Local application of rapamycin reduces epidural fibrosis after laminectomy via inhibiting fibroblast proliferation and prompting apoptosis

Abstract

BackgroundEpidural fibrosis is a common complication after laminectomy. It is associated with intractable lower back pain and additional complications. To date, no study has evaluated whether the local application of rapamycin (RAPA) can inhibit fibroblast proliferation and reduce epidural scar adhesion after laminectomy. The results of the present study showed that the local application of RAPA reduces epidural fibrosis after laminectomy in rats.MethodsIn this study, 32 male Sprague-Dawley rats were randomly divided into four groups (0.2 mg/ml RAPA-treated group, 0.1 mg/ml RAPA-treated group, 0.05 mg/ml RAPA-treated group and physiological saline group). Laminectomy was performed at the level of lumbar segment 1 to 2, and different concentrations of RAPA or saline were applied to the laminectomy sites for 10 min. Four weeks after laminectomy, the rats were sacrificed, and the degrees of epidural adhesion in each group were evaluated. Macroscopic assessment, analysis of hydroxyproline content, and histological analysis were used to determine the therapeutic effect of the local application of RAPA on the inhibition of fibroblast proliferation and the reduction of epidural fibrosis after laminectomy. Next, we cultured fibroblasts from epidural scar tissues of rats that had undergone laminectomy. Fibroblasts were exposed to the indicated concentrations of RAPA, and western blotting and TUNEL assays were used to assess the effects of RAPA on inhibiting fibroblasts proliferation and promoting fibroblast apoptosis.ResultsThe results of macroscopic assessments, analysis of hydroxyproline content, and histological analyses indicated that RAPA significantly inhibited fibroblast proliferation and reduced epidural fibrosis in the treated groups in the rat model. The western blotting results indicated that the expression levels of the pro-apoptotic proteins cleaved-PARP and Bax were up-regulated, whereas those of Bcl-2 were reduced. TUNEL assay indicated that the apoptosis rates of fibroblasts were significantly increased after exposure to the indicated concentrations of RAPA.ConclusionsThe local application of RAPA reduced epidural fibrosis after laminectomy by inhibiting the proliferation of fibroblasts, stimulating their apoptosis, and decreasing collagen synthesis. This protocol may be used in new clinical treatment strategies to reduce epidural fibrosis after laminectomy.

Prospective application of stem cells to prevent post‐operative skeletal fibrosis

“…Local immune response is considered critical for each type of post‐operative skeletal fibrosis and some immunosuppressant drugs used to prevent post‐operative skeletal fibrosis have achieved success, including rapamycin, tacrolimus, pimercrolimus, azithromycin, methotrexate and cyclosporin A . Results showed that they could prevent post‐operative skeletal fibrosis by inhibiting fibroblast proliferation, suppressing interleukin 2 (IL‐2) and TGF‐β1 expression, regulating miR‐429 and RhoE expression and activating autophagy and the ER stress signaling pathway …”

Effect of Platelet-Rich Plasma, Fat Pad and Dural Matrix in Preventing Epidural Fibrosis

“…are the prominent experimental materials. 2,[4][5][6][7]9,12,13,15,16,18,19 Some of these materials have been used clinically. Although most studies are animal experiments, as of today, satisfactory clinical studies confirming that these materials can help attenuate fibrosis in clinical applications have not been achieved yet.…”

Laminin α5 modulates fibroblast proliferation in epidural fibrosis through the PI3K/AKT/mTOR signaling pathway

“…Cells were cultured at 37˚C in 5% CO 2 with DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 15% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin & streptomycin (Gibco; Thermo Fisher Scientific, Inc.). A total of 1x10 6 fibroblasts were seeded in petri dishes with a variety of specifications overnight until a confluence of 70% was attained; then the dishes were washed twice with PBS. One third of the fibroblasts were set as the lY group and were treated with the Pi3K inhibitor lY294002 (Medchemexpress) diluted in dimethylsulfoxide to 50 µM for 24 h. Another third of the fibroblasts were treated with siRNA for knockdown or lentiviral vectors for overexpression.…”

“…Fibroblasts in the overexpression and scramble control group were cultured until they reached 70% confluence. Subsequently, 1x10 6 fibroblasts/well were transfected with 2x10 7 Tu (multiplicity of infection of 20) laminin α5 GV418 lentiviral vector or GV418 scramble control empty vector overnight in the presence of 2 mg/ml polybrene (Gibco; Thermo Fisher Scientific, Inc.) and then replaced with fresh complete medium. After 48 h-transfection at 37˚C, cells were cultured in puromycin (Sigma-aldrich; Merck KGaa) at a concentration of 2 µg/ml for 72 h for the preliminary screening to eliminate non-transfected cells.…”

“…additionally, further studies have illustrated that the excessive proliferation of fibroblasts in the operative region is the most significant element of local fibrosis formation (4,5). Therefore, the methods of inhibiting fibroblast proliferation to reduce fibrosis have become an attractive area of study and previous studies have demonstrated positive results (6)(7)(8). However, there are numerous limitations for clinical application, hence further research is still required to solve the problem completely.…”