cetuximab was applied to the treatment group. After 6 weeks, rats were sacrificed and examined histopathologically and immunohistochemically; EF tissue was also graded. Statistical significance was accepted at P < 0.05. RESULTS: Fibroblast counts and fibrosis density, determined by histopathologic examination, and EF, according to immunohistochemical assessment based on CD105, were found to be higher in the treatment group than in the control group, and this was statistically significant (P < 0.001). Based on OPN staining, the results were consistent with classical methods, and no significant difference was detected among the groups (P = 0.358). CONCLUSION: Our study revealed that cetuximab inhibits the development of EF and that CD105, and not OPN, is a reliable marker for grading EF. In addition, cetuximab did not result in toxic, systemic side effects in surrounding tissues. LEVEL OF EVIDENCE: N/A.

Emodin promotes fibroblast apoptosis and prevents epidural fibrosis through PERK pathway in rats.

BACKGROUND: Laminectomy is usually classed as a common orthopedic surgery, but postoperative epidural fibrosis often leads to less-than-desirable clinical outcomes. As demonstrated by prior studies, emodin (EMO) exerts an anti-fibrotic effect. Here, we carried out investigation into the inhibitory effect created by EMO application on epidural fibrosis after laminectomy in rats. METHODS: The paper conducts a series of experiment. In vitro, we observed the effect of EMO on fibroblasts by Cell Counting Kit-8 (CCK-8) assay. Apoptosis of fibroblasts induced by EMO was detected by western blot, TUNEL assay, and flow cytometry. The results revealed that EMO was capable of inducing fibroblast apoptosis, and the proteins of PERK pathway also changed accordingly. In vivo, the effect of EMO on epidural fibrosis in 12 male Sprague-Dawley rats was observed by histological staining. RESULTS: CCK-8 assay indicated that EMO was effective in reducing fibroblast viability in a time- and a dose-dependent manner. TUNEL assay and flow cytometry analysis have demonstrated that the apoptotic rate of fibroblasts increased as the EMO concentration rose. Western blot analysis proved that EMO promoted the relative expression of p-perk and p-eIF2α and that the expression of its downstream proteins CHOP and GRP78 was also enhanced. The expression of apoptotic protein Bax and cleaved PARP was upregulated, whereas the expression of anti-apoptotic protein Bcl-2 was downregulated. In addition, histological and immunohistochemical analysis demonstrated that EMO functioned to inhibit epidural fibrosis and increase GRP78 expression in fibrous tissue by promoting apoptosis of fibroblasts. CONCLUSIONS: EMO could have inhibitory effect on epidural fibrosis in a concentration-dependent manner. The potential mechanism might be through PERK signaling pathway to promote fibroblast apoptosis. It has a possibility to be taken as a novel method for the treatment of epidural fibrosis.

Apigenin inhibits fibroblast proliferation and reduces epidural fibrosis by regulating Wnt3a/ß-catenin signaling pathway.

BACKGROUND: Failed back surgery syndrome (FBSS) is a common complication after the laminectomy. Epidural fibrosis is the major cause of lower back pain and other complications. Numerous studies have shown that apigenin (API) could treat various fibrotic diseases by regulating various signaling pathways, whereas no study has discussed whether API can inhibit fibroblast proliferation and reduce epidural fibrosis after the laminectomy by regulating Wnt3a/ß-catenin signaling pathway. METHODS: Human fibroblasts were cultured and treated with API in different concentrations for 24 h. CCK-8 detection and EdU incorporation assay were performed to detect cell viability and cell proliferation. Western blotting analysis was applied to detect expressions of proliferative proteins, Wnt3a, and its downstream proteins. Moreover, the Wnt3a gene was overexpressed in fibroblasts to define the relationship between Wnt3a/ß-catenin signaling pathway and fibroblast proliferation. Wnt3a overexpressed fibroblasts were treated with API to verify if it could reverse the effects of API treatment. Twenty-four Sprague-Dawley rats were randomly divided into four groups. Laminectomy was performed and the rats were gavaged with different doses of API or 5% sodium carboxyl methyl cellulose (CMC-Na) solution for 1 month. The abilities of API to inhibit fibroblast proliferation and to reduce epidural fibrosis were evaluated using histological and immunohistochemical analysis. RESULTS: CCK-8 detection and EdU incorporation assay demonstrated that API could inhibit the viability and proliferation rate of fibroblasts in a concentration-dependent manner. The Western blotting analysis revealed that API could inhibit the expressions of PCNA, cyclinD1, Wnt3a, and its downstream proteins. The overexpression of Wnt3a in fibroblasts could upregulate the expressions of proliferative proteins such as PCNA and cyclinD1. The inhibitory effect of API on PCNA, Wnt3a, and its downstream proteins was partially reversed by overexpression of Wnt3a. Moreover, the results of the histological and immunohistochemical analysis revealed that API could reduce the epidural fibrosis in rats by inhibiting fibroblast proliferation in a dose-dependent manner. CONCLUSIONS: API can inhibit fibroblast proliferation and reduce epidural fibrosis by suppressing Wnt3a/ß-catenin signaling pathway, which can be adopted as a new option to prevent epidural fibrosis after the laminectomy.

The effect of 10-hydroxycamptothecine in preventing fibroblast proliferation and epidural scar adhesion after laminectomy in rats.

Little is known about the influence of 10-hydroxycamptothecin (HCPT) on fibroblast proliferation and pathological changes in epidural scar tissue after laminectomy. Here we illustrated the effect of HCPT on fibroblast proliferation and epidural scar adhesion after laminectomy in rats. In the present study, seventy-two rats underwent laminectomies at Lumbar-1 level, then HCPT in various concentrations (0.1, 0.05, and 0.01 mg/ml) or saline (9 mg/ml) were applied to the laminectomy sites. Four weeks later the rats were killed and the epidural adhesion was evaluated. The area of epidural scar tissue and the number of fibroblasts were also determined. The degree of epidural adhesion was classified according to Rydell standard. The results showed that no or little epidural adhesions were seen in the laminectomy sites treated with 0.1 mg/ml HCPT. The Rydell classification, the area of epidural scar tissue and the number of fibroblasts in 0.1 mg/ml HCPT group were significantly less than those of 0.05 mg/ml HCPT group, 0.01 mg/ml HCPT group and saline group. Moderate epidural adhesions were noted in the laminectomy sites of 0.05 mg/ml HCPT group. The Rydell classification, the area of scar tissue and the number of fibroblasts were less than those of 0.01 mg/ml HCPT group and saline group. However, dense epidural adhesions were found in 0.01 mg/ml HCPT group and saline group. The Rydell classification, the area of scar tissue and the number of fibroblasts showed no significant difference compared with those of saline group. In conclusion, topical application of 0.1 mg/ml HCPT could effectively prevent fibroblast proliferation and reduce epidural adhesion after laminectomy in rats.