Journal of Huazhong University of Science and Technology ［Med Sci］ 26 (6): 2006

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Effect of Straight-leg-raising Movement on Epidural Fibrosis in Early Stage after Laminectomy in a Rabbit Model

ZHAO Jijun (赵继军)1, YANG Shuhua (杨述华)1*, LI Zhengwei (李正维)2, HU Yong (胡 勇)1 1Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2Department of Orthopedics, the First Hospital Affiliated to Dalian Medical University, Dalian 116023, China

Summary： To determine the effect of straight-leg-raising (SLR) movement on epidural fibrosis after laminectomy, 40 adult New Zealand rabbits were selected as laminectomy models in the study. They were divided into 2 groups: a SLR group (group S) and a control group (group C) randomly, with each group having 20 animals. All rabbits were subjected to total laminectomy in the site of S1. Every 5 rabbits in each group selected randomly were killed at the 1st, 2nd, 4th, and 8th week after the sur-gery. Segments of spines from L7 to S2 were removed en bloc. After gross evaluation, specimens were sliced up. The slices were stained by HE and Masson’s trichrome methods respectively for histologi-cal examination. Our results showed that formation process of scar in group S was retarded as com-pared with that of group C at the time of the 2nd-week, but there was no statistical difference between groups in the adhesion degree (P≥0.05). At the 4th and 8th week, the epidural fibrosis of group S was more serious than that of group C. Since the 2nd-week, the area of scar in group S was larger than that of group C. The number of fibroblasts and inflammatory cells in group S were larger than those of group C at early stage. But in later stage, there was no statistical significance between the two groups. It is concluded that SLR movement after laminectomy may promote the formation of epidural fibrosis and retard the maturity of scar. SLR movement can also aggravate scar adhesion. Key words：laminectomy; nerve root; scar; straight-leg-raising movement; fibrosis DOI 10.1007/s 11596-006-0623-3

The formation of scar may compress dura sac and nerve roots, and it is one of the factors inducing recurrent radicular pain after laminectomy. Some doctors believe that straight-leg-raising (SLR) movement can prevent epidural fibrosis after laminectomy. However, there is no experimental evidence supporting such a point of view. The purpose of this study is to determine the effect of SLR movement on epidural fibrosis after laminectomy in a rabbit model.

1 MATERIALS AND METHODS

1.1 Animals Forty adult New Zealand rabbits (20 males and 20

females), weighing 2.3–3.0 kg, were used in the experi-ment. 1.2 Operative Procedure

All the animals underwent total laminectomy in the site of S1 after being anesthetized with 10 g/L pentobar-bital sodium (2 mL/kg) administered intravenously. The skin of rabbits in lumbar and sacrum area was prepared with a clipper. The operative field was then prepared in a sterile manner using povidone-iodine solution and then draped in a standard fashion. A midline longitudinal in-cision was made down to the spinous process. The paraspinous muscles were stripped away from the lamina

ZHAO Jijun, male, bone in 1975, M.D., Ph.D. *Corresponding author

and spinous process. The muscles were retracted bilater-ally with retractors. The lamina of S1 was identified and then total laminectomy was performed, including exci-sion of the spinous process and ligamentum flavum untill underlying dura was exposed. Hemostasis was achieved by bipolar electrocautery. At last the wound was closed in a standard manner. 1.3 Grouping

The rabbits were randomly divided into 2 groups: SLR group (group S) and control group (group C) and each group had 20 animals. Each rabbit was raised in a single cage. The animals in group S repeated SLR movement passively 3 times a day from the third day after the surgery; each animal repeated the action for 20 times each time. Animals in group C were not given any treatment. Every 5 rabbits of each group selected ran-domly were killed at the time of the 1st, 2nd, 4th and 8th week after the surgery. 1.4 Gross Evaluation

The spine segments of L7 to S2 were removed en bloc including paraspinal tissues. The bone of the right site in specimen was ectomized. The defects were used for observation of scar. At the time of dissection, the postoperative scar tissue was grossly evaluated on the degree of the adhesion to dura mater and nerve roots according to Rydell criteria: Grade 0: no adhesion; Grade 1: loose adhesion, easily segregated; Grade 2: extensive adhesion, needing blunt dissection without dura mater injury, the movement of nerve roots was limited obvi-ously; Grade 3: tight adhesion, needing sharp dissection, easily injuring dura mater, and nerve roots was affixed in

26 (6): 713-715, 2006 Journal of Huazhong University of Science and Technology ［Med Sci］

华 中 科 技 大 学 学 报 ［ 医 学 （ 英 德 文 ） 版 ］

Journal of Huazhong University of Science and Technology ［Med Sci］ 26 (6): 2006

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scar. 1.5 Histological Analysis

After gross evaluation, specimens were soaked in 10 % formalin for 5 days, and later placed in decalcify-ing solution until complete decalcification was achieved. The specimens were then dehydrated in alcohol and em-bedded in paraffin. Cross-section slices of 3 to 5 µm thick at the site of laminectomy defects were obtained and stained by hematoxylin and eosin and Masson’s trichrome methods. Each specimen was scored for extent, density, and arachnoidal involvement of scar. The extent of fibrosis along the dura was scored according to He’s Classification: Grade 1, only thin fibrous band(s) be-tween the scar tissue and dura mater were observed; Grade 2, continuous adherence was observed but occu-pied less than two-thirds of the laminectomy defect; and Grade 3, large fibrosis adherence was observed, occupy-ing more than two-thirds of the laminectomy defect and/or extending to the nerve roots.

The Masson’s trichrome slices at the 2nd, 4th, and 8th week were used for area assessment of epidural scar by a computer image analysis system (Image-pro plus 4.5, USA). The area index of scar is defined as the ratio of scar intruded in vertebral canal and the cross-section area of canal. The numbers of fibroblasts and inflamma-tory cells were counted in three fields locating in the middle and margins of the laminectomy wihtin the same field of high magnification (×400). The average number of cells in the three fields was then calculated. 1.6 Statistical Analysis

SPSS 12.0 statistical software was employed to perform all statistical comparisons. A two-tailed Wil-coxon-Mann-Whitney test was used to determine statis-tical significance between groups in terms of adhesion degree; one-way ANOVA test was employed to compare the numbers of fibrolasts and inflammatory cells, the area index scar and histologic score. Significance level was set at 5 %.

2 RESULTS

2.1 General Conditions All of the animals were active, ambulatory, and

healthy-appearing at the time they were killed. The wounds were well-healed in all laminectomy sites with-out evidence of infection. 2.2 Gross Evaluation

There were a lot of granulation tissues formed be-hind dura mater and nerve roots with less blood clot one week after surgery in group C. While there were much more blood clots with less granulation tissue in group S than in group C. The soft granulation tissues in group C did not accompany blood clot, while there were still some blood clots in group S two weeks after the surgery. Scar formed at the 4th week in both groups. But scar in group S was more extensive and tighter than that of group C. Scar was tenacious in both groups at the 8th week. Adhesion and extension of scar were more serious in group S than those in group C. The statistical results of gross adhesion grades were shown in table 1. Area index of the cases negative for MDM2 (8.36±2.03) was much higher than that of the MDM2-positive cases

(4.19±1.67, t=7.80, P<0.01). The similar result was found in the two different types.

Table 1 Gross adhesion grading

1st week 2nd week 4th week 8th week Grade

C S

C S

C S

C S

0 2 5 0 0 0 0 0 0 1 3 0 5 5 3 0 2 0 2 0 0 0 0 2 4 3 1 3 0 0 0 0 0 1 0 4

There was no statistically significant difference between groups at the 1st and 2nd week (P>0.05); There was statistically sig-nificant difference between groups 4 and 8 weeks after surgery (P<0.05); Adhesion of scar in group S was more serious than that of group C 2.3 Histology

The results of histological assessment were consis-tent with gross evaluation. In the early stage, maturity progress of scar in group S was more hysteretic than that of group C. But in the later stage, the scar in group S was more tenacious than that of group C (fig. 1). The number of fibroblasts and inflammatory cells in group S were larger than those of group C at early stage (fig. 2). The statistical results were shown in tables 2, 3, 4.

Fig. 1 The histology at first week (A, B) (HE×100) and at 8th week (C, D) (Masson×100) A, C: Group S; B, D: Control group

Fig. 2 At 2nd week, the fibroblasts and inflammatory cells in group S (A) were more than those in group C (B) (HE×400)

Table 2 Histological scores of scar

Time (week) Group S Group C

1 0.30±0.27 0.10±0.22

2 1.10±0.42* 0.60±0.22

4 1.90±0.42* 0.80±0.57

8 2.80±0.27* 2.10±0.42 *P<0.05 as compared with group C

Journal of Huazhong University of Science and Technology ［Med Sci］ 26 (6): 2006

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Table 3 Area index of scar

Time (week) Group S Group C

2 0.2303±0.0361* 0.1656±0.0229

4 0.2912±0.0448* 0.2505±0.0474

8 0.3274±0.0407* 0.2982±0.0457 *P<0.05 as compared with group C

Table 4 Count of cells

Fibroblasts Inflammatory cells Time (week) Group S Group C Group S Group C

1 50.00±10.42* 38.60±3.03 21.80±3.70* 16.93±1.75 2 54.73±8.06* 41.07±8.08 28.00±3.54* 22.27±3.01 4 46.67±6.33 39.2±3.18 10.33±1.35 8.73±2.89 8 23.87±6.30 22.13±3.25 3.20±0.89 2.33±1.00

*P<0.05 as compared with group C

3 DISCUSSION

Failed back surgery syndrome (FBSS) is a clinical condition with which patients who undergo one or more surgical procedures for lumbosacral disease obtain un-satisfactory long-term relief of symptoms, with persistent or recurrent low back and/or leg pain[1, 2]. In the interna-tional specialized literature, the frequency of its occur-rence ranged between 1 % and 48 %[3], and the rate of necessary reinterventions after primary discectomy were between 5 % and 18 %[4]. Approximately 5 % to 24 % of all cases of FBSS were the results of epidural fibrosis[1, 2,

5]. Ross et al believe that for every 25 % increase in scar-ring, the risk of recurrent radicular pain increases 2.02 times[6].

Clinically, many doctors believe that SLR move-ment can inhibit adhesion of scar in the early stage after laminectomy. But the conclusions of this study are com-pletely different to traditional point of view. We found that granulation tissues formed in controls. However, there were still mainly blood clots in group S in early stage after surgery. It indicates that the movement of nerve roots aggravates bleeding and retard the formation of granulation. The situation of gross adhesion grade is the same between the two groups at the 2nd-week. But areas of scar in group S were larger than controls. At the 4th and 8th week, there were no significant differences with respect to the degree of maturity between the two groups. But the situation of scar adhesion and immobili-zation of nerve roots by scar in group S were more seri-ous than in group C. So we are led to to conclude that SLR movement can increase the area of scar and aggra-vate the adhesion of scar and the immobilization of nerve roots. The number of fibroblasts and inflammatory cells in group S were larger than those of controls C at early stage, suggesting that SLR movement can aggravate in-juries in operated sites and inflammatory reactions fur-ther more.

SLR movement causes not only the stretch of never roots but also the micro-movement of lumbar. The mi-cro-movement of the operated site is a kind of disadvan-tage in preventing the formation of scar. LaRocca et al presumed that hematoma could lead to the extension of scar into vertebral canal and TGF-β, PDGF and FGF of

hematoma could aggravate fibrosis and adhesion[7]. Son-ger et al presumed that hematoma was the initiation stage of fibrosis[8]. So reduction of scar depends on reducing the formation of hematoma. Granulation tissue is vul-nerable because its collagen fibers are ratites and its ves-sels are very fragile. Movement of nerve roots and lum-bar necessarily injure the granulation and then bleeding is inevitable.

Inflammation is another important factor contribut-ing to the formation of scar after laminectomy. Instability of the operated site might aggravate lesions of wound, as a result, inflammatory reactions become much more in-tense. Though inflammatory reactions could lyze and clear debris and provide suitable surroundings for tissue repair, mediators of inflammation and growth factors delivered by macrophages attract fibroblasts aggregating in the wound and stimulate the synthesis of collagen. Hematoma becomes the medium of fibroblasts and pro-vides a framework for collagen. Thus a great amount of scar comes into being.

Furthermore, the movement of nerve roots probably can facilitate the diffusion of inflammatory cells, media-tors of inflammation and fibroblasts. The result is fibro-sis aggravated.

Since SLR movement could aggravate the forma-tion and adhesion of scar in early stage after laminec-tomy, it is harmful to perform the movement in order to loose the adhesion when scar is matured. Because nerve root is very tender and its movement is limited, the force produced by the movement can’t loose the adhesion. On the contrary, it may lesion nerve root by itself to do so.

REFERENCES 1 Gerszten P C, Moossy J J, Bahri S et al. Inhibition of

peridural fibrosis after laminectomy using low-dose ex-ternal beam radiation in a rat model. Neurosurgery, 1999,43:597-602

2 Gerszten P C, Moossy J J, Flickinger J C et al. Inhibition of peridural fibrosis after laminectomy using low-dose external beam radiation in a dog model. Neurosurgery, 2000,46:1478-1485

3 Ganzer D, Giese K, Völker L et al. Two-year results after lumbar microdiscectomy with and without prophylaxis of a peridural fibrosis using Adcon-L. Arch Orthop Trauma Surg, 2003,123:17-21

4 Fristch E W, Heisel J, Rupp S. The failed back surgery syndrome: reasons, intraoperative findings, and long-term result: a report of 182 operative treatments. Spine, 1996,21:626-633

5 Alkalay R N, Kim D H, Urry D W et al. Prevention of postlaminectomy epidural fibrosis using bioelastic materi-als. Spine, 2003,28:1659-1665

6 Ross J S, Robertson J T, Frederickson R C et al. Associa-tion between peridural scar and recurrent radicular pain after lumbar discectomy: magnetic resonance evaluation. Neurosurgery, 1996,38:855-863

7 LaRocca H, Macnab I. The laminectomy membrane. Studies in its evolution, characteristics, effects and pro-phylaxis in dogs. J Bone Joint Surg(Br), 1974,56:545-550

8 Songer M N, Carson E W, Pandit S M. Analysis of peridural scar formation and its prevention after lumbar laminotomy and discectomy in dogs. Spine, 1995, 20:571-580

(Received Oct. 20, 2006)