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

Thirty-two healthy male Sprague-Dawley rats (mean weight of 350 g) were purchased from Shanghai Laboratory Animal Center. The animals received care in compliance with the principles of International Laboratory Animal Care, and the experimental protocol was approved by the Animal Care and Research Committee of Yang Zhou University (Yangzhou, China). RAPA, which was purchased from Astellas Ireland Co, Ltd. (Killorhlin, Co. Kerry, Ireland), was dissolved in dimethyl sulfoxide (DMSO) purchased from Sigma-Aldrich (St Louis, MO, USA) and stored at −20 °C. The RAPA solution was diluted with saline or cell culture medium such that the DMSO comprised < 0.1%. Specific antibodies against cleaved-PARP, Bcl-2 and Bax were purchased from Cell Signaling Technology (Cambridge, MA, USA). All rats were randomly divided into four groups (eight rats in each group): 0.05 mg/ml RAPA, 0.1 mg/ml RAPA, 0.2 mg/ml RAPA, and saline groups. Before the experiment, the rats were housed for 1 week and allowed to adapt to the conditions of the laboratory.

A primary fibroblast cell line was cultured from the epidural scar tissue of rats that had undergone laminectomies. Briefly, the scar tissues at L1–L2 level were anatomically separated and rinsed immediately with phosphate-buffered saline, then cut into 1 × 1 mm² sections and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA, USA) supplemented with 10 % foetal bovine serum, 0.1 U/l penicillin, and 50 μg/ml streptomycin (Gibco, CA, USA) at 37 °C in 5 % CO₂. Culture media were replaced every 2 days until fibroblasts reached approximately 80 % confluence. The cells of passages 3 to 6 were used in all experiments, the cells were transfered into a serum-free medium overnight and then subjected to various reagent treatments with varying doses of RAPA, as indicated in the figure legends.

All other reagents were purchased from Sigma Aldrich (St Louis, MO, USA) unless stated otherwise.

A rat laminectomy model was used to determine the effects of RAPA on epidural fibrosis. Briefly, each rat was anaesthetized with an intraperitoneal injection of 1 % pentobarbital sodium solution (4 ml/kg body weight) and then fixed in the prone position. The animal’s back hair at the level of L1 and L2 was shaved, and skin was sterilized with iodophor three times. The laminectomy model was constructed according to methods described in previous studies [28, 29]. All procedures were performed under sterile conditions with basic surgical tools, surgical microscopes and an electrical drill. A median incision of the dorsal skin at the level of L1–L2 was made, and the paraspinal muscles were separated. A rongeur was used to remove the spinous process and vertebral plate, and the dura mater at the L1 to L2 level was exposed. Then, cotton soaked with different concentrations of RAPA (0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml) or saline was applied to the surgical site for 10 min (the volume of application was approximately 1 ml). The surrounding tissues were protected from contact with the agent. Satisfactory haemostasis was achieved by using gauze; bone wax and cauterization after laminectomy were not needed. All procedures were performed with care to avoid injury to the neural tissues.

Four weeks after the surgery, four rats in each group were randomly selected, and the surgical sites were reopened carefully. The degree of epidural fibrosis at the L1 to L2 level was evaluated by three professional blinded pathologists according to the Rydell classification [30] (grade 0 = epidural scar tissue was not adherent to the dura mater; grade 1 = epidural scar tissue was adherent to the dura mater but was easily dissected; grade 2 = epidural scar tissue was adherent to the dura mater and was difficult to dissect without disrupting the dura mater; and grade 3 = epidural scar tissue was firmly adherent to the dura mater and could not be dissected).

After macroscopic assessment of epidural fibrosis, the scar tissue (wet weight = approximately 4 mg) was collected from L1 to L2 area surrounding the laminectomy site. The content of hydroxyproline in the scar tissue of different groups was determined according to the methods described in previous study [31, 32]. Briefly, the samples were lyophilized, ground and hydrolysed with 6 mol/l HCl at 130 °C for 12 h separately. The samples were then neutralized with 2.5 N NaOH with methyl red as an indicator. Chloramine T (1 ml) was added to the hydrolysed samples along with four hydroxyproline standards of known concentrations. After incubation for 20 min at room temperature, the hydroxyproline developer was added to the samples and standards. The absorbance of the solution was measured at 558 nm with a spectrophotometer, and the hydroxyproline content per milligram of scar tissue was calculated according to the standard curve, which was constructed with serial concentrations of commercial hydroxyproline.

Four rats randomly selected from each group were sacrificed after 4 weeks for histological evaluation. The entire L1–L2 spine column, including the paraspinal muscles and epidural scar tissues, was collected and immersed in 10 % buffered formalin for 5 days. The specimens were decalcified in 10 % EDTA (Sigma Aldrich, St Louis, MO, USA) for 3 weeks and embedded in paraffin. Then, 4-mm transverse sections were made through the L1–L2 vertebra, and this was followed by staining with haematoxylin–eosin (HE) and Masson’s trichrome. Three counting areas (100 × 100 μm each) were selected at the middle and at the margins of the laminectomy sites, and the number of fibroblasts was calculated.

To further determine the effects of RAPA on the proliferation of fibroblasts, the fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA, USA) supplemented with 10 % foetal bovine serum, 0.1 U/l penicillin, and 50 μg/ml streptomycin (Gibco, CA, USA) at 37 °C and 5 % CO₂. Briefly, fibroblasts were plated in six-well plates at a density of 8 × 10⁵ cells/well and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA, USA) supplemented with 10 % foetal bovine serum, 0.1 U/l penicillin, and 50 μg/ml streptomycin (Gibco, CA, USA) at 37 °C in 5 % CO₂. After reaching approximately 80 % confluence, the cells were transferred into serum-free medium overnight and then subjected to treatment with different concentrations of RAPA (0, 0.01, 0.1, and 1 μg/ml) for 48 h, as indicated in the figure legends. Then, the number of fibroblasts and changes in cell morphology were observed under a light microscope.

To further determine the effect of RAPA on the expression of apoptotic proteins, such as cleaved-PARP, Bax, and Bcl-2, fibroblasts were seeded in six-well plates at a density of 5 × 10⁵ and cultured in DMEM supplemented with 10 % foetal bovine serum and 1 % penicillin. When the cells were confluent, they were transfered into serum-free medium overnight and then pre-treated with or without different concentrations of RAPA for 48 h. Then, total proteins were extracted from cultured cells using radioimmuno-precipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were determined using a bicinchoninic acid assay (BCA; ThermoFisher, MA, USA). Thirty micrograms of each protein lysate was added to each lane and resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Sigma Aldrich, St Louis, MO, USA) on 12 or 6 % gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The primary antibodies were diluted in 1 % (w/v) skimmed milk powder in TBS-Tween and incubated overnight at 4 °C. Membranes were then washed and incubated with the appropriate secondary antibodies conjugated with IRDye 800CW (molecular weight = 1162 Da). Antibody reactivity was detected after exposure in an Odyssey infrared imaging system (LI-COR, Nebraska, USA).All the experiments were repeated three times.

To evaluate the apoptotic rate of RAPA on fibroblasts, the cells were transferred into a serum-free medium overnight and then pre-treated with or without different concentrations of RAPA for 48 h, the apoptotic rate of fibroblasts was analysed using a TdT-mediated dUTP-biotin nick-end labelling (TUNEL) test system (Roche, USA) according to the manufacturer’s instructions. Following staining, the apoptotic features of cell death were examined via fluorescence microscopy. The obtained images were merged and analysed using Image J software. The percentage of TUNEL-positive cells was defined as the number of TUNEL-stained cells divided by the number of DAPI-stained cells. The cells were counted by a naive observer.

The surgery was well tolerated by all rats, without the occurrence of wound infection, neurological deficits or cerebrospinal leaks. The results of macroscopic observation indicated that soft or weak scar adhesion was found in the 0.2 mg/ml RAPA group. Moderate scar adhesion was seen in the 0.1 mg/ml RAPA group; the tissues could be dissected by manual traction with less bleeding. However, severe epidural adhesions were observed in both the 0.05 mg/ml RAPA group and control group; dissection of the scar tissue was difficult and was accompanied by bleeding and disruption of the dura mater. The levels of epidural fibrosis were evaluated according to the Rydell classification, and the results are shown in Table 1.

The typical images of HE staining and masson staining of epidural scar tissues at L1–L2 levels were shown in Figs. 1 and 2, respectively. In both the control group and the 0.05 mg/ml RAPA group, dense epidural scar tissue and compact collagen tissues were found in the laminectomy sites, and the scar tissues were widely adhered to the dura mater and dorsal muscle (Figs. 1d and 2d; Figs. 1c and 2c). Moderate epidural scar adhesion and collagen tissues were found in the 0.1 mg/ml RAPA group (Figs. 1b and 2b). However, loose scar adhesion and collagen tissues were formed in the 0.2 mg/ml RAPA group (Figs. 1a and 2a).

The hydroxyproline content of epidural scar tissue is shown in Fig. 3. The hydroxyproline content of the 0.2 mg/ml RAPA group was 33.08 ± 2.23 μg/mg, which was significantly lower than those of the 0.1 mg/ml RAPA group (43.39 ± 2.01 μg/mg), the 0.05 mg/ml RAPA group (55.08 ± 1.41 μg/mg) and the control group (58.14 ± 3.19 μg/mg) (#p < 0.05). The hydroxyproline content of the 0.1 mg/ml RAPA group was also lower than those of the 0.05 mg/ml RAPA group and the control group (*p < 0.05). However, there was no significant difference between the 0.05 mg/ml RAPA group and the control group (p = 0.203).

The fibroblasts of epidural scar tissue and fibroblast counts in each group are shown in Figs. 4 and 5, respectively. The fibroblast counts were 19 ± 2.65 in the 0.2 mg/ml RAPA group, 34.67 ± 2.52 in the 0.1 mg/ml RAPA group, and 50.33 ± 2.08 in the 0.05 mg/ml RAPA group. The 0.2 mg/ml RAPA group and the 0.1 mg/ml RAPA group showed significantly lower fibroblast counts than the control group (54.67 ± 3.21) (*p < 0.05), but there was no significant difference between the 0.05 mg/ml RAPA group and the control group (p = 0.122). The number of fibroblasts in the 0.2 mg/ml RAPA groups was also significantly lower than those of the other RAPA groups (#p < 0.05).

As shown in Fig. 6, after fibroblasts were transfered into serum-free medium overnight and treated with different concentrations of RAPA (0, 0.01, 0.1, and 1 μg/ml) for 48 h, the proliferation of fibroblasts was clearly inhibited. The number of fibroblasts decreased with increasing doses of RAPA.

To evaluate the effect of RAPA on the apoptosis of fibroblasts, western blotting was used to determine the expression of the pro-apoptotic proteins cleaved-PARP and Bax and the anti-apoptotic protein Bcl-2 after fibroblasts were treated with different concentrations of RAPA. With increasing doses of RAPA, the expression levels of cleaved-PARP and Bax increased. However, the expression level of the anti-apoptotic protein Bcl-2 decreased. The results are shown in Fig. 7.

As shown in Fig. 8, the results of the TUNEL assay indicated that few apoptotic fibroblasts were found in the control group. After fibroblasts were treated with different concentrations of RAPA, the number of apoptotic fibroblasts increased. As shown in Fig. 9, compared with those of the control group, the rates of TUNEL-positive cells increased significantly in the fibroblasts of RAPA-treated groups (*p < 0.05).

All animals received care according to the principles of Laboratory Animal Care and international recommendations, and the experimental protocol was approved by the Animal Care and Research Committee of Central South University, China.