Verapamil inhibits scar formation after peripheral nerve repair in vivo.

The calcium channel blocker, verapamil, has been shown to reduce scar formation by inhibiting fibroblast adhesion and proliferation in vitro. It was not clear whether topical application of verapamil after surgical repair of the nerve in vivo could inhibit the formation of excessive scar tissue. In this study, the right sciatic nerve of adult Sprague-Dawley rats was transected and sutured with No. 10-0 suture. The stoma was wrapped with gelfoam soaked with verapamil solution for 4 weeks. Compared with the control group (stoma wrapped with gelfoam soaked with physiological saline), the verapamil application inhibited the secretion of extracellular matrix from fibroblasts in vivo, suppressed type I and III collagen secretion and increased the total number of axons and the number of myelinated axons. These findings suggest that verapamil could reduce the formation of scar tissue and promote axon growth after peripheral nerve repair.

Introduction

During peripheral nerve repair, fibroblasts proliferate and release extracellular matrix, mainly as collagen, which forms scar tissue at the injury site. Excessive scar tissue at the cut ends of axons hinders axonal regeneration, affects peripheral nerve regeneration and nerve conduction (Ngeow, 2010). Several studies have confirmed that inhibiting the inflammatory reaction and fibroblast proliferation could noticeably suppress scar growth surrounding the site of injury (de Luca et al., 2014; Gong et al., 2014; Grinsell and Keating, 2014).

The calcium channel blocker, verapamil, has been shown to inhibit in vitro cultured fibroblast adhesion and proliferation (Lundborg, 2000), contribute to extracellular matrix degradation and reduce scar formation (Terenghi et al., 2011). Whether topical application of verapamil after nerve repair in vivo could inhibit the formation of excessive scar tissue and promote nerve regneration remains unclear. Thus, we sought to verify the inhibitory effect of verapamil on scar formation after peripheral nerve repair in in vivo tests.

Materials and Methods

Ethical approval

The experiments were approved by the Animal Ethics Committee of Huazhong University of Science and Technology of China. The animal studies were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Precautions were taken to minimize suffering and the number of animals used in each experiment.

Animals

Sixty specific-pathogen-free male Sprague-Dawley rats aged 3–5 months and weighing 180–200 g were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology of China [animal license No. SCXK (E) 2008-0004]. All rats were housed at 18–25°C and at a relative humidity of 50%. The rats were equally and randomly assigned to experimental and control groups.

Sciatic nerve injury and treatment with verapamil

The rats were intraperitoneally anesthetized with 1% sodium pentobarbital (50 mg/kg). The four limbs were fixed in the prone position. After shaving, a longitudinal incision was made on the dorsal side of the right lower limb to expose the biceps femoris. The biceps femoris was bluntly dissociated to expose the sciatic nerve. The superior segment of the sciatic nerve was transected, and then an epineurial neurorrhaphy was carried out with No. 10-0 Prolene suture. The stoma was wrapped with gelfoam soaked in 200 μM verapamil solution (Approval No. GYZZ H23020293; Heilongjiang Duoduo Pharmaceutical Co., Ltd., Jiamusi, China), forming a tube around the repair. The wrapped stoma was placed under the biceps femoris. The biceps femoris was sutured with absorbable suture and the skin was sutured with silk sutures. After disinfecting, the wound was dressed (Yang et al., 2006). In the control group, the stoma was wrapped with gelfoam soaked in physiological saline. After regaining consciousness, all rats were fed with benzylpenicillin sodium solution (North China Pharmaceutical Co., Ltd., Shijiazhuang, Hebei Province, China).

Wet weight and atrophy index of the gastrocnemius muscle

Four weeks after injury, the rats were decapitated. The skin of the posterior leg on the injury side was cut open to expose the gastrocnemius muscle. The gastrocnemius muscle was completely dissociated by breaking from the Achilles tendon. Blood was removed with filter paper. Sample weight was weighed with a analytical balance (Keep Apex, Wuxi, Jiangsu Province, China). Gastrocnemius muscle atrophy index was calculated as the wet weight of gastrocnemius muscle/body weight.

Sample collection

Four weeks after injury, the rats were decapitated. After removal of gastrocnemius muscle, the sciatic nerve was exposed. Taking the suture site as the center, 1.5 cm of sciatic nerve was obtained, dehydrated through a graded alcohol series, cleared with xylene, embedded in paraffin and sliced into 4-μm-thick sections. The sections were dried at 50°C for further use.

Toluidine blue staining for axons and myelinated nerve fibers

All nerve sections were dewaxed, hydrated, fixed in 4% paraformaldehyde for 1 hour, washed once or twice with phosphate buffered saline (PBS), treated with preheated 0.5% toluidine blue (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), stained in a water bath at 55°C for 20 minutes, washed three times with distilled water, and placed in 0.15% ethanol and HCl for 3 seconds. The samples were dehydrated with 90% ethanol, counter-stained with 0.25% eosin for 5 seconds, decolorized with 95% ethanol, dehydrated, cleared with xylene, mounted, and observed with a light microscope (Precise, Beijing, China). The number of myelinated nerve fibers was counted at 200× magnification with HPLAS-1000 high-resolution color pathological image analysis system (China Daheng Group, Inc. Beijing Image Vision Technology Branch, Beijing, China).

Van Gieson staining for morphology of collagen

Nerve sections were dewaxed, hydrated, stained with Weigert's iron hematoxylin for 20 minutes, washed with running water, differentiated with 95% ethanol, and washed with distilled water for 10 minutes. The samples were stained with Van Gieson's solution for 1 minute, washed, differentiated with 95% ethanol, dehydrated with absolute alcohol, cleared with xylene, and mounted. Collagen deposition was observed with a light microscope.

Western blot assay for types I and III collagen expression

Each sciatic nerve sample was homogenized and total protein was extracted. Protein concentrations were measured. Ten samples of each group underwent electrophoresis, and then transferred onto the polyvinylidene fluoride membrane. Membranes were blocked with 5% skimmed milk, incubated with rabbit anti-rat type I collagen monoclonal antibody (1:200; Abcam, Cambridge, UK) and rabbit anti-rat type III collagen monoclonal antibody (1:400; Abcam) in a wet box overnight at 4°C, followed by 0.5 hours at room temperature. Then they were incubated with CY3 goat anti-rabbit secondary antibody (1:200; Abcam) in the dark for 1 hour at room temperature, and visualized with X-ray. Optical densities of types I and III collagen were analyzed with Image-Pro Plus 5.0.2.9 software (Media Cybernetics, Bethesda, MD, USA). Results were presented by the ratio of optical density of collagen to β-actin.

Statistical analysis

The data are expressed as the mean ± SD and analyzed with SPSS 13.0 software (SPSS, Chicago, IL, USA). Two-sample t-test was used to compare the data between two groups. A value of P < 0.05 was considered statistically significant.

Results

Verapamil alleviated the injury of gastrocnemius muscle in rats with sciatic nerve injury

Compared with the control group, wet weight of gastrocnemius muscle was higher, and the atrophy index was larger in the experimental group (P < 0.05; ).

Verapamil improved the morphology of axons in the sciatic nerve of rats with sciatic nerve injury

Toluidine blue staining results reveal that at 4 weeks after injury, the total number of axons and the number of myelinated axons were higher in the experimental group than in the control group (P < 0.05; , ).

Verapamil effects on the morphology of axons in rats with sciatic nerve injury (toluidine blue staining, × 100).

(A) Axons were regular and dense in the experimental group; (B) axons were disordered and irregular; collagen was visible in the control group. Arrows show axons; triangles show Schwann cells.

Verapamil effects on collagen fiber morphology at the site of injury in rats with sciatic nerve injury

Van Gieson staining results demonstrate that at 4 weeks after injury, proliferation of newborn capillaries, accumulation of fibroblasts and scar tissue formation were observed at the injury site. Collagen deposition was less at the injury sites in the experimental group than in the control group ().

Verapamil effects on collagen deposition at the injury site in rats with sciatic nerve injury (Van Gieson staining, × 40).

After sciatic nerve injury, the broken end was intervened with verapamil solution in the experimental group, with physiological saline in the control group. (A) Experimental group: less collagen was observed in the nerve stem and below the epineurium. (B) Control group: the epineurium was plump; collagen deposition was more within the nerve stem. Arrows present collagen deposition.

Verapamil inhibited the expression of types I and III collagen at the site of injury in rats with sciatic nerve injury

Western blot assay results show that four weeks after injury, compared with the control group, the expression of types I and III collagen at the site of injury was lower in the experimental group than in the control group (P < 0.05; ).

Verapamil effects on the expression of types I and III collagen at the injury site in rats with sciatic nerve injury (western blot assay).

The expresssion of types I and III collagen was presented by the ratio of optical density of types I and III collagen to β-actin. After sciatic nerve injury, the broken end was intervened with verapamil solution in the experimental group, with physiological saline in the control group. The data are expressed as the mean ± SD, with 10 rats in each group. Two-sample t-test was used to compare the data between two groups. *P < 0.05, vs. control group.

Discussion

After peripheral nerve injury, fibrin exudation was induced by proinflammatory cytokines. Fibroblasts accumulated, divided, and secreted extracellular matrix (mainly types I and III collagen) around the injury site. Lee and Ping (1990) first reported the effect of calcium channel blockers in the reduction of scar formation. Doong et al. (1996) found that intracellular calcium concentration first increased and then decreased after treatment with verapamil, which inhibited the functions of various Ca2+ -mediated cells. Iannetti et al. (2009) identified that calcium channel blockers inhibited fibroblast growth possibly by suppressing the protein kinase C pathway.

Calcium channel blockers can prevent the flow of calcium into fibroblasts, depolymerize actin, destroy the cytoskeleton, induce spherical change in fibroblasts, block the movement of vesicles during protein synthesis, cause collagen precursor to gather in cells and inhibit the secretion of collagen. On the one hand, calcium channel blockers reduce the biological activity of cells by inhibiting signal transduction inside and outside fibroblasts. On the other hand, calcium channel blockers suppress synthesis and secretion of collagen and extracellular matrix by changing fibroblast morphology and ultrastructure, reduce tissue adhesion and scar formation. Types I and III collagen and mRNA levels were higher in scar tissue than in skin. Verapamil has an inhibitory effect on types I and III collagen and mRNA synthesis. Inhibiting mRNA at the genetic level is the main pathway by which calcium channel blockers inhibit collagen synthesis (Huang et al., 2014).

Roth et al. (1996) considered that calcium channel blockers not only suppressed extracellular matrix secretion, but also promoted extracellular matrix degradation. Several studies have confirmed that collagen synthesis was reduced and collagenase secretion was increased when fibroblasts became more spherical (Baccei and Kocsis, 2000; Özgenel, 2003; Gordon et al., 2009). Calcium channel blockers can induce spherical changes in fibroblasts. After this effect is activated, even if the drug is removed, the synthesis and secretion of collagenase still remained. Spherical changes induced synthesis and secretion of collagenase are probably associated with gene expression and regulation.

In summary, topical verapamil application reduces the formation of types I and III collagen in the sciatic nerve at the site of injury and, after epineurial repair, diminishes the formation of scar tissue and indirectly promotes axon growth. Whether verapamil impacts anti-tensile force and anti-tensile strength during the healing of nerves requires further investigation.

Table 1

Effects of verapamil on gastrocnemius muscle morphology in rats with sciatic nerve injury

Table 2

Verapamil effects on the number of axons in the sciatic nerve of rats with sciatic nerve injury