Application of expanded inert polytetrafluorethylene membrane in prevention of post laminectomy fibrosis in wistar rats

Estudo do uso da membrana de politetrafluoroetileno inerte expandido para a prevenção da fibrose pós laminectomia em ratos wistar

ORIGINAL ARTICLE

Application of expanded inert polytetrafluorethylene membrane in prevention of post laminectomy fibrosis in wistar rats

Estudo do uso da membrana de politetrafluoroetileno inerte expandido para a prevenção da fibrose pós laminectomia em ratos wistar

Tarcísio Eloy Pessoa Barros Filho^I; Cláudia Regina G. C. M. Oliveira^II; Reginaldo Perilo de Oliveira^III; Erika Meirelles Kalil Pessoa de Barros^IV; Raphael Martus Marcon^V; Alexandre Fogaça Cristante^VI

^IHead, Wistar Rats Discipline

^IIHead, Laboratory of Pathological Anatomy

^IIIAssistant MD

^IVCoordinator, MD

^VCoordinator Physician

^VIPhysician Preceptor

^{Correspondence}

SUMMARY

Post laminectomy fibrosis in spine surgery has been responsible for a high rate of failure in a short or long term. Many of this patients develop chronic or recurrent pain after surgery of discal herniation, canal stenosis etc.

Although there some doubts about the etiology of this problem, it was known that fibrosis interfere with the normal mobility of roots and spinal cord. This factor is considered as the most important aspect in the genesis of the surgical failures.

To avoid postoperative fibrosis, a lot of materials of interposition between posterior spine muscles and dura mater were studied, without good response.

This paper studied comparatively post laminectomy fibrosis in Wistar rats with the addition of the following materials to avoid it: control group (without interposition) and expanded inert polytetrafluoroethylene membrane (Preclude Spinal Membrane^®).

It was observed that accurate surgical technique apparently is an important factor to prevent the formation of abundant scar tissue and that the expanded inert polytetrafluoroethylene membrane is an inert material.

Key Words: Laminectomy; Fibrosis; Wistar rats; expanded inert Polytetrafluoroethylene membrane.

RESUMO

A fibrose pós laminectomia em cirurgias da coluna vertebral tem sido responsabilizada por um grande percentual das falhas nestas cirurgias, tanto a curto quanto a longo prazo. Muitos dos pacientes desenvolvem sintomas de dor crônica ou recorrente após tratamento cirúrgico de hérnias de disco, estenose de canal, etc.

Apesar de ainda existirem dúvidas quanto à fisiopatologia do problema, sabe-se que a fibrose interfere na mobilidade normal das raízes e da medula espinal, fator este responsabilizado como o principal na gênese das falhas cirúrgicas.

Para se evitar a fibrose pós-cirúrgica, vários materiais de interposição entre a musculatura posterior da coluna e a dura-máter foram estudados, sem grandes resultados.

Neste trabalho foi feito o estudo da membrana de politetrafluoroetileno inerte expandido (Preclude Spinal Membrane^®) comparando-se a mesma com grupo controle para a prevenção da fibrose pós laminectomia.

Observou-se que a técnica cirúrgica apurada parece ser fator importante na prevenção da formação de tecido cicatricial abundante e que a membrana de politetrafluoroetileno inerte expandido mostrou ser material inerte.

Descritores: Laminectomia; Fibrose; Ratos Wistar, Membrana de Politetrafluoroetileno inerte expandido.

INTRODUCTION

Post-surgical fibrosis has been held responsible as a possible cause for persistent pain following spinal cord surgeries. Also, the cicatricial tissue increases the technical difficulty and the risks for subsequent procedures. Poor early results in spinal cord surgeries characterize by persistent or recurrent pain and account for over 20% of all cases of patients who were submitted to laminectomy and discectomy, where poor results in the long range may reach up to 40% according to some authors⁽³⁾. Recurrent discal herniation, postooperative degenerative changes, arachnoiditis, poor patient selection, improper surgery and extradural fibrosis formation were identified as possible causes of poor surgical results^(3,4,6).

The association of peridural fibrosis and pain is controversial. Postoperative fibrosis may cause strain to the nerve roots which may lead to nutritional or conduction disorders resulting in pain. The normal mobility of the spinal cord and nerve roots is limited by perineural fibrosis⁽²⁾. Kusch et al. have studied the response to pain in patients during decompression for recurrent discal herniation or canal stenosis performed under local anesthesia, showing that invariably perineural fibrosis will fix the painful root in the spinal canal⁽¹²⁾. This study suggests that perineural fibrosis increases the vulnerability of nerve roots towards compression or strain that may occur in a recurrent discal herniation or in a canal stenosis. Adhesive neuritis was also described as an etiological pain factor that recurs in the carpal tunnel syndrome⁽¹⁰⁾, besides being the cause of pain in a variety of other peripheral nerves following surgeries and injuries^(1,17,20).

For many years investigators have studied ways of preventing peridural fibrosis following laminectomy and discectomy^{(5,8,9,13,19)}. Peridural fibrosis is simply the replacement of peridural fat by fibrous tissue, that may hold the dura mater and the nerve roots against the surrounding structures, both anteriorly and posteriorly⁽¹⁹⁾. It was never proven that peridural fibrosis formation will directly cause any postoperative radicular pain and/or back pain. However, as mentioned above, peridural adhesions will limit the mobility of nerve roots and increase their susceptibility towards recurrent discal herniations and stenosis⁽⁷⁾. Besides, fibrosis formation will make future surgeries more difficult⁽¹⁶⁾.

The last studies showed that fibrosis may occur both anteriorly and posteriorly and correlates with the surgical dissection area⁽¹⁹⁾. This does not agree with the previously accepted idea that fibrosis was caused by an invasion of fibroblasts that originate posteriorly in the erector muscle of the spine to form the so-called "laminectomy membrane"⁽¹⁵⁾. Also, in early studies, it was thought that fibrosis was only the result of annulus fibrosus surgical injury⁽¹¹⁾.

Based on the theory that adhesion derived from the spinal cord posterior muscles, several materials have been inserted between these muscles and the dura mater in order to evaluate their potential for reducing fibrosis formation. Geofoam, Gelfilm, Silastic membrane, bone wax, steroids, hemostatic agents, Dacron and other materials such as carboxymethylcellulose, elastase and polylactic acid have been used, although they have not proven to be effective against fibrosis formation around nerve roots⁽⁵⁾. Langenskiold and Kiviluoto⁽¹³⁾ were the first to use fat-free grafts and show that this type of graft partially prevents posterior epidural adhesions. The authors have also shown that such grafts remain feasible for many years following their implantations ⁽¹⁴⁾. Also, some studies have shown that sodium hyalouronate, a natural polymer of the glucosamineglycan family (mucopolysaccharide acid), with its semi fluid properties, covers the nerve roots and the dura mater both anteriorly and posteriorly, thus reducing fibrosis at the critical region (anteriorly) where adhesions are formed between the nerve roots and the annulus fibrosus⁽⁵⁾. Sodium hyalouronate was shown to be superior to other materials both in fibrosis extension and in the strength of the adhesions, with local anti-inflammatory properties, reduction of free radicals, fagocytosis inhibition by monocytes and adherence, and leukocyte migration^{(18 ,19)}.

OBJECTIVE

The purpose of this study is to analyze and quantify the fibrosis that develops after laminectomy and the effect exerted by the insertion of the expanded inert polytetrafluoroethylene membrane – e-PTFE (Preclude Spinal Membrane^®) on the prevention of fibrosis formation.

MATERIALS AND METHODS

Rats

Wistar rats were used after their ages and weights were standardized. In all, 6 rats were utilized.

Anesthetic procedures

Rats were anesthetized with intraperitoneal pentobarbital. The female rats received a 35-55 mg/kg dosis, while the males received a 55-75 mg/kg. The recommended doses were 35 mg/kg and 55 mg/kg, respectively for females and males. Such pentobarbital doses should keep each rat anesthetized for about 2 hours.

Laminectomy

The spinal cord was exposed by a laminectomy performed with the aid of a surgical microscope:

• An opening was made in the skin at the dorsal median line in order to expose the T8-12 vertebrae of the spinal cord.

• The muscles inserted in the T9-11 spinal processes were cut or separated and the articular processes of the vertebrae were exposed using a bipolar coagulant agent to stop hemorrhages as needed.

The T-10 lamina, as well as the distal half of the T9 spinal process was removed with the aid of a punch. The operation was performed most carefully to prevent injuring the dura mater. The area of the laminectomy performed in this way was of approximately 2 x 2 mm.

Insertion material

The rats were divided into two groups. In the first group no insertion material was used, while in the second group, in the gap formed by the laminectomy, a fragment of expanded inert polytetrafluoroethylene membrane (Preclude Spinal Membrane ^®) long enough to cover the laminectomy was used as insertion material.

Postoperative procedures

The musculature was brought back by means of simple stitches using mononylon 4-0 thread. The skin was closed by continuous stitches using mononylon 2-0 thread. A prophylactic 25mg/kg dosis of Kefalotin (Neutral Keflin^®) was administered peritoneally. All rats remained alive during 43 days, a time long enough for the initial hematoma to have been already replaced by fibrous tissue as shown in previous studies⁽¹⁸⁾.

Euthanasia and tissue sample collections for analysis

Euthanasia was performed at 43 days following the laminectomy, when the outcomes had already been established. The euthanasia and tissue sample removal were performed according to the following procedures:

• 43 days after the laminectomy and after the euthanasia the body weight was measured.

• The rat was anesthetized with 40 mg/kg i.v. pentobarbital.

• The aorta was catheterized by toracotomy and an incision was performed in the right atrium. Then the rat was exsanguined by infusing 500 ml saline solution through the catheter, followed by 500 ml of a 10% formol solution as a fixative for the tissues.

• The cord was removed from C5 to L5, while the paravertebral musculature was preserved.

• The cervical, lumbar and toracic segments were identified with the injured site.

• Samples were stored in a 10% formol solution at room temperature and left for 3 days until they were used.

Material preparation and structural analysis

The region of two discs adjacent to the laminectomy site, plus the 1-cm proximal region and a 1-cm caudal region (controls) were axially cut in four zones. Each sample was imbedded in paraffin and decalcified in 7% nitric acid for 2 days. Axial cuts 6- to 8 mm in length of each region were prepared and stained with hematoxylin and eosin (HE). An experienced pathologist using an optical microscope studied all cuts; the pathologist was blinded to the randomization of the rats. After analyzing the cuts he analyzed the following:

- Hyperemia: congestion or local vascularization increase.

- Hemorrhage: local red blood cell overflow

- Fibrosis: presence of dense connective tissue

- Inflammatory response: inflammatory infiltrate of polymorphonuclear or mononuclear leukocytes, with some presence or no presence of granulomatous inflammatory response of the foreign body type.

These factors were scored according the scale below.

0 – absent

1 – discreet

2 – moderate

3 – marked

Additionally, it was checked whether the surrounding structures were invaded by fibrosis.

Rats were excluded that presented abnormal proximal and distal sites.

Statistical analysis of outcomes

The scores obtained from the rats for the different parameters were analyzed by group for averages, standard deviations and mean square errors. Also, an intergroup comparison was made based on the original hypothesis that the groups were not equal, so the unilateral Mann Whitney U-test was used (a < 0,05).

RESULTS

Four female rats (66%) and 2 male rats (33%) were used. The mean age was 20.33 weeks (age group 20 to 22 weeks). The average weight was 251.66 g (minimum weight 200 g, maximum weight 280 g). The rats were killed at 43 days. The body weight of all rats increased during the trial, the average weight gain being 19.83 g (minimum 1 g, maximum 39 g).

No deaths, infections, autophagia or pressure ulcers were observed in none of the rats.

The histological studies did not show any spinal canal invasion by fibrosis in none of the groups. Fibrosis was limited to the posterior region of the spinal cord (Figure 1).

Table 1 below shows the scores obtained in histological analysis for hyperemia, hemorrhage, fibrosis and inflammatory response at the lami-nectomy site.

In rat 4 (ePTFE membrane) foreign body giant cells were observed (Figure 2). In the other rats little inflammatory response of the foreign body type was observed.

No changes were noticed at the proximal and distal control sites that could support the exclusion of any of the rats.

Both groups were analyzed for each anatomico-pathological item, together with the averages, standard deviations and mean square errors for each item. Also, a comparison was made between groups for each variable, using the unilateral Mann Whitney U-test (a £ 0,05). No statistical difference was noticed between the control group and the group treated with expanded inert polyterafluoro-ethylene membrane (ePTFE - Preclude Spinal Membrane^®).

DISCUSSION

Post-surgical fibrosis has been held responsible as a possible cause of persistent pain following spinal cord surgeries. Also, the cicatricial tissue increases the technical difficulty and the risks for subsequent procedures.

The association of peridural fibrosis to pain is controversial. Postoperative fibrosis may strain the nerve roots, leading to nutritional or conduction disorders resulting in pain. The normal mobility of the spinal cord and nerve roots is limited by perineural fibrosis⁽²⁾.

For many years, investigators have studied ways of preventing peridural fibrosis following laminectomy and discectomy^{(5,8,9,13,19)}. Peridural fibrosis is simply the replacement of peridural fat by fibrous tissue, which can hold the dura mater and nerve roots against the surrounding structures, both anteriorly and posteriorly⁽¹⁹⁾.

The last studies show that fibrosis will occur both anteriorly and posteriorly and correlates with the surgical dissection area⁽¹⁹⁾. This is not in agreement with the previously accepted idea that fibrosis was caused by invasion of posteriorly originated fibroblasts.

Based on the theory that adhesion derives from spinal posterior muscles, various types of materials have been inserted between these muscles and the dura mater in order to evaluate their potentials in the reduction of fibrosis formation. This study investigated the expanded inert polytetrafluoroethylene membrane - ePTFE (Preclude Spinal Membrane^®) as an insertion material.

We decided to use Wistar rats due to their availability in this country and also for the fact that their handling is technically easier. Female and male rats were used because it was difficult to obtain one-gender rats from our bioterium, but this does not invalidate our study, since the literature does not mention that cicatricial changes will be different according to sex. All rats were considered adults, since their age variations were not significant. The weights of all rats were consistent with their ages and all of them gained weight after the trial, which shows that they recovered well, with no significant occurrences during the period.

The fact that no deaths, infection, autophagia or pressure ulcers have occurred in the rats is explained by the postoperative prophylaxis with antibiotics, the sterile surgical environment and the individual care given to each rat.

The most surprising outcome was that no histological signs were seen in any of the fibrosis invasion groups in the spinal canal, although the 43-day postoperative time was enough for that to happen, as shown in the literature. This may be explained mainly in the accurate surgical technique used, since in all groups and in all rats the hemorrhage index was equal to zero. Possibly this allowed the local healing to be smoother and better organized that expected. Also, polytetrafluoroethylene showed to be able to create a mechanical barrier against fibrosis.

No changes were observed at the proximal and distal control sites that might cause the withdrawal of any of the rats.

Only in Rat 4 (ePTFE membrane) the presence of granulomatose inflammatory response of the foreign body type was observed, which was not expected for a group with material insertion where a higher presence of such kind of response would be expected. Probably this has occurred because an inert material was used.

Since no fibrosis invaded the spinal cord canal in both groups, the laminectomy site was evaluated for anatomicopathological factors such as hyperemia, hemorrhage, fibrosis and inflammatory response, as factors capable of predicting a higher level of local cicatricial tissue in the future.

No statistically significant differences were observed between the control group and the expanded inert polytetrafluoroethylene membrane group for all anatomicopathological variables. This might indicate that this is an inert material, but the small number of subjects in each group should be taken into account as the cause of absence of statistical differences.

CONCLUSIONS

• An accurate surgical technique seems to be an important factor in the prevention of abundant cicatricial tissue formation.

• An expanded inert polytetrafluoroethylene membrane showed to be an inert material capable of creating a mechanical barrier against fibrosis.

Work performed at the Laboratory of Rachiomedular Trauma and Peripheral Nerves Studies of the Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo

Trabalho recebido em 20/03/2003

Aprovado em 28/03/2003

• 1. Acus RW, Flanagan JP. Perineural fibrosis of superficial peroneal nerve complicating ankle sprain: a case report. Foot Ankle 11:233-235, 1991.
• 2. Brieg A, Marions O. Biomechanics of the lumbosacral nerve roots. Acta Radiol 1:1141-1160, 1963.
• 3. Burton CV. Lumbosacral arachnoiditis. Spine 3:24-30, 1978.
• 4. Chauchoix J, Ficat C, Girard B. Repeat surgery after disc excision. Spine 3: 256-259, 1978.
• 5. Emery I, Gilding D. An analysis of silicone membrane implants and adjacent non-osseous tissue insection around the lumbar dura and nerve roots. In: ANNUAL MEETING OF THE BRITISH ORTHOPAEDIC ASSOCIATION - Liverpool, England, 1977.
• 6. Fager CA, Friedberg SR. Analysis of failures and poor results of lumbar spine surgery. Spine 5:87-94, 1980.
• 7. Gaughan E, Nixon A, Krooh L et al. Effect of sodium hyaluronate on tendon healing and adhesion formation in horses. Am J Vet Res 52:764-773, 1991.
• 8. Gill G, Schech M, Kelly ET, Rodrigo JJ. Pedicle fat graft for prevention of scar in low back surgery. Spine 10:662-667, 1985.
• 9. Gill GG, Sakovich L, Thompson E. Pedicle fat graft for the prevention of scar formation after laminectomy. Spine 4:176-185, 1979.
• 10. Hunter JM. Recurrent carpal tunnel syndrome, epineural fibrosis fixation, and traction neuropathy. Hand Clin 7:491-504, 1991.
• 11. Key JA, Ford LT. Experimental intervertebral disc lesions. J Bone Joint Surg Am 30:621-630, 1948.
• 12. Kusch SD, Ulstrom CL, Michael CJ. The tissue origin of low back pain and sciatica: a report of pain response to tissue stimulation during operations on the lumbar spine using local anesthesia. Orthop Clin North Am 22:181-187, 1991.
• 13. Langenskiod A, Kiviluoto O. Prevention of epidural scar formation after operation on lumbar spine by means of free fat transplants. Clin Orthop 115: 82-85, 1976.
• 14. Langenskiod A, Valle M. Epidurally placed free fat graft visualized by CT scanning 15-18 years after discectomy. Spine 10:97-98, 1985.
• 15. Larocca H, Macnab I. The laminectomy membrane. J Bone Joint Surg Br 56: 545-550, 1974.
• 16. Pheasant HC. Source of failure in laminectomies. Orthop Clin North Am 6: 319-329, 1985.
• 17. Schwarzman RJ. Brachial plexus traction injuries. Hand Clin 7:547-556, 1991.
• 18. Songer M, Ghosh L, Spencer D. Effects of sodium hyaluronate on peridural fibrosis after lumbar laminectomy and discectomy. Spine 15:550-554, 1990.
• 19. Songer M, Rauschning W, Carson E, Pandit SM. Analysis of peridural scar formation and its prevention after lumbar laminotomy and discectomy in dogs. Spine 20:571-580, 1995.
• 20. Wiglis EFS, Murphy R. The significance of longitudinal excursion in peripheral nerves. Hand Clin 2:741-766, 1986.

Correspondence to

Rua Dr. Ovidio Pires de Campos, 333 - 3º andar

Cerqueira Cesar - CEP 05403-010 - São Paulo - SP

Publication Dates

• Publication in this collection
  03 June 2003
• Date of issue
  Apr 2003

History

• Received
  20 Mar 2003
• Accepted
  28 Mar 2003