Introduction
Laminectomy is commonly performed to treat the spine pathologies (1). Depending on the dimensions of a laminectomy, removal of the soft tissues from the epidural space can cause many complications. Epidural fibrosis (EF) is one such common complication (2). This EF tissue can also be described as a healing tissue; however, this healing tissue increases in the epidural space after laminectomy, and may cause adhesion between tissues, and compression around neural tissue (3,4). These excessive healing tissue can also lead to the reappearance of the patient’s existing symptoms before surgery (5,6). Therefore, reducing adhesion via EF is critical for reducing the success rate of surgery.
In the literature, many agents have been used (1-8). However, no effective treatment has been reported (2). Additional surgical interventions for the treatment of EF may also lead to many complications (5,7-9). Additionally, many surgical interventions after the first surgery can also cause more fibrotic tissue than was formed in the first operation (8). Therefore, the preventing the formation of EF is the best approach (9).
EF formation is occurs as a result of an inflammatory process (10). This complex inflammation process ends with a process in which cellularity decreases but the extracellular matrix component accumulates excessively (7,10). Basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-β) have been showed to have a critical role in the formation of fibrosis (7,10,11). Therefore, researchers have focused on altering fibroblast proliferation and trans differentiation.
Pirfenidone, which is known as 5-methyl-N-phenyl-2-(1H)-pyridone, is an antifibrotic medicine (11-13). It has been approved for the treatment of idiopathic pulmonary fibrosis (11-14). According to previous studies, pirfenidone reduced the deposition of collagen and inflammatory cells, and inhibited the proliferation and differentiation fibroblasts by TGF-β1 (14). Therefore, pirfenidone, which has antifibrotic properties, can also be a potential agent for reducing EF formation.
Here we performed an experimental laminectomy in rats to show the effect of the pirfenidone on EF formation.
Materials and Methods
Animals
The experimental procedure was reviewed and approved by the animal Ethics Committee of Aydın Adnan Menderes University Animal Experiments Local Ethics Committee (approval no: 64583101/2019/017, date: 26.03.2019).). Twenty-four female Wistar albino rats, the average weight of which was 350±10 mg, were used in this study. Environmental conditions of the experimental study were 20-24 °C, 50±10% humidity and a 12-hour light/dark cycle. The subjects were ensured easy access to food and water.
Experimental groups
Group 1: Laminectomy; only T11 and T12 laminectomies were performed (n=8).
Group 2: Twenty-five mg/kg pirfenidone; T11 and T12 laminectomies and after hemostasis, surgical layers were closed in accordance with the normal anatomic layer. Then 25 mg/kg pirfenidone was given orally once a day for 30 days (n=8).
Group 3: Fifty mg/kg pirfenidone; the same procedure as group 2 was performed, and then 50 mg/kg pirfenidone was given orally once a day for 30 days (n=8).
Anesthesia and Surgical Procedure
A mixture of ketamine (50 mg/kg, Ketasol, Richter Pharma, Austria) and xylazine (10 mg/kg, Bioveta Plc, Czech Republic) was given intraperitoneally to the subjects in the groups for anesthesia. They were set up in the prone position. The laminectomy areas of the rat were wetted for shaving and then povidone-iodine was applied. After a longitudinal midline incision, the T11-T12 laminae were exposed and laminectomies were performed at the same levels. After hemostasis, the surgical wound was closed in accordance with anatomic layers. Cervical dislocation was performed under deep anesthesia for sacrifice at the end of the postoperative 30th day. Then, the vertebral column was removed en bloc.
Histopathologic Evaluation
After appropriate samples were taken from the materials, they were placed in cassettes, and routine tissue follow-up was performed in an automatic tissue tracking device within 14-16 hours. Serial sections, 3-4 μm in size were taken from the samples embedded in paraffin blocks. The slides were stained with Masson’s Trichrome Hematoxylin-Eosin (H&E), and then they were analyzed with a microscope.
The extent of fibrous tissue and its relationship with dura mater were scored according to He schame (15). The arachnoid involvement was also noted.
The cell density of scar tissue observed in the EF area was evaluated according to the Hinton method (16). The density of Vimentin were evaluated during the immunohistochemical examination.
Immunohistochemistry of Vimentin
Immunohistochemical staining was performed with a DAKO brand automatic staining device using an Avidin-Biotin complex system. Evaluations for vimentin immunohistochemical staining were performed under the light microscopy at x40, x100, x200, and x400 magnifications. If the staining rate was 10% or more, the staining was accepted as positive. The degree of staining was evaluated according to the staining rates that can be observed at different magnifications. The staining visible at x200 magnification was considered light, the stainingvisible at x100 magnification was considered medium, and the staining visible at x40 magnification was considered strong staining.
Western Blot Analysis for Proteins
Protein analysis was performed using the western blot analysis method previously defined by Arabacı Tamer et al. (17). The blotted membrane was blocked with 3% Bovine serum albumin for two hours at room temperature. The membrane was incubated with primary antibody (1:500 dilution monoclonal antibody anti-TGF-β, anti-FGF, anti-Bactin, Santa Cruz Biotechnology, Heidelberg, Germany) for 24 hours at a temparture of 4 °C temperature. Afterwards, the membrane was washed twice in TBST (Tris-buffered saline containing 0.1% Tween-20) and membrane was incubated with secondary antibody (1:2000 anti-mouse IgG1-HRP and anti-rabbit IgG-HRP Santa Cruz Biotechnology) for two hours at room temperature. Band analysis was performed using NIH-ImageJ software. For normalization, β-actin was used with respect to each band.
Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) Analysis
The quantitative real-time PCR (qPCR) analysis method was used for gene analysis (18). The primers used were as follows: GAPDH Forward 5’-ATGACTCTACCCACGGCAAG-3’, GAPDH Reverse 5’-CTGGAAGATGGTGATGGGTT-3’: TGF-β Forward 5’-ACCTGCAAGACCATCGACATG-3’, TGF-β-Reverse 5’-CGAGCCTTAGTTTGGACAGGAT-3’, FGF Forward 5’-AAGCCCGTCGGTGTCCATGG-3’, FGF Reverse 5’-GATGGCACAGTGGATGGGAC-3’. It was made using Applied Biosystem Software for the analysis. Relative quantitative data were calculated by the 2-ΔΔCt method. The calculation of gene expression ratios was expressed with GAPDH as a reference gene.
Statistical Analysis
Whether the quantitative variables were normally distributed among the groups was checked using the Kolmogorov-Smirnov test. In group comparisons, one-way analysis of variance (ANOVA) and Kruskal-Wallis H test was used. Pairwise comparisons of groups were made using the Tukey HSD and Bonferroni corrected multiple comparison test for ANOVA and Kruskal-Wallis H test, respectively. Descriptive statistics were expressed as mean ± standard deviation for variables with normal distribution and median (25th-75th percentile) for variables without normal distribution. P<0.05 value was evaluated as statistically significant.
Results
Effect of Pirfenidone on Epidural Fibrosis in Histopathological Analysis
When the groups were evaluated in according to EF, the grade 3 (Figure 1A and 1B) EF were dominant in group 1, grade 2 (Figure 1C and 1D) and grade 3 EF were predominant in group 2, and grade 1 (Figure 1E and 1F) and grade 2 EF were dominant in group 3. According to these results, group 3 showed a statistically significant difference between other groups (p˂0.001) (Table 1). In addition to this, arachnoid involvement was detected in three rats in group 1 and two rats in group 2, but in group 3, no arachnoid involvement was detected (Table 2).
Effect of Pirfenidone on Average Fibroblast Numbers in Histopathological Analysis
When groups were compared in terms of fibroblast count, the highest number of fibroblasts was in group 1, while the least number of fibroblasts was in group 3. The number of fibrolasts in group 2 was found to be between group 1 and group 3 (Table 1). According to these results, a 50 mg/kg dose of pirfenidone provided a statistically significant in decreasing the fibroblasts number compared to other groups (p˂0.001).
Effect of Pirfenidone on Immunostaining in Histopathological Analysis
To compare the results of immunostaining, a comparison was made by scoring 3 for severe staining (Figure 2A), 2 for moderate staining (Figure 2B), and 1 for low staining (Figure 2C). When groups were compared in terms of vimentin staining, it was found that severe staining was dominant in group 1, and severe and moderate staining was dominant in group 2, whereas in group 3. According to these results, there was no statistically significant difference between group 1 and group 2, but there was also a statistically significant difference between group 3 and other groups (p˂0.001) (Table 1).
Western Blot Analysis Results
TGF-β1 protein expression levels were decreased with pirfenidone treatment in group 2 and group 3 (p<0.001) compared with group 1. Moreover, bFGF protein expression levels were suppressed in group 2 and group 3 (p<0.001). Both protein expression results showed that the treatment of a 50 mg/kg dose of pirfenidone in laminectomy suppressed bFGF and TGF-β1 protein expression levels more (Figure 2D) (Table 3).
qRT-PCR analysis Results
qRT-PCR results revealed that TGF-β1 gene expression significantly decreased in the pirfenidone treated groups when compared with group 1 (p<0.001). bFGF gene expression levels were also significantly decreased in the pirfenidone-treated groups when compared with the group 1 (p<0.001). TGF-β1 and bFGF gene expression results showed that administration of a 50 mg/kg dose of pirfenidone suppressed increasing gene levels in EF, thus showing a better effect (Table 1).
Discussion
Our study is the first to use pirfenidone at 25 mg/kg and 50 mg/kg doses in a laminectomy model and to examine its effect on the EF formation. According to the results of our study, it was revealed that, when pirfenidone was administered orally at 50 mg/kg doses, it statistically significantly decreased EF formation compared with controls and 25 mg/kg doses of pirfenidone. In addition, this study revealed that pirfenidone reduced fibroblast proliferation and showed this effect by reducing TGF-β1 and FGF expression in fibrotic tissue.
In addition to decreasing TGF-β expression, many factors may be effective in the emergence of this antifibrotic effect of pirfenidone. In previous studies related to EF, it was revealed that reducing the effect of factors such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, platelet-derived growth factor, FGF and vascular endothelial growth factor (VEGF) reduced EF formation (8,19-21). When the studies related with pirfenidone were examined, a study conducted by Oku et al. (11) revealed that pirfenidone decreased lung fibrosis by reducing the interferon (IF)-γ, bFGF and TGF-β expression. In another study conducted by Guo et al. (14) in a silica-induced lung fibrosis model, the authors reported that the use of pirfenidone reduced the expression of IL-1β, TNF-α and IL-6 in lung tissue. In addition to these, in one in vitro study conducted by Liu et al. (22), the authors reported that pirfenidone showed an antifibrotic effect by reducing angiogenesis by decreasing the expression of VEGF/VEGFR2. This reveals that pirfenidone has an active role in reducing EF formation in the proliferation phase, and may also have an effect as an anti-inflammatory agent.
Another possible factor related with the emergence of antifibrotic activity may be that pirfenidone uses different pathways in different tissues. Analyzing pirfenidone from this point of view, Sun et al. (13) reported that pirfenidone showed antifibrotic properties by using the TGF-β1/Smad/CTGF pathway. Liu et al. (23) suggested that pirfenidone could have an antifibrotic effect by antagonizing the MAPK signaling pathway. In another study, Sun et al. (24) reported that pirfenidone showed antifibrotic activity by antagonizing the Smad and PI3K/AKT pathway. In addition to these studies, in a study conducted by Guo et al. (14), the authors reported that pirfenidone showed antifibrotic activity by antagonizing the TGF-β/smad2/3 pathways. The fact that pirfenidone has an antifibrotic activity over different pathways reveals that pirfenidone has a pleiotropic feature. Therefore, pirfenidone may also be used in different diseases beyond lung fibrosis, such as EF.
Another factor in the emergence of its anti-inflammatory and antifibrotic activity may be due to its dose-related effect. Many studies have revealed the dose-dependent effect of pirfenidone. One of these experimental studies was by Sun et al. (13), who reported that pirfenidone decreased collagen accumulation and fibroblast proliferation both in vivo and in vitro depending on the dose. In another study was by Liu et al. (23), pirfenidone was shown to reduce TGF-β, type-3 collagen, α-SMA and fibronectin in a dose-dependent manner. In addition, Shi et al. (25) revealed that pirfenidone reduced fibroblast proliferation and collagen accumulation in a dose-dependent manner. In our study, pirfenidone reduced the EF grade, fibroblast proliferation and TGF-β1 and bFGF expression in a dose-dependent manner. The results of our experiment are supported by the literature.
In this study, although we show for the first time that the use of oral pirfenidone has a positive effect on reducing the formation of EF, the study has some limitations. First, only two different doses of pirfenidone were administered. Second, in this initial study, molecular mechanisms were not investigated to reveal the mechanism of action of pirfenidone. Finally, a small sample size was used.
Conclusion
According to the results of our study, it was revealed that the use of oral pirfenidone decreased EF formation. In the emergence of this effect of pirfenidone, it has been demonstrated that at least two growth factors, namely TGF-β1 and bFGF, reduce the level of both gene expression and protein production depending on the dose. Therefore, pirfenidone can be a potential agent for EF.
Ethics
Ethics Committee Approval: The experimental procedure was reviewed and approved by the animal Ethics Committee of Aydın Adnan Menderes University Animal Experiments Local Ethics Committee (approval no: 64583101/2019/017, date: 26.03.2019).
Informed Consent: Informed consent is not required.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: Z.K., N.K.Ç., S.A., Concept: Z.K., S.A., Design: Z.K., Data Collection or Processing: Z.K., Ö.Ç., Analysis or Interpretation: N.K.Ç., Ö.Ç., H.Ö., Literature Search: Z.K., N.K.Ç., Ö.Ç., Writing: Z.K.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: ADUBAP-TPF-19047.