Extracellular Vesicles Derived from Epidural Fat-Mesenchymal Stem Cells Attenuate NLRP3 Inflammasome Activation and Improve Functional Recovery After Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating event which caused high mortality and morbidity. Recently, nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome has been showed to act a critical t role in the secondly injury phase of SCI. In current study, we aimed to investigate the effect and underlying molecular mechanisms of extracellular vesicles derived from epidural fat (EF)- mesenchymal stem cells (MSCs) for the treatment of SCI. Ninety-six Sprague-Dawley rats were used for current study and randomly divided into four groups: sham group, SCI group, SCI + Saline group, SCI + Extracellular vesicles group. Basso-Beattie-Bresnahan (BBB) scores was applied to evaluate the neurological functional recovery. Cresyl violet-stained was conducted evaluate the protective effect of EF-MSCs-Extracellular vesicles on lesion volume after SCI. ELISA, immunohistochemistry assay, TUNEL assay and western blotting were conducted to investigate the underlying molecular mechanisms. Our results demonstrated that the administration of EF-MSCs-Extracellular vesicles via tail vein injection improved neurological functional recovery and reduced the lesion volume after SCI. And systemic administration of EF-MSCs-Extracellular vesicles significantly inhibited NLRP3 inflammasome activation and reduced the expression of inflammatory cytokines. Additionally, the expression levels of proapoptotic protein Bax was decreased and antiapoptotic Bcl-2 was upregulated with the treatment of EF-MSCs-Extracellular vesicles after SCI. In summary, in current study, we demonstrated for the first time that the EF-MSCs-Extracellular vesicles can improve neurological functional recovery after SCI, and the underlying molecular mechanisms may partly through the inhibition of NLRP3 inflammasome activation.

Effect of Methyl Palmitate on the Formation of Epidural Fibrosis in an Experimental Epidural Fibrosis Model.

AIM: To investigate the effects of local and systemic administration of methyl palmitate on the formation of epidural fibrosis. MATERIAL AND METHODS: Twenty-eight rats were randomly divided into four equal groups (control, Spongostan, local methyl palmitate and orally methyl palmitate) and laminectomy was performed between T11 and L1 in all rats. Local methyl palmitate (300 mg/kg) was applied with Spongostan; methyl palmitate (300 mg/kg) was given orally three times per week on different days for a total period of 4 weeks. Four weeks later, the vertebral column from T9 to L3, including the paraspinal muscles and epidural scar tissue, was removed en bloc and epidural fibrosis and arachnoidal involvement was graded and evaluated histopathologically. Kruskal-Wallis and Pearson Chi-Square test were used for statistical analysis. A statistically significant p-value was determined as p < 0.05. RESULTS: The grading of epidural fibrosis was lower at a statistically significant level in orally-administrated methyl palmitate groups compared to the control and spongostan groups (p < 0.05). CONCLUSION: The findings of this study show that oral methyl palmitate decreases the formation of epidural fibrosis and that this effect of methyl palmitate could be mediated by reducing the functions of inflammatory cells such as macrophages, neutrophils and fibroblasts, including anti-inflammatory and antioxidant effects.

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.

Detection of epithelial cell transfer in spinal areas by light microscopy and determining any tissue coring via cell culture during combined spinal-epidural interventions.

BACKGROUND AND OBJECTIVES: Epithelial tissue coring by spinal needles during subarachnoid injections may cause intraspinal epidermal tumors. Previous studies have investigated tissue transfer with different needle types during subarachnoid or epidural injection. This study deals with the transfer of epithelial tissue during combined spinal-epidural (CSE) anesthesia. METHODS: We studied 68 American Society of Anesthesiologists I to III adult patients. CSE anesthesia was induced under aseptic conditions at the L2-3 or L3-4 interspace with patients in the lateral decubitus position. Cerebral spinal fluid, spinal needle stylet, fluid used to flush the interior of the spinal needle, fluid used to wash the exterior of the spinal needle, fluid used to flush the interior of the epidural needle, and fluid used to wash the exterior tip of the epidural needle were examined under light microscopy (n = 30 patients) or incubated in a cell-culture medium (n = 38 patients). Samples were incubated in cell-culture medium alone (n = 13) or in a cell-culture medium for 3 weeks and then in a medium with epidermal growth factor (n = 25). As a positive control, skin tissue samples were taken by punch biopsy from 10 randomly chosen patients who underwent CSE interventions. These samples were incubated in an enriched medium serum. RESULTS: Light microscopy revealed that there was cell transfer in all phases in various rates: samples 1, 2, 3, 4, 5, and 6 contained epithelial cells and debris in ratios of 6.9%, 20.7%, 6.9%, 20.7%, 26.7%, and 33.3%, respectively. Epithelial cell colonization was detected in the cell-culture samples taken from the control group but not in the samples taken from the CSE group. CONCLUSIONS: We could not reproduce the cells or cell debris obtained during the CSE interventions in vivo, which can be explained by a possible structural deformation of cells or the inadequacy of the amount of cells that were transferred.

Morphofunctional changes in spinal cord neurons after epidural lidocaine.

Morphofunctional and histoenzymological changes in spinal cord neurons of mongrel dogs were studied after epidural administration of isobaric 2% lidocaine solution. Control animals received epidural 0.9% sodium chloride. The results obtained from these studies provide evidence for the absence of pathological structural-metabolic changes in nerve tissue after treatment with lidocaine. The occurrence of certain morphofunctional rearrangements in spinal cord neurons were typical of animals of both the experimental and control groups. The changes recorded varied within the limits of physiological variation and provided evidence predominantly of the functional response of these nerve tissue structures to epidural injections of both sodium chloride and lidocaine.

Cytopathologic examination of epidural catheter for postoperative analgesia. Pathophysiology and clinical management.

AIM: The authors performed a prospective study in a series of patients undergoing combined general and epidural anaesthesia for major abdominal surgery in order to define if the epidural catheter inserted for postoperative analgesia induced in the short-term (7-8 postoperative days) any cytopathologically appreciable inflammatory response. METHODS: From April to September 2001, 20 consecutive patients undergoing combined general and epidural anaesthesia for major abdominal surgery at the National Cancer Research Institute and Villa Scassi Hospital (Genoa), were recruited after obtaining Institutional Ethics Committee approval and written consent from the patients. The standard technique for epidural anaesthesia was adopted. Preoperatively, all patients received peridurally a dose test of 3 ml of 2% lidocaine (60 mg) followed by 5 ml of ropivacaine 0.75%, and a continuous infusion of ropivacaine 0.375% (5-10 ml/h; maximal dose=20 ml) intraoperatively. As regards the therapeutic management of postoperative analgesia, patients received a continuous infusion of ropivacaine 0.2% for at least 48 hours and supplemental bolus (2 mg/die) of morphine hydrochloride. The epidural catheter was always removed between the 7th and 8th postoperative day, and it was examined by the pathologist according to the Thin Prep 2000 procedure. RESULTS: The cytopathologic examination of the tip of the epidural catheter gave the following findings: amorphous material without cells (n=10); rare granulocytes and histiocytes (n=6); stromal cells (n=3), and rare lymphocytes (n=1). CONCLUSION: We were unable to detect any cytopathologically appreciable inflammatory response at the tip of the epidural catheter which could have suggested the occurrence of inflammation in the epidural tissues. Given the positive results of prophylactic epidural administration of small doses of corticosteroids in the reduction of postepidural anaesthesia back pain and their direct membrane action on nociceptive C-fibers, this kind of backache seems to be related to the stimulations of such nociceptors more than to a catheter-related inflammatory response of epidural tissues with possible evolution in peridural fibrosis, as reported following surgical intervention for lumbosacral disease.

[Morpho-functional changes of the spinal cord neurons after epidural administration of lidocaine]. / Morfofunktsional'nye izmeneniia v neironakh spinnogo mozga posle epidural'nogo vvedeniia lidokaina.

The analysis of morphological and histoezymological changes of spinal cord neurons has been performed in outbred dogs following epidural infusion of isobaric 2% lidocaine solution. In the control group the animals received epidural infusion of 0.9% saline. The results obtained indicate the absence of pathological structural and metabolic changes in the nervous tissue after lidocaine application. Certain signs of morpho-functional reorganization were noted in spinal cord neurons of animals in both experimental and control groups. The registered changes were found to vary within the physiological fluctuation limits and are rather indicative of the functional reaction of the studied structures of the nervous tissue to the epidural injections of either saline and lidocaine.

Influence of bioresorbable, unsintered hydroxyapatite/poly-L-lactide composite films on spinal cord, nerve roots, and epidural space.

The effect of forged unsintered hydroxyapatite/poly-L-lactide (u-HA/PLLA) composite films on spinal cord and nerve roots and its degradation behavior and osteoconductivity in epidural space were compared with those of calcined HA (c-HA)/PLLA and unfilled PLLA films. Partial laminectomy was performed on 20 rabbits, and u-HA/PLLA and PLLA films were implanted in the intervertebral space. Total laminectomy was performed on 30 rabbits to implant u-HA/PLLA, c-HA/PLLA, and PLLA films in both epidural and subcutaneous spaces. For up to 50 weeks, there were no histological changes in the spinal cord or nerve root, and no inflammatory cell infiltration into the epidural space around the films. The rate of decrease in viscosity average molecular weight of both composite films was initially higher than that of PLLA but eventually became lower, although there was no difference in the degradation behavior of the three films in either the epidural or subcutaneous spaces after 50 weeks. Scanning electron microscopic and energy-dispersive X-ray analysis indicated calcium phosphate deposits on the surface of composite films with new bone formation from 4 weeks. The u-HA/PLLA composite film therefore has good biocompatibility, osteoconductivity, and fast primary degradation rate, which may prove compatible with application to spinal surgery.

Comparison of tissue reaction with chondroitinase ABC and chymopapain in rabbits as the basis of clinical application in chemonucleolysis.

Although chemonucleolysis with chymopapain is an approved treatment for lumbar intervertebral disk herniation, recent serious complications have raised doubt concerning its safety. It is therefore necessary to search for a safer and more selective agent than chymopapain for chemonucleolysis. Experimental chemonucleolysis with chondroitinase ABC was thus tested and compared with chymopapain. Acute tissue reactions to chondroitinase ABC were investigated and compared with chymopapain. The epidural space, yellow ligament, sciatic nerve, knee joint, and Achilles tendon were examined. Chymopapain damaged nervous and ligamentous tissues as well as cartilaginous tissue. Chondroitinase ABC did not damage nervous and ligamentous tissue. Chondroitinase ABC affected only cartilaginous tissue, and its action was chiefly limited to digestion of the matrix. Chondroitinase ABC has high enzymatic specificity for matrix in vivo. In addition, chondroitinase ABC is less toxic to noncartilaginous tissues than chymopapain. Chondroitinase ABC might be a more suitable and safer enzyme than chymopapain for chemonucleolysis.

[The posterior intervertebral spaces of the craniovertebral joint]. / Les espaces intervertébraux postérieurs de la jointure crânio-rachidienne.

Closing of the posterior intervertebral spaces of the craniovertebral joint is not performed by the classical posterior atlanto-occipital and atlantoaxial membranes. In the atlanto-occipital space, the connective laminae come from the occipital periosteum and from the anterior fascia of the rectus capitis posterior minor muscle, and pass round the anterior side of the posterior arch of the atlas to reach the spinal dura mater. In the atlantoaxial space, the anterior fasciae of the rectus capitis posterior major muscle and of the inferior oblique muscle, as well as the periosteum of the posterior arch of the atlas, extend to reach the spinal dura mater. Thus, the epidural space is sealed posteriorly by the connective laminae of the atlantoaxial space, and lets above a superior recessus containing the ganglia of the spinal nerves C1 and C2 and in which the vertebral artery transits.