Comparative Preventive Efficacy of Recombinant Tissue Plasminogen Activator ( rt-PA ) , Urokinase , Hyaluronic acid and Enoxaparin in Post Laminectomy Epidural Fibrosis in Rats

Aim: We aimed to determine the effects of rt-PA and enoxaparin on post laminectomy epidural fibrosis and to compare the effects of these two medications with hyaluronic acid and urokinase in an experimental model of spinal epidural fibrosis. Method: Forty Spraque-Dawley rats were anaesthetized by intraperitoneal anesthesia (ketamin 8 mg/100 g) and randomly allocated to five groups. All groups were underwent L1-2 total laminectomies. Topical medications were applied to group 2-5 right after the laminectomy space. Hence, in group 1 (G1=control) only laminectomy was constituted, in group 2 (G2) laminectomy + topical Urokinase, in group 3 (G3) laminectomy + topical rt-PA, in group 4 (G4) laminectomy + topical enoxaparin and in group 5 laminectomy + topical Hyaluronic acid (G5) application were performed. The potentials of local urokinase, rt-PA, hyaluronic acid and enoxaparin in preventing the production of epidural fibrosis were assessed. Result: In inter group comparison, G1-G2, G1-G3, G1-G4, G1-G5 groups differed from each other for total scar score, middle scar score and deep scar score (p<0.05). However, G2-G3, G2-G4, G2-G5, G3-G4, G3-G5 and G4-G5 were not different from each other. Conclusion: In rats, the topical thrombolysis with fibrinolitic agents is safe and efficacious in preventing of post laminectomy epidural fibrosis. Thrombolytic therapy with topical hyaluronic acid, rt-PA, urokinase and enoxaparin may come to play an important role in the prevention of post operative spinal epidural fibrosis and arachnoiditis. Based on the results we found that rt-PA and enoxaparine inhibit the formation of epidural scar adhesions and arachnoiditis compared with control group.


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The first group was the control group in which total laminectomy of L1-2 was achieved and following durotomy dura mater was closed without any medication.In the second group all interventional procedures were done in a similar fashion and Urokinase (25 IU-Laboratories Serono S.A. CH-1170 Aubonnel, Switzerland) was inserted beneath and over the dura mater at laminectomy area.Used dose of Urokinase for this study was based on the published knowledge in the literature (3).
In the third group, after the total laminectomies of L1-2 rt-PA (Actilyse, Boehringer Ingelheim) was inserted beneath and over the dura mater following durotomy in laminectomy area and then dura mater was closed.The rt-PA is supplied as a lyophilized powder that has a concentration of 1 mg rt-PA/ml, pH of 7.3.The dose of rt-PA chosen for this study was 0.25 mg.The chosen dose of rt-PA for this study was based on the published knowledge in the literature (6,7).In the fourth group all interventional procedures were done in a similar fashion and enoxaparine (Clexane R 10 mg/0.1 ml amp, Eczacibasi, Istanbul, Turkey; 1.5 mg/kg) was inserted beneath and over the dura mater at laminectomy area.Used dose of enoxaparine for this study was based on the published knowledge in the literature (21).In the fifth group all procedures were done in a similar fashion and Hyaluronic acid (8 mg/kg) (Synvisc 16 mg/2ml, Wyeth, USA) was applied.The chosen dose of Hyaluronic acid for this study was based on the published knowledge in the literature (1).All rats were followed up in a temperature-controlled room for 6 weeks.Neurological function was evaluated weekly.At the end of follow up, the animals were sacrificed by a lethal dose of pentobarbital (60 mg/kg).
Spinal columns were harvested en block immediately after sacrifice and placed in buffered formaldehyde for at least three days.All fixation and dehydration procedure were performed at room temperature.Each specimen was then decalcified with boric acid approximately 3 weeks.Three blocks tissue from each laminectomy site to be processed.Four-micron sections were examined after staining with Haematoxylen and Eosin (H&E).All sections were examined with a light microscope (Olympus BX-50) to measure the tissue response to the two groups.All specimens were evaluated in a blinded manner by two different pathologists.The objective criteria of adhesion were graded according to following classification as Total scar score in Table 1 (22).Additionally we also used scoring system for gross evaluation of peridural fibrosis to investigate the quantity of the peridural fibrosis with another scoring system as stated in previous report in the literature (Table 2) (19).
The histopathological scores for each rat total scar, intermuscular scar, middle scar, deep scar, dural adhesion and new bone growth recorded in the slices of the laminectomy area, and were again retrieved and analyzed.Furthermore, according to the dural adhesion score in the rats; score 0 and 1 were accepted as absence of

INTRODUCTION
It is well known that postoperative significant epidural fibrosis and/or adhesive arachnoiditis are one of the most common causes of failed back surgery syndrome after spine surgery however characteristics of patients selected is a very important factor (1)(2)(3)(4).Failures of spine surgery characterized by persistent or recurrent pain accounts for up to 40% of all patients after laminectomy and discectomy in long term follow up (1).Adhesive arachnoiditis may occur with any chemical or physical injury to the leptomeninges (1,2,10,(16)(17)(18)(19).It was also stated that the amount of scar formation after lumbar discectomy related to the clinical outcome, the size of surgical exposure, and some fibrinolytic factors (5).Post operative fibrosis causes nerve root traction that leads to perfusion or conduction disturbances resulting in pain (1).
Up to date there are different studies aiming the prevention of post laminectomy scarring using by different mechanical barriers, chemical regulators of scar formation and prevention of local hematomas (1,3,5,8,10,(14)(15)(16)18,19,22).Still many substances have been under investigation to remedy the detrimental effects of post laminectomy epidural fibrosis.Hyaluronic acid (1) rt-PA (9) and urokinase (3) have been studied separately and found useful.In the literature we did not find any report investigating the comparative efficiency of antifibrinolytic agents on arachnoiditis and postoperative scar formation.We aimed to evaluate the effects of rt-PA, enoxaparin, hyaluronic acid and urokinase on post laminectomy epidural fibrosis and arachnoiditis, and to compare their effects in an experimental model of spinal epidural fibrosis and arachnoiditis.

MATERIALS AND METHODS
Fourty Spraque-Dawley rats, aged 6 months, weighing average of 300 g, were utilized in this randomized experimental study.We certify that all applicable institutional and governmental regulations concerning the ethical use of animals were followed during the course of this research.We investigated for prevention of epidural fibrosis on post laminectomy area, and drugs were locally administered in surgical space.
Surgical procedure was performed under intra-peritoneal anesthesia (ketamine hydrochloride 8 mg/100 g).Lower half of the back was sawed and preoperative dose of intramuscularly cefotaxime (100 mg/kg) was given.The rats were positioned prone on the operating table.Above the dorsal area was prepared povidine iodine (Betadine R ) soap and solution.The area was draped in an aseptic fashion and a midline incision was made along the spinous process of the lumbar area.The fascia and thoracolumbar muscle were opened bilaterally and total laminectomies of L1-2 were performed.Bleeding was controlled.The dura over the spinal cord was opened and then closed microsurgically.The animals were allocated randomly in the five groups of 8 rats each.

RESULTS
Fourty rats were included in this randomized study program.Rats were divided into five groups to investigate the effects of topical urokinase, rt-PA, enoxaparin and hyaluronic acid in setting which experimental post laminectomy spinal epidural fibrosis.The animals reported in this study were healthy and ambulatory without severe neurological deficit at the time of sacrifice.Three rats died of anesthesia complication in the early postoperative period.Four rats had severe neurological deficit.These rats replaced in the study.Active epidural bleeding was encountered in two rats at the time of surgery and controlled by cotton compression without using bipolar-coagulation.In the control animals, there was severe hemorrhage within surgical area in one rat.One rat had moderate hematoma within surgical area in rt-PA group and two rats had also severe hematoma in urokinase group.These four rats were excluded from the study and four new rats were included instead of them.Comparative results with control and rt-PA, urokinase, hyaluronic acid, enoxaparin for each parameter were showed separately as grade and P values in table 3-8.
Inter-groups comparison was statistically perform ed.
The control animals showed heavy scar formation at all laminectomy levels, including thick dural adhesions associated with deep scar (Table 6).The histological observation showed a dens fibrous tissue layer in control defects (Figures 1,2,3).Treatment with all these drugs did not affect the healing of skin subcutaneous tissue or fascia and primarily inhibit peridural scar formation adjacent to the dura and the laminectomy site.We detected lower grade for all evaluated parameters such as middle scar (p=0.04),deep scar (p=0.001) and dural adhesion (p=0.004),total scar (p=0.007)but new bone formation and intermusculer scar were not significantly affected by rt-PA (Table 3-8).(Figures 1,2,3).The presence of arachnoiditis was less in rt-PA and urokinase group (p=0.04).

DISCUSSION
Postoperative significant epidural fibrosis and or adhesive arachnoiditis are the one of the most common causes of failed back surgery syndrome after spine surgery (1-4,10,13).Numerous factors are involved in pathogenesis of postoperative epidural adhesions (15).The review of the literature reveals the presence of many hypotheses and treatment attempts regarding epidural fibrosis (3,4,(18)(19)(20).Epidural fat destruction, local hematoma, migration of fibroblasts from the raw surface of the erector spinae musculature was stated the source of postoperative scar tissue (11)(12)(13)22).Furthermore, foreign body reaction from surgical swab debris, as well as a systemic fibrinolytic defect, has also been reported as possible etiologies of post laminectomy scar formation (4,12).It was also stated that the amount of scar formation after lumbar discectomy may be related to the size of the surgical exposure, and some fibrinolytic factors (5).
Prevention of post laminectomy scarring has been attempted using different mechanical barriers, chemical regulators of scar formation and prevention of local hematomas (1,3,5,(8)(9)(10)14,15,17,18,19,22).Still many substances have been under investigation to remedy the detrimental effects of post laminectomy epidural fibrosis.Hyaluronic acid (1), rt-PA (9) and urokinase (3) have been studied separately and found useful on the prevention of post laminectomy epidural fibrosis and arachnoiditis.However, there was no study investigating their comparative responses in post laminectomy epidural fibrosis.Herein we investigated the effects of rt-PA, enoxaparin, hyaluronic acid and urokinase on post laminectomy epidural fibrosis, and compared the effects of these medications.As seen in results section, all medications used in this study were found efficacious on the prevention of post laminectomy epidural fibrosis and arachnoiditis.But we did not find any significant difference among them.For this reason we thought that choosing of preventive medication in spine surgery may be based on the availability and cost of medication.
In conclusion, in rats, the topical thrombolysis with fibrinolitic agents is safe and efficacious in preventing of post laminectomy epidural fibrosis.Thrombolytic therapy with topical hyaluronic acid, rt-PA, urokinase and enoxaparin may come to play an important role in the prevention of post operative spinal epidural fibrosis and arachnoiditis.Based on the results we found that rt-PA and enoxaparine inhibit the formation of epidural scar adhesions and arachnoiditis compared with control group.Any statistical significance between the effectiveness of these medications was found.However, according to the statistical significance level, the efficacies of these medications were ranked as urokinase, rt-PA, enoxaparine and hyaluronic acid respectively.

Table 1 .
Scoring system for gross evaluation of epidural fibrosis as total scar score.Grade 0 When the dura mater was free of the scar tissue.Grade 1 When only thin fibrous bands between the scar tis sue and the dura mater were observed.

Table 2 .
Scoring system for detailed evaluation of epidural fibrosis.

Table 3 .
Analysis of the groups with control according to Total Scar Score

Table 4 .
Analysis of the groups with control according to Intermusculer Scar Score

Table 5 .
Analysis of the groups with control according to Middle Scar Score

Table 6 .
Analysis of the groups with control according to Deep Scar Score

Table 7 .
Analysis of the groups with control according to dural adhesion score

Table 8 .
Analysis of the groups with control according to New Bone Growth Score