Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 58, Num. 4, 2010, pp. 530-536

Neurology India, Vol. 58, No. 4, July-August, 2010, pp. 530-536

Original Article

Protective effects of the calcium-channel blocker flunarizine on crush injury of sciatic nerves in a rat model

Su Jian-Hua, Chen Yu-Fang, Tang Jin-Rong, Wu Le, Zhang Ping, Yu Long-Bin, Niu Qi, Xiao Hang

Department of Neurology, the First Affiliated Hospital of Nanjing Medical University, Nanjing- 210 029
Correspondence Address:Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, Nanjing - 210 029, zdgaoyang@medmail.com.cn

Date of Acceptance: 10-Jun-2010

Code Number: ni10143

PMID: 20739787

DOI: 10.4103/0028-3886.68665

Abstract

Keywords: c-fos, crush injury, dorsal root ganglion, flunarizine, nerve conduction velocity, sciatic nerve

Introduction

The protein c-fos is widely expressed in neurons and acts as the third messenger in the signal transduction pathway in neurons. Recent studies have investigated its role in the transduction of harmful signals. Dragunow and Faull ^[1] reported that c-fos is located basally in neurons and that its expression can increase after behavioral stress. Kaczmarek and Nikofajew ^[2] reported that increase in c-fos messenger ribonucleic acid (mRNA) and/or protein expression is caused by the action of neurotransmitters on membrane receptors which, in turn, is induced by physiological stimuli. According to Shortland and Molander ^[3] stimulation of A-beta afferents induce expression of c-fos in postsynaptic cells. In addition, c-fos is expressed in the axons involved in the conduction of nociceptive stimulus soon after nerve trunk injuries. Other studies have proposed that nerve lesions caused due to various stimuli can be investigated by detecting the loci and level of c-fos expression. ^{[4],[5],[6],[7],[8]} Various factors, such as ischemia, inflammation, and trauma, can cause calcium influx, which in turn can damage neurons and axons. Neural damage thus induced can be mitigated by administering calcium-channel blockers (CCBs). ^[9],[10] However, the mechanism of action of CCBs is not yet fully understood. Calcium influx can also be induced by the overstimulation of N-methyl-d-aspartate (NMDA) receptor. ^[11],[12]

We investigated the mechanism of the protective effect of CCB after crush injury to peripheral nerves, expression of c-fos protein at an early stage of crush injury, pathological changes of sciatic nerve trunks, and the nerve conduction velocities (NCVs) of sciatic nerves at week 4 after sciatic nerve crush injury in rats. The effect of a CCB, flunarizine was also examined on c-fos expression and repair of neural lesions.

Materials and Methods

Sciatic nerve injury in rats

We obtained 256 Sprague-Dawley (SD) rats that were of the specific-pathogen-free (SPF) grade (128 males and 128 females; body weight: 178-224 g) from the Animal Center of Nanjing Medical University, Nanjing, China. After 12 h fast the rats were randomly divided into sham-operated, saline, low-dose flunarizine (FI), and high-dose flunarizine (FII) groups. Each group included 64 rats (32 males and 32 females). All study procedures were performed in accordance with the animal care guidelines followed at the Nanjing Medical University, which conform to the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised, 1985). The rats were fixed onto rat plates after they were anesthetized with intraperitoneally (IP) administration of 3% pentobarbital (10 mL/kg). An incision was made in the right hind limb and the sciatic nerve was exposed. In the sham-operated group, the incision was closed after exposing the sciatic nerve. The sciatic nerves of the rats in other groups were clipped with artery forceps (surgical hemostatic forceps, straight, 14 cm Cr, Zhangjiagang Shuangyin Apparatus Co., Ltd., Zhangjiagang, China) by applying pressure equal to half the body weight of the rats with accurate electronic scales (OCS-XZ-GSC, Nanjing Lianyu Measuring Equipment Co., Ltd., Nanjing, China) for 30 s; the pressure was then released and the incisions were closed. The rats were administered antibiotics to prevent infections. The rats in the low-dose group were given 1 mg/(kg·d) of flunarizine [It was equivalent to 10 mL/(kg·d) solution after dilution; batch number 030118687; Xi′an-Janssen Pharmaceutical Company, Xi′an China] and the high-dose group received 2 mg/(kg·d) intraperitoneally. The rats in the saline group were administered an equivalent volume of saline.

Separation of dorsal root ganglia and sciatic nerve trunks

The rats were killed and their dorsal root ganglia (DRG) and sciatic nerve trunks (which comprise the distal tibial nerve) were immediately separated by the Li′s method ^[13] . The lumbar and sacral segments of the spinal cord and cauda equina were removed. The section was made along the midline of the body. The spinal segments, DRG, neural roots, and the sciatic nerve trunks were placed in culture dishes containing oxygen-enriched, saturated Dulbecco′s modified Eagle′s medium (DMEM; pH 7.4; osmotic pressure, 340 mOsm/L). The DRG, nerve roots (anterior and posterior roots), and sciatic nerve trunks were removed from the neural canal of the rats. The membrane of the peripheral connective tissue, which connects to the nerve trunk, was removed under a stereoscopic microscope, using fine corneal scissors and wire forceps.

Detection of c-fos mRNA in DRG cells by using reverse transcription-polymerase chain reaction

Eight rats from each group were culled after administering flunarizine at 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and their spinal cord, DRG, neural root (anterior and posterior roots), and sciatic nerve trunk were separated. RNA was extracted from the separated DRG and reverse transcribed using SuperScript II™ reverse transcriptase, according to the manufacturer′s instructions, to make up a total reaction volume of 20 μL. First-strand complementary RNA was synthesized from 4 μg of total RNA by using 0.5 μg of oligo (dT) primers, 1Χ first-strand buffer, 0.01 M dithiothreitol (DTT), 0.5 mM deoxyribonucleotide (dNTP) mix, and 200 U of SuperScript II™ at 42°C for 50 min. The reaction was stopped by heating at 70°C for 15 min. Each sample was amplified by performing 35 cycles of polymerase chain reaction (PCR) using oligonucleotide primers: c-fos primer 5′ATGATGTTCT-CGGGTTTCAA-3′ (forward) and 5′-TGACATGGTCTTCACCACTC-3′ (reverse), amplifies a 348-bp fragment; β-actin primer 5′-CCACGAGAAGATGACCCAGAT-3′, 677-bp fragment (Beijing Sanboyuan Biotechnology Limited-liability Company, Beijing China). The amplification conditions were as follows: Initial denaturation was performed at 95°C for 2 min, followed by 35 cycles of denaturation (at 95°C for 1 min), annealing (at 55.5°C for c-fos/56°C for c-jun) for 1 min, extension (at 72°C for 1 min), and final elongation (at 72°C for 2 min). The amplified products were resolved on agarose (1%) by performing homeothermic gel electrophoresis at 80 v for 30 min. The bands were excised and eluted from the gel, purified, precipitated overnight with ethanol, and sequenced. The electrophoresis results were observed under an ultraviolet lamp and a density scan of the positive bands was performed. Then, the refractive index (RI) of c-fos mRNA was calculated using the formula RI = c-fos mRNA density/β-actin density Χ 100%.

Detection of c-fos protein in DRG by Western blot

Eight rats from each group were culled after administering flunarizine at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h, and their spinal cord, DRG, the connected neural root (anterior and posterior roots), and sciatic nerve trunk were isolated. The isolated DRG were infused with 4% paraform, and dehydration and paraffin embedding were performed using routine methods. The specimens were cut into 50-μm sections, and avidin-biotin peroxidase complex (1:150; ABC; Sigma, USA) immunohistochemical stain was used to detect c-fos expression in DRG. Then, the number of c-fos-positive cells in 5 microscopic fields Χ 200 LM was counted to determine the average number of cells in each field.

Pathological examination

The rats in each group were sacrificed at week 4 after crush. The DRGs and sciatic nerves were separated according to the method previously described. The sciatic nerve trunk was stained by Weil′s medullary sheath staining method. Pathological examination of the sciatic nerves was performed under a light microscope.

Nerve conduction velocities (NCV) of sciatic nerves

Eight rats from each group were killed at week 4 after the crush injury. Then a 6-cm length of sciatic nerve (which comprise the distal tibial nerve) trunk was separated. The nerve conduction velocities (NCVs) of the sciatic nerves were detected at 2 min, 3 min, and 4 min using D95 Super NCV lab determinator (Medical Electrons Institute of Academy of Jiangsu Biomedical Engineering, Nanjing, China). The characteristics of each stimulus impulse were as follows: 0.04 ms; 2.7 v; and scanner speed, 50000 mm/s. The recording electrodes were placed 3.5 cm apart on every sciatic nerve trunk and the temporal changes in the action potential were recorded.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS; version 11.5; Bizinsight, Beijing, China). P<.05 was considered to be significant. Intergroup data were compared using analysis of variance (ANOVA). The quantities of c-fos mRNA and protein were consistent with normal distributions and were analyzed using Student-Newman-Keul′s (SNK) test.

Results

c-fos mRNA expression in DRG

The levels of c-fos expression in the sham-operated group were low at all the post-injury time points of measurement. In the saline, FI, and FII groups, the baseline levels (at 0 h) of c-fos mRNA expression were lower than that at the 30-min time point (P<.01) and were the highest at the 1-h time point (P<.01), after which they tended to decrease. However, in all these three groups, the expression levels of c-fos mRNA at 2 h after the injury were higher than those at baseline (P<.01). The c-fos mRNA expression levels in the other groups at 30 min, 1 h, and 2 h after the crush injury were obviously higher than those in the sham-operated group (P<.01). The c-fos mRNA expression levels in the FI and FII groups at 30 min, 1 h, and 2 h after the crush injury were significantly lower than those in the saline group (P<.01). The c-fos mRNA expression level in the FII group was significantly lower than that in the FI group (P<.01) [Figure - 1] a, b, c, d and e,[Table - 1].

c-fos protein expression in DRG

The levels of c-fos expression in the sham-operated group were low at all the post-injury time points of measurement. In the saline, FI, and FII groups, c-fos expression level (at 0 h) tended to increase after the 1-h time point (P<.01) and reached peak expression levels at the 2-h time point (P<.01). The c-fos expression level started to decrease after the 2-h time point, but remained higher than the baseline level even 24 h after the injury (P<.01). The c-fos protein expression levels in the other groups at 1 h, 2 h, 4 h, and 24 h after the crush injury were obviously higher that those in the sham-operated group (P<.01). The c-fos protein expression levels in the FI and FII groups at 1 h, 2 h, 4 h, and 24 h after the crush injury were significantly lower than those in the saline group (P<.01). The c-fos protein expression level in the FII group was significantly lower than that in the FI group (P<.01) [Figure - 2] a, b, c, d and e,[Table - 2].

Pathological changes of sciatic nerve trunks detected by light microscopy

There was extensive and severe myelinoclasis and vacuolar degeneration of the sciatic nerve trunk in the saline group. There was segmental and mild myelinoclasis and a few instances of vacuolar degeneration of the sciatic nerve trunk in the FI and FII groups; this was milder in the FII group. There were no pathological changes in the sham-operated group [Figure - 3] a, b, c, and d.

NCVs of sciatic nerves

At week 4 after the crush injury, the NCVs of the sciatic nerves in the sham-operated and FII groups were greater than that in the FI and saline groups (P<.01). There was no difference between the sham-operated and FII groups or between in the saline and the FI groups (P>.05) [Table - 3].

Discussion

As has been explained by Narita, c-fos expression in the nerve trunk is the response of axons to nociceptive stimuli. ^[4] The results of our study showed that the c-fos expression in the saline and flunarizine groups significantly increased at 30 min after the crush injury of the sciatic nerves in the SD rats. These observations are similar to Curran′s results, where maximal levels of c-fos mRNA expression were detected at 30 min after treatment with growth factors, c-fos protein expression was high for about 2 h after the injury, and cell morphology remained normal at the latter time point. ^[14] It was reported by Morano that a single toe pinch in rats produced nuclei- and condition-specific neuronal responses in the anterior region of the bed nucleus of the stria terminalis. Particularly, acute noxious stimulation increased c-fos expression in the dorsal medial and fusiform nuclei of the bed nucleus. Chronic neuropathic pain induced by chronic constriction injury of the sciatic nerves led to decrease in the number of c-fos-positive cells after acute mechanical stimulation in the dorsal medial and fusiform nuclei and increased c-fos immunoreactivity in the ventral medial aspect of the bed nucleus of the stria terminalis. ^[15] In our study, the c-fos expression in the FI and FII groups was less than that in the saline group (the difference was more significant in the FII (high-dose) group (P<.01). It was reported that pretreatment with l-type CCB - capsaicin - completely prevented mechanical hyperalgesia induced by disc compression. Tang et al. ^[16] reported that the obvious expression of c-fos-like immunoreactive neurons in the dorsal horn of the spinal cord, which they brought about by direct compression of the L5 nerve root, was markedly decreased by pretreatment with capsaicin. In addition, pretreatment with an l-type CCB suppressed the haloperidol-induced c-fos expression throughout the neostriatum and the nucleus accumbens when evaluated 2 h after the injection of the CCB. ^[17] However, c-fos protein expression observed only in the lateral part of the neostriatum at 5 h after the injection of haloperidol in rats pretreated with l-type CCB was higher than that in rats pretreated with the vehicle alone. In addition, pretreatment with the l-type CCB prolonged the duration of haloperidol-induced catalepsy in rats. Infusion of the l-type CCB directly into the neostriatum mimicked the patterns of changes caused by haloperidol-induced c-fos expression. Ca ⁺⁺ acting as a second messenger stimulated the expression of c-fos, ^[5] and flunarizine downregulated the expression of c-fos by blocking the calcium influx. ^[4],[5]

In our study, the pathological changes of the sciatic nerve trunk in the FI and FII groups [especially, in the FII (high-dose) group] were milder than those in the saline group at week 4 after the crush injury. The NCVs of the sciatic nerves in the FII group were greater than the NCVs of the sciatic nerves in the saline and FI groups (P<.01). The spaces between the toes of the rats in the FI and FII groups were significantly greater than that seen in the rats in the saline group (P<.01). Patro et al. ^[18] reported that flunarizine administration markedly reduced the extent of DRG neuron loss. In their experiment, PRTS was 89 and 95.7% in the sciatic nerve crush (SNC) group and the SNC + flunarizine group, respectively. Similarly, the data on 2-4-toe spread suggested a protective action of flunarizine. The PRTS was 94.5 and 99% at the end of the experiment in the SNC and the SNC + flunarizine groups, respectively. The treatment improved the percentage relative toe-spread also significantly greater than the untreated injured rats. The dosages of flunarizine in this experiment was higher than that used in Patro et al.′s ^[18] experiment, but the toe spaces of the saline, FI, and FII groups (which was 68.03%, 87.08% and 96.95%, respectively, of that in the sham-operated group) was less than that found by Patro et al. ^[18] It might be that tested toe spaces was different. Why did not flunarizine completely inhibit the pathological lesions caused by crush injury of the sciatic nerves in rats? Was the development of lesions influenced by other ion channels? Mert et al. found that the slow and fast K ⁺ channels and slow Na ⁺ currents affect the membrane potential and depolarization of the action potential of neurons. Myelin damage, even if it is minimal, might markedly affect subsequent impulse generation and the pattern of action-potential activity. ^[19]

Varejao et al. reported that there was good correlation between sciatic functional index and toe-out angle measurements in predicting functional recovery. ^[20] Luis et al. ^[21] proposed that the combined functional and morphological analyses should be performed in experiments aimed at predicting functional recovery. Our results suggest that the expression of c-fos in the early stage after crush injury could affect subsequent sciatic function. Higher c-fos expression, low NCV, and administration of the CCB flunarizine may lead to the downregulation of c-fos expression soon after crush injury, thereby decreasing the pathological damage and increasing NCVs by blocking the calcium influx. The results confirmed the opinion of Yang and Averbeck that calcium influx can be induced by various factors and can impair neurocyte function. ^[7],[10] The findings reported by Matthews indicated that activity of the voltage-dependent Ca ⁺⁺ channel was important for sustaining the release of neurotransmitters and excitability of neurons and that early use of flunarizine might protect against the loss of important functions. ^[22] Recently, Galtrey and Fawcett ^[23] opined that the final degree of functional recovery achieved was associated with retrograde axonal regeneration. The effects of retrograde axonal regeneration on neuronal function are most clearly evaluated by skilled paw-reaching and grip-strength tests. The lesion model and functional tests would be useful in testing therapeutic strategies for the effects of inappropriate axon regeneration following peripheral nerve injury in humans.

In conclusion the overexpression of c-fos soon after crush injury to the peripheral nerves can induce pathological damage and adversely affect neural functions. CCBs block calcium influx into neurons, which in turn may inhibit expression of c-fos, thereby mitigating neuronal damage and improving neural functions.

Acknowledgment

This work was supported by a grant from the Medical Research Council [grant number: Natural Science of Jiangsu Province BK2001116, Item of Changzhou Board of Health 2002-202-17 and 2004-182-01, Item of Jintan Science and Technology Bureau 2002-28-25 ].

References

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