ORIGINAL ARTICLE

Calcium channel blockers in reduction of epidural fibrosisand dural adhesions in laminectomy rats

Zhiqiang Wang • Yuguang Wang • Peng Xie •

Wei Liu • Sai Zhang

Received: 6 November 2013 / Accepted: 16 December 2013

� Springer-Verlag France 2013

Abstract

Objective To investigate the effect of verapamil on the

prevention of epidural fibrosis in laminectomy rats.

Methods A controlled double-blinded study was con-

ducted in sixty healthy adult Wistar rats that underwent

laminectomy at the L1–L2 levels. All rats were divided

randomly into three groups according to the treatment

method, with 20 in each group: (1) VP treatment group; (2)

vehicle treatment group; and (3) sham group (laminectomy

without treatment). All rats were killed 4 weeks post-

laminectomy. The hydroxyproline content, Rydell score,

vimentin cells density, fibroblasts density, inflammatory

cells density and inflammatory factors expressions were

evaluated.

Results The histological evaluation showed less epidural

scar adhesions in verapamil group than other two groups.

The hydroxyproline content, Rydell score, vimentin cells

density, fibroblasts density, inflammatory cells density and

inflammatory factors expressions all suggested better

results in verapamil group than other two groups.

Conclusion Topical application of verapamil could inhi-

bit fibroblasts proliferation and TGF-b1 and IL-6 expres-

sions and prevent epidural scar adhesion in post-

laminectomy rat model.

Keywords Epidural fibrosis � Verapamil �Laminectomy � Failed back syndrome � Rat

Introduction

Failed back surgery syndrome (FBSS) is characterized by

recurrent persistent low back pain and chronic nerve

radicular in the patient post-lumbar laminectomy [1]. It is

reported to occur in 8–40 % of patients who undergo

lumbar disk surgery [2]. Epidural fibrosis (EF) is thought to

significantly contribute to FBSS [3]. EF, as a scar tissue

adjacent to the dural mater following lumbar laminectomy,

can cause extensive adhesions around the nerve roots and

dural mater. Thus, EF can lead to spinal stenosis, restric-

tion of nerve root mobility and dural compression. There-

fore, EF is still a great challenge to surgeons.

Most authors believe the best way of avoiding EF is to

cut its formation. A number of attempts have been con-

ducted to prevent EF, such as modified meticulous surgery

[4], topical application of anti-inflammatory agents [5],

animal collagen membranes [6] and traditional Chinese

medical agents, such as pseudo-ginseng and Angelica sin-

ensis [7]. Although some of them have achieved moderate

success in animals, there is still no single agent or bio-

materials which have gained success in clinical application

or wide acceptance.

Verapamil (VP) is a widely applied calcium channel

antagonist, which has been shown to inhibit synthesis/

secretion of extracellular matrix molecules, including col-

lagen, glycosaminoglycans and fibronectin [8]. Also, it is

reported that VP is able to show beneficial effect in the

control of cell growth and matrix accumulation of keloids.

Literatures have indicated the anti-inflammatory [9], anti-

fibrosis [10], anti-scar [11], neuroprotective [12] and anti-

cancer [13] effects in several fields of VP. At the same

time, several literatures reported that VP has shown its

ability on inhibiting keloid formation [8, 9, 14, 15]. Con-

sidering EF, as a post-operative scar tissue adjacent to

Z. Wang � Y. Wang � P. Xie � W. Liu � S. Zhang (&)

Department of Orthopedics, Pingjin Hospital, Logistics

University of Chinese People’s Armed Police Forces, 220th

Chenlinzhuang Road, Hedong District, Tianjin 300162, China

e-mail: zhangsai2015@gmail.com

123

Eur J Orthop Surg Traumatol

DOI 10.1007/s00590-013-1395-7

dural mater formed by the deposition of extracellular

matrix proteins, has a similar formation mechanism with

keloid. The present study was designed.

In the present study with laminectomy rat, VP was

employed to investigate its efficacy on the prevention of

EF. Specifically, the macroscopic assessment, the histo-

logical analysis, the expressions of interleukin 6 (IL6),

transforming growth factor-b1 (TGF-b1) and hydroxypro-

line were used to evaluate.

Materials and methods

Experiment design

A total of seventy-two adult healthy male Wistar rats

(mean weight 250 g) were purchased from Radiation Study

Institute-Animal Center, Tianjin, China. In compliance

with the principles of International Laboratory Animal

Care and with the European Communities Council Direc-

tive (86/809/EEC), animals were housed in the laboratory

that were under the condition of 20–25 �C room tempera-

ture, a 12-h light–dark cycle and accessible clean food and

water ad libitum. Before experiment, the rats were housed

for 1 week to adjust them to the environment. Animals

were randomly divided into three groups (24 rats in each

group): (1) VP treatment group (VP 2.5 mg/ml, diluted in

vehicle); (2) vehicle treatment group (vehicle composition:

propylene glycol-5 %, alcohol-50 % and distilled water-

45 %); (3) sham group (laminectomy without treatment).

Reagents and antibodies

Verapamil was purchased from Sigma-aldrich Corporation

(St. Louis, MO, USA). Cal-EX II solution for decalcifica-

tion and dehydration was purchased from Thermo Fisher

Scientific (Waltham, MA, USA). b-dimethylamino-

benzaldehyde was purchased from Sigma-aldrich Corpo-

ration (St. Louis, MO, USA). Reverse Transcriptase was

purchased from Promega (Madison MA, USA). Primary

antibody (ab92547) was purchased from abcam (Cam-

bridge, UK). Secondary antibodies were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Surgical procedure

Rat laminectomy model was performed under sterile con-

ditions with basic surgical tools, surgical microscopes and

electrical drill. Rats were anesthetized by intra-peritoneal

injection of 10 % chloral hydrate (0.3 ml/100 g body

weight). After being restrained on a warm pad in the prone

position, all the rats were numbered individually. Before

the surgery, the fur of each rat was shaved around L1 and

L2, and the exposed skin was sterilized. L1–L2 total lam-

inectomy was performed employing electrical drill. Pro-

cedures were performed carefully to keep the dura and the

nerve root from being traumatized.

Topical application of verapamil

Verapamil (2.5 mg/ml, diluted in vehicle, composition:

propylene glycol-5 %, alcohol-50 % and distilled water-

45 %) or vehicle was administered to the laminectomy

sites with cotton pads (5 9 4 mm2, application volume is

about 0.8 ml) for 5 min separately [16]. Wet gauze was

used to cover the surrounding tissues to prevent the contact

of agent. After the cotton pads were removed from the

surgical field, the laminectomy sites were irrigated imme-

diately with saline to eliminate surplus VP. Then, the

wound site was surgically closed.

Macroscopic assessment of EF

Macroscopic assessment was performed 4 weeks post-

operatively, and six rats were randomly selected from each

group and anesthetized. Then, the surgical sites were reo-

pened. The epidural scar adhesion evaluation was made by

assistants under double-blind trials based on the Rydell

classification. (Grade 0 = epidural scar tissue was not

adherent to the dura mater; Grade 1 = epidural scar tissue

was adherent to the dura mater, but easily dissected; Grade

2 = epidural scar tissue was adherent to the dura mater and

difficultly dissected without disrupting the dura matter;

Grade 3 = epidural scar tissue was firmly adherent to the

dura mater and could not be dissected).

Histological analysis

Histological analysis was performed 4 weeks post-opera-

tively. Six rats in each group were selected. The whole L1–

L2 vertebral column including the paraspinal muscles and

epidural scar tissue was removed en bloc and fixed in 10 %

phosphate-buffered formaldehyde solution. Decalcification

and dehydration were performed with Cal-Ex II solution

for 2 days. Five micrometers axial sections of the lami-

nectomy site were cut and were stained with hematoxylin–

eosin (H&E).

The epidural scar adhesion was evaluated under the light

microscope. The number of fibroblasts and inflammatory

cells were calculated based on previous study: Grade 1\100

fibroblasts/inflammatory cells per 9400 field; Grade 2

100–150 fibroblasts/inflammatory cells per 9400 field;

Grade 3 more than 150 fibroblasts/inflammatory cells per

9400 field [17]. Three different counting areas were selected

at the middle and at the margins of the laminectomy sites.

The cells were counted and mean was calculated.

Eur J Orthop Surg Traumatol

123

To further quantify the density of fibroblasts, the

vimentin immunohistochemistry was performed with

application of the monoclonal anti-vimentin antibody, and

the number of vimentin was evaluated. Three different

counting areas were selected at the middle and at the

margins of the laminectomy sites. The positive vimentins

were counted and mean was calculated.

Hydroxyproline content (HPC) analysis

HPC analysis was performed 4 weeks post-operatively. Six

rats in each group were selected. The scar tissue approxi-

mately 5 mg wet weight was collected around the lami-

nectomy site. The samples were rinsed, homogenated,

centrifuged and hydrolyzed. One milliliter hydroxyproline

developer (b–dimethylaminobenzaldehyde solution) was

added to the samples and the standards. The absorbance at

550 nm was read employing a spectrophotometer. HPC per

milligram of scar tissue was calculated.

Analysis of IL-6 and TGF-b1 concentrations

The mRNA analyses of IL-6 and TGF-b1 were performed

4 weeks post-operatively. Six rats in each group were

selected. The scar tissues were collected from the lami-

nectomy sites, and the total RNA was extracted. The RNA

(2 lg) was transcribed into cDNA employing AMV

Reverse Transcriptase. Quantitative real-time PCR (RT-

PCR) was performed based on previous study using the

Bio-rad MYIQ2 (USA) [18]. The following primers were

used: TGF-b1 (148 bp), forward, 50-GCCCTGCCCCTA

CATTTGG-30, reverse, 50-CTTGCGACCCACGTAGTAG

ACGAT-30; IL-6 (131 bp), forward, 50-ACCCCAACTTC

CAATGCTCT 30, reverse, 50-TGCCGAGTAGACCTCAT

AGTGACC-30; GAPDH (169 bp), forward, 50-TCACC

ACCATGGAGAAGGC-30, reverse, 50-GCTAAGCAG

TTGGTGGTGCA-30 [19]. GAPDH amplification was

employed as an internal control.

Statistical analysis

The statistical analysis was performed employing SPSS

13.0 statistical package (SPSS Inc., Chicago, IL, USA).

Data are expressed as mean ± standard deviation values.

The single-factor analysis of variance (ANOVA) and q test

were applied to evaluate three independent samples. Sta-

tistical significance was assumed at P \ 0.05.

Results

Macroscopic evaluation of epidural scar adhesion

The recovery of all rats was uneventful post-operations. All

rats did not show any sign of wound infection, neurological

deficit or disturbance of wound healing.

In the laminectomy sites of rats treated with VP, soft or

weak fibrous adhesion was observed. However, in the

laminectomy sites of rats treated with vehicle or nothing,

severe epidural adhesions were seen. The dissection of

epidural scar tissue would lead to serious bleeding and the

risk of disruption of the dura mater or nerve root injury. It

was impossible to re-expose the dura mater completely.

The grades of epidural scar adhesion in rats were evaluated

according to the Rydell standard (Table 1).

Hydroxyproline content (HPC)

HPC concentration in epidural scar tissue in the VP group

(35.41 ± 3.66 lg/mg) showed a significant reduction

compared with that of the vehicle group (49.44 ± 2.14 lg/

mg, P = 0.004) and sham group (51.02 ± 3.02 lg/mg,

Fig. 1 H&E staining for the epidural adhesion tissues in the

laminectomy sites applied with VP (a), vehicle (b) and nothing (c).

a Loose scar tissues without adherence to dura mater were seen in VP

group. b, c Dense scar tissues adhered to dura maters were observed

in the vehicle and sham group. The magnification was 9100. SC

spinal cord, L laminectomy defect, D dura mater, EF epidural fibrosis

Eur J Orthop Surg Traumatol

123

P = 0.001). The content in vehicle group showed no sig-

nificant difference compared with that of sham group

(P = 0.126).

Histological analysis

In the laminectomy sites of the VP group, loose or little

epidural scar adhesion was seen (Fig. 1a). However, in the

laminectomy sites of the vehicle group and sham group,

dense epidural scar tissue with widespread adhesions to

dura mater was observed (Fig. 1b, c).

Effect of VP on fibroblasts and inflammatory cells

The fibroblasts and inflammatory density grades of epidural

scar tissue in each group are set out in Table 2. The fibrotic

and inflammatory cell densities in VP group were less than

those of the vehicle group and sham group. Both fibroblasts

and inflammatory cells densities did not show significant

difference between the vehicle group and sham group.

The immunohistochemistry analysis for vimentin pro-

teins showed that less vimentin was observable in the

VP group (72.19 ± 11.28 lg/mg) versus the vehicle

(145.27 ± 21.37 lg/mg, P = 0.006) and sham group

(155.69 ± 24.61 lg/mg, P = 0.004). The content in

vehicle group showed no significant difference compared

with that of sham group (P = 0.279). Representative sec-

tions are shown in Fig. 2.

Effect of VP on IL-6 and TGF-b1

The results of mRNA expression levels of both TGF-b1

and IL-6 are shown in Fig. 3: the VP group was lower than

those of the vehicle group (P = 0.013) and sham group

(P = 0.007); the expressions between vehicle group and

sham group did not show significant differences

(P = 0.133).

Discussion

After extensive literature reviews, among various agents

studied to prevent EF, mitomycin C(MMC) was found to

be able to reduce the amount of fibrosis by inhibiting

collagen synthesis in fibroblasts in post-laminectomy rats

[16, 20–22]. However, MMC, as a toxic agent, is doubted

to be available for use in clinical patients. Whether MMC

would result in an increase in rate of infection had drawn

some researchers’ attention [23]. What’s more, a latest

report on the topical application of 0.5 mg/ml mitomycin C

in 62 patients who underwent microendoscopic discectomy

was presented. Though, neither serious drug adverse effects

nor clinically significant laboratory adverse effects were

observed. The benefit of the application of MMC cannot be

observed clinically [24].Therefore, the agent with a series

of advantages of little or no toxicity is calling on.

Potential abilities of VP that could reduce epidural scar

adhesion and inhibit fibrotic and inflammatory cells pro-

liferation of laminectomy sites were suggested in the

present study. Multiple parameters including the Rydell

classification, the Hydroxyproline content, the histological

analysis, the density grade of fibrotic and inflammatory

cells and RT-PCR suggested a good efficacy of VP on

inhibiting EF in rats. Previous literatures reported the anti-

fibrotic, anti-inflammatory and anti-proliferative properties

of VP employed in different medical fields [9–13]. In the

present study, its properties were proved again in post-

laminectomy rats. VP showed its superior effect in

Table 1 Grades of epidural scar adhesion in rats, according to the

Rydell standard

Group Grade

0 1 2 3

VP 5 1 0 0

Vehicle 0 0 1 5

Sham 0 0 0 6

Fig. 2 Immunohistochemistry analysis of vimentin proteins in epi-

dural scar tissues applied with VP (a), vehicle (b) and nothing (c).

The density of vimentin proteins in the VP group (a) was less than

those of other 2 groups. The density of vimentin proteins in the

vehicle group was similar with that of sham group. The magnification

was 9400

Eur J Orthop Surg Traumatol

123

aforementioned multiple parameters analyses, with the

inhibition of inflammatory cytokines, such as IL-6 and

TGF-b1, which are suggested to be involved in the pro-

motion and/or development of EF [19]. Accordingly, both

the depositing collagen fibrosis and epidural scar adhesion

decreased. Previous literatures and the present data may

explain some if not all of the possible mechanisms that

make VP effective in preventing EF [8, 25]. We hypothe-

size that the major mechanism of VP in preventing EF was

its role in reducing inflammatory cytokines and inducing

apoptosis of macrophages through blocking NF-jB path-

way [25]. Undoubtedly, this needs further research.

Despite recent advances in the understanding of wound

healing and scar formation, the treatment of EF is still

controversial. Calcium channel blockers were first clini-

cally used in collagen matrix on the connective tissue

remodeling by Lee and Ping [26]. The first successful

results of the intralesional application of VP were pre-

sented in burn scars [11]. Four years after that, the litera-

ture reported a novel successful treatment of earlobe

keloids with VP and its cure rate of 55 % [27]. A latest

report suggest that VP, with an extremely low cost and

fewer adverse effects, is a good choice for flattering the

raised scars [28]. As far as we know, the present study is

the first study looking into VP’s suppressive effects on EF

by down-regulating the expression of TGF-b1 and IL-6 and

also reducing hydroxyproline deposition in rats. Undoubt-

edly, more research on drug safety, effectively safe con-

centration, long-term effects and possible side and other

adverse effects of VP are all warranted before clinical trials

and application.

Conclusion

Based on this preliminary study, verapamil may be an

effective treatment to reduce the formation of epidural

fibrosis in post-laminectomy rats.

Conflict of interest None.

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