Effect of topical rifamycin application on epidural fibrosis in rats.
Rifamycin SV is a well-known semisynthetic antituberculosis agent which has been used widely in various surgical procedures owing to its bactericidal effects against wide spectrum of Gram-negative and Gram-positive microorganisms with a tendency to Staphylococcus aureus.  Peritoneal lavage with rifamycin SV is suggested to be effective in the treatment of intraabdominal infections and reducing adhesion formation, as well.  Furthermore, the topical administration of rifamycin SV during spinal neurosurgical procedures via intraoperative irrigation of the surgical field is increasing. Surgical site irrigation for the prevention of both superficial and deep wound infections is mostly preferred in cases with prolonged operative times requiring foreign material implantations. Although there are many data in the literature which discuss the role of rifamycin SV in the treatment of wound infections, [10,11] data about the effects of topical rifamycin SV on epidural fibrosis following spinal procedures is limited.
In the present study, we aimed to investigate the effects of topical rifamycin SV irrigation on epidural fibrosis after laminectomy in a rat model. We hypothesized that rifamycin SV would be effective in the alleviation of epidural fibrosis after laminectomy. If this hypothesis is true, it might lead to new studies searching the dose-dependent effect of rifamycin SV as well as studies comparing rifamycin SV and other materials which were previously proven to be effective in the prevention of epidural fibrosis, in higher-order animal models.
MATERIALS AND METHODS
All the experimental procedures were performed with the permission of the Committee of Ethics for Animal Studies of Abant Izzet Baysal University to minimize the discomfort of the animals during the surgery and recovery periods. In this experimental study which was conducted between March 2015 and April 2015, a total of 20 adult, female Wistar rats, weighing 250 to 350 g were used.
The rats were randomly divided into two groups providing 10 rats in each group. The control group (n=10) did not receive any treatment but laminectomy. In the treatment group (n=10), surgical field was irrigated with 1 mL rifamycin SV (Rif[R] 250 mg/3-mL ampoule).
Anesthesia and surgical procedure
The animals were anesthetized with intraperitoneal (i.p.) Xylazine (10 mg/kg; Bayer, Istanbul, Turkey) and ketamine hydrochloride (60 mg/kg; Parke-Davis, Istanbul, Turkey) prior to the surgical procedures. The lower back of each rat was shaved, and the surgical area was sterilized with povidone. A vertical midline skin incision was made over the L3-L5 levels with the aid of operative microscope under a magnification of 16x (Zeiss OPMI 1; Carl Zeiss Meditec, Oberkochen, Germany). The lumbosacral fascia was incised, and the L3-5 laminae were exposed after subperiosteal dissection of the paraspinal muscles. The L3, L4 and L5 laminas were totally removed. Dura was clearly and completely exposed after the removal of ligamentum flavum and epidural fat tissue of the relevant surgical area. Hemostasis was achieved by cotton pads. The treatment was administered to the treatment group and the wounds were closed in anatomical layers.
The animals were sacrificed on postoperative Day 30 with a lethal dose (60 mg/kg; IE Ulagay, Istanbul, Turkey) of pentobarbital. The vertebral columns of the rats were removed en bloc, including the whole laminectomy area between the L1 and L5 levels. The specimens were, then, placed in 10% buffered formalin.
Evaluation of epidural fibrosis
The specimens were dehydrated and embedded in paraffin after complete decalcification with ethylenediaminetetraacetic acid (EDTA, R&D Systems Inc., Minneapolis, MN, USA) for histological examination. Sections of 10 [micro]m were obtained on the axial plane and stained with the Masson's trichrome which stains nuclei black, collagen blue and muscle fibers, cytoplasm and keratin red. The sections were examined with the aid of Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) and photographed by using a Nikon DS-Fi1 camera (Nikon Corporation, Tokyo, Japan). All the laminectomy sections were evaluated for fibrosis density and arachnoid involvement by a single pathologist who was blind to the study groups. Quantitative morphometric analysis of the sections was performed by the Nikon Nis elements D 3.1 Digital Analyzing System (Nikon Corporation, Tokyo, Japan). A magnification of 100x was used for the measurements. The thickness of the fibrosis was measured at three different points. As previously described in the literature, the first sample was obtained from the midpoint of the laminectomy defect, while the second and third samples were obtained 2 mm from the right and left sides of the first sample, respectively.  Epidural fibrosis was graded according to the following scheme suggested by He et al.  Grade 0, there is no scar tissue on dura mater; Grade 1, there is only a thin fibrous band between the scar tissue and the dura mater; Grade 2, there is scar tissue in less than two-thirds of the laminectomy defect; and Grade 3, there is scar tissue involving more than two-thirds of the laminectomy defect or extending to the nerve roots. The mean values were used for statistical analysis. The presence of arachnoid involvement was recorded.
Statistical analysis was performed using the IBM SPSS version 22.0 software (IBM Corp., Armonk, NY, USA). Descriptive data were expressed as the median (25-75%) for the comparison of fibrosis grades between the groups. The Fisher's exact test was used to analyze the contingency tables (p<0.05). A p value of <0.05 was considered statistically significant.
No adverse effects of rifamycin SV (erythema) and procedural complications (wound infection, hematoma, cerebrospinal fluid leakage) were observed. There was no mortality, and all the rats were ambulatory at the time of sacrifice. There was no statistically significant difference between the treatment and control groups in terms of epidural fibrosis (p=0.164) and arachnoid involvement (p=0.303).
In the treatment group, Grade 2 epidural fibrosis was seen in 50%, whereas Grade 3 epidural fibrosis (Figure 1) was seen in 40% of the axial sections stained with the Masson's trichrome. In the control group, Grade 2 epidural fibrosis was seen in 20% and Grade 3 epidural fibrosis (Figure 2) was seen in 80%; however, no Grade 1 epidural fibrosis was seen. On the other hand, in the treatment group, Grade 1 epidural fibrosis was seen in 10% of the sections. The fibrosis grading of the groups is shown in Table 1. Although the number of Grade 3 epidural fibrosis was significantly lower in the treatment group, there was no statistically significant difference between the treatment and control groups (p=0.164).
Arachnoid involvement was observed 10% and 40% of the sections in the treatment and control groups, respectively. However, there was no statistically significant difference between the treatment and control groups (p=0.303). The values regarding the arachnoid involvement are shown in Table 2.
Epidural fibrosis is the result of the proliferation of fibroblasts transforming into myoblasts and accumulation of disorganized extracellular matrix proteins,  being a natural response of the body to surgery. The scar formation causes traction on dura mater and compression or tethering of the nerve roots leading to one of the most devastating causes of failed-back surgery with persistent lower back and leg pain. [14-16] Unfortunately, 8 to 48% of patients with previous lumbar disc surgery suffers from failed-back surgery syndrome. [17-19]
Key and Ford  firstly reported epidural fibrosis following lumbar disc surgery. Since then, the administration of various kinds of drugs and materials such as autologous fat grafts, Adcon-L, polytetrafluoroethylene membrane, Gelfoam, and fibrinolytic agents have been administered desperately to the surgical field to act as a barrier or reduce the epidural fibrosis formation around the dura matter and nerve roots. ([4,21-25]) However, the effective and accepted treatment of epidural fibrosis has not established, yet. Reoperation is complicated and dangerous due to the high likelihood of intraoperative nerve root injury and dural tear, as well.  Therefore, preventing or minimizing the epidural scar formation is essential. Congenital root anomalies, excessive traction on the nerve roots and extreme blood loss during surgery, intraoperative use of sponges, and foreign tissue reactions are all shown to aggravate epidural fibrosis formation. [27,28] Minimizing surgical dissection and performing meticulous hemostasis have been suggested to decrease scar tissue formation.  Besides preventing epidural fibrosis, protecting the surgical area against wound infection should be one of the main goals of surgery.
Meticulous hemostasis was the main effort to prevent epidural fibrosis before certain agents and materials were introduced to the surgical practice. With the introduction of these materials, surgeons have left scrupulous hemostasis. Hemostasis and washing surgical site are two most important factors for preventing epidural fibrosis. The content of washing water is crucial. A bactericidal liquid may be far more suitable than a simply sterile water.
Rifamycin SV irrigation of the surgical site, which has been well-studied and preferred by most of the neurosurgeons, is widely used to avoid superficial and deep wound infections.  Additionally, rifamycin SV is found to have an inhibitory effect on cytokine and chemokine synthesis on monocytes and macrophages, leading to its usage in inflammatory bowel disease, rheumatoid arthritis, and gut inflammation. ([11,31-34]) The detractive effect of rifamycin SV on the number of adhesions in the treatment of intraabdominal infections has been shown, as well.  However, rifamycin SV has been studied only once in terms of its effects on epidural fibrosis reporting decreased formation of epidural fibrosis without a statistically significant difference, compared to the control group in a rat model. 
In the present study, we investigated the effects of topical administration of rifamycin SV via the irrigation of the surgical area on epidural fibrosis formation in a rat model. Our data demonstrated that irrigating the laminectomized vertebrae with rifamycin SV decreased the grade of epidural fibrosis and arachnoid involvement. Grade 3 epidural fibrosis was found in four of the rats (40%) in the treatment group, while eight of the rats (80%) in the control group had Grade 3 epidural fibrosis. The median value of fibrosis grade was dramatically decreased in the treatment group. The median value of fibrosis grade did not decrease to Grade 1 in the control group. Although we observed clinically significance between the groups, there were no statistically significant differences between the groups which can be attributable to the small sample size, being the major limitation of the study.
In conclusion, our study results suggest that rifamycin SV can be used to avoid the formation of epidural fibrosis given the anti-inflammatory effects of the agent, although not statistically significant. However, further investigations in larger study samples are warranted.
Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.
The authors received no financial support for the research and/or authorship of this article.
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Cem Dinc (1)[iD], Cengiz Tuncer (1)[iD], Mehmet Erhan Turkoglu (2)[iD], Mehmet Tokmak (3)[iD], Pinar Ocak (1)[iD], Uygur Er (1)[iD]
(1) Department of Neurosurgery, Duzce University School of Medicine, Duzce, Turkey
(2) Department of Neurosurgery, Ministry of Health, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey
(3) Department of Neurosurgery, Medipol University, School of Medicine, Istanbul, Turkey
Received: December 01, 2017 Accepted: February 01, 2018 Published online: January 31, 2019
Corresponding author: Cem Dinc, MD. Duzce Universitesi Tip Fakultesi Beyin Cerrahisi Anabilim Dali, 81620 Duzce, Turkey.
Table 1. Histopathological results of epidural fibrosis grades of study groups Groups Fibrosis Grade n % 1 1 10.0 Treatment 2 5 50.0 3 4 40.0 Total 10 100.0 1 0 0.0 Control 2 2 20.0 3 8 80.0 Total 10 100.0 p=0.165. Table 2. Comparison of the arachnoid involvement of study groups (*) Groups Arachnoid involvement (-) Arachnoid involvement (+) n % n % Treatment 9 90.0 1 10.0 Control 6 60.0 4 40.0 Total 15 75.0 5 25.0 (*) Fisher's exact test was used; p=0.303.
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|Title Annotation:||Original Article|
|Author:||Dinc, Cem; Tuncer, Cengiz; Turkoglu, Mehmet Erhan; Tokmak, Mehmet; Ocak, Pinar; Er, Uygur|
|Publication:||Turkish Journal of Physical Medicine and Rehabilitation|
|Date:||Mar 1, 2019|
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