Vasoactive agent buflomedil up-regulated expression of vascular endothelial growth factor in a rat model of sciatic nerve crush injury.

OBJECTIVES: To study the effect of Buflomedil on the morphological repair on crush injury of sciatic nerve and also the expression of vascular endothelial growth factor (VEGF).
MATERIALS AND METHODS: Rat sciatic nerves were crushed by pincers. All of the 400 Sprague Dawley rats were randomly divided into: Sham-operated; saline; saline + VEGF-antibody; Buflomedil; and Buflomedil + VEGF antibody groups. The expression of VEGF in dorsal root ganglia (DRGs), following crush injury to sciatic nerves, was studied by RT-PCR, immunohistochemistry. The effects of Buflomedil on expression of VEGF and repair of neural pathology were also evaluated.
RESULTS: VEGF mRNA was significantly increased in Buflomedil and Buflomedil + VEGF-antibody groups, compared with other groups. The number of VEGF-positive neurons was significantly increased in the Buflomedil and the saline groups. Besides, Buflomedil also caused less pathological changes in DRGs.
CONCLUSIONS: The vasoactive agent Buflomedil may decrease the pathological lesion and improve the functional rehabilitation of peripheral nerves, which may correlate to upregulation of the expression of VEGF, following crush injury to the peripheral nerves.

Introduction

It is known that crush injury to peripheral nerves can adversely affect neural microcirculation and capillary occlusion,[1] and can result in dysfunction of neural conduction. It has been reported by clinical and experimental studies that vasoactive treatment can alleviate the effects of lesions in peripheral nerves,[23] and Buflomedil is an effective agent for treating disorders of peripheral nerves.[4-8] However, its mechanism is incompletely understood. In addition, it is known that the vascular endothelial growth factor (VEGF) can protect against neuronal lesions in brain and spinal cord disorders.[9-11] In order to explore the protective mechanisms involved in vasoactive treatment on peripheral nerves after injury, the sciatic nerves of rats were crushed, and the effect of the vasoactive agent, Buflomedil, on expression of VEGF was evaluated.

Materials and Methods

Sciatic Nerve Injury In Rats

Total of 400 Sprague Dawley (SD) rats (200 males and 200 females, SPF grade, body weight 180–220 g, Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into: sham-operated (S-O); saline (S); saline + VEGF antibody (S + Ab); Buflomedil (B); and Buflomedil + VEGF antibody (B + Ab) groups. Each group included 80 rats (40 males and 40 females). All procedures were performed in accordance with the animal care guidelines of 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 10 ml/kg of 3% pentobarbital through intraperitoneal injection (ip). An incision was made in the right hind limb to expose the sciatic nerve.[12] In the S-O group, the incision was closed after exposure of the sciatic nerve. The sciatic nerves of the rats in other groups were clipped with artery forceps for 30 s, the pressure was then released and the incisions were closed. Routine antibiotics were given to prevent infections. The rats in the Buflomedil [Nanjing Jinling Pharmaceutical Co. Ltd., batch number 021104, Nanjing, China] and VEGF antibody [Beijing Zhongsan Jinqiao Biotechnology Ltd., Beijing, China] groups were administered drugs (20 mg/kg/d, approximately equal to five times in adult doses, ip) and rabbit IgG polyclonal VEGF antibody (diluted with saline to 1:100, 2 ml/d, ip), and an equivalent volume of saline (ip) was given to the rats in the saline group.

Separation of Dorsal Root Ganglia (DRGs)

The Dorsal root ganglia (DRGs) were separated immediately after the rats were sacrificed. The ribs were sheared along both sides of the spine, and the spine's lumbar and sacral segments and cauda equina were removed. The section was cut along the median line, the spine segments and DRGs were placed in culture dishes containing oxygen-enriched, saturated Dulbecco's Modified Eagle's Medium (DMEM), pH 7.4, the osmotic pressure was 340 mOsm/L DRGs.

Detection of vascular endothelial growth factor mRNA in DRG cells

The DRGs were taken at time points including: 0, 3, 6, 12, 24, 48, 72, and 96 h, as well as on day 7 after crushing the sciatic nerves. Reverse transcribed-polymerase chain reaction (RT-PCR) was performed as guide of reverse transcribed kit (Promega, Madison, WI, USA, TRIzol from Invitrogen, Carlsbad, CA, USA). The oligonucleotide primers for VEGF165 were 5′-GAAGTGGTGAAGTTCATGGATGTC-3′ (forward) and 5′-CGATCGTTCTGTATCAGTCTTTCC-3′ (reverse), and amplified a 541 bp fragment; and for β-actin the primers were 5′-CGCTGCGCTGGTCGTCGACA-3′ (forward) and 5′-GTCACGCACGATTTCCCGCT-3′ (reverse), amplified a 619 bp fragment (Shanghai Shenggong Biotechnology limited-liability company, Shanghai, China). Amplification products were resolved by agarose (2%) gel homeothermic electrophoresis at 80 V for 30 min. The predicted sizes of the amplification products were observed after electrophoresis, under an ultraviolet lamp, and densitometry was done. Then, the index of mRNA (RI) was calculated (RI = VEGF mRNA density / β-actin density × 100%).

Detection of VEGF-positive neurons in DRGs

Immunohistochemistry: The DRGs of each group were taken at 0, 3, 6, 12, 24, 48, 72, and 96 h, and also on day 7 after crushing the sciatic nerves. They were treated by immunohistochemistrical technique (pv-6001 / 6002 immunohistochemistry kit from Beijing Zhongsan Jinqiao Biotechnology Ltd., China).

Image analysis: Two specimens were randomly selected from each group at each time point and immunohistochemistry of VEGF was performed. A light microscope (× 400) was used to observe the number of cells with positive expression in each sample (five fields were randomly taken from each section to count the positive cells and the average value was calculated), and a comparison was conducted.

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 DRGs was stained by Hematoxylin–Eosin method. Examination of the DRGs was performed by light microscope.

Statistics

Statistical Package for the Social Sciences (SPSS, Bizinsight, Beijing, China) 11.5 was used, and P values < 0.05 were considered to be significant. Analysis of variance (ANOVA) was used for comparisons. The quantitative VEGF mRNA and the number of cells expressing VEGF were consistent with normal distributions. The Student–Newman–Keuls analysis (SNK) method was used.

Results

vascular endothelial growth factor mRNA by RT-PCR

There was little expression of VEGF in all groups, 0 h after crushing, and no significant difference among them (P > 0.05). Compared with 0 h: (1) the VEGF mRNA levels were not different from the S-O group (P > 0.05); (2) VEGF mRNA from other groups increased at 3 h and 6 h (P < 0.01 at 6 h) after crushing, and the VEGF mRNA from the S, S + Ab, B and B + Ab groups at 6, 12, 24, 48, 72, and 96 h were more than that at 0 h (P < 0.01); (3) VEGF mRNA peaked at 72 h and then decreased to baseline, such that there was no difference between 0 h and day 7 in each group (P > 0.05); (4) VEGF mRNA from the S, S + Ab, B and B + Ab groups were significantly more than that of the sham-operated group at 6, 12, 24, 48, 72, and 96 h (P < 0.01); and (5) VEGF mRNA from the B and B + Ab groups were significantly more than that of the S group at 6, 12, 24, 48, 72, and 96 h (P < 0.05), but there was no intragroup difference (P > 0.05). There was no difference between the S + Ab group and the S group (P > 0.05) [Figures 1a, b].

Figure 1

(a-a) VEGF mRNA in sham-operated group (M marker, N negative) (a-b) VEGF mRNA in saline group (M marker, N negative) (a-c) VEGF mRNA in saline + VEGF-antibody group (M marker, N negative) (a-d) VEGF mRNA in Buflomedil group (M marker, N negative) (a-e) VEGF mRNA in Buflomedil + VEGF-antibody group (M marker, N negative) (a-f) b-actin (M marker, N negative) (b) VEGF mRNA by RT-PCR

VEGF-positive neurons in DRGs

There were only a few VEGF-positive neurons in the DRGs in the S-O group. In the B group, the number of VEGF-positive neurons began to increase at 6 h (P < 0.05), but at 12 h in the other groups (P < 0.01); the numbers peaked at 72 h (P < 0.01) and then started to decrease, returning to normal level on day 7. The VEGF-positive neurons in the B group were more compared with those of S group and B + Ab group (P < 0.05); however, the VEGF-positive neurons in S + Ab group were less than that of the S group (P < 0.05) [Figures 2a, b].

Figure 2

(a-a) VEGF-positive neurons in DRGs in sham-operated group (a-b) VEGF-positive neurons in DRGs in saline group (a-c) VEGF-positive neurons in DRGs in saline + VEGF-antibody group (a-d) VEGF-positive neurons in DRGs in Buflomedil group (a-e) VEGF-positive neurons in DRGs in Buflomedil + VEGF-antibody group (b) VEGF-positive neurons in DRGs

Pathological changes of DRGs light microscopically

On week 4 after crushing, in the S and S + Ab groups, many neurons were lost, the neuronal degeneration, for example, the grumose chromosomes and nuclear pyknosis appeared, the nucleoli or even nuclear structure disappeared. The tigrolysis, vacuolar degeneration, and many apoptosis body appeared in the cytoplasm and glial cells proliferated. The pathological changes of the S + Ab group were more severe than those of the S group. In the B group, the mild nuclear pyknosis appeared, few apoptosis bodies appeared in the cytoplasm, and the other pathological changes were also less than those in the S group. The pathological changes in B + Ab group were severe than those in the B group. There was no pathological change of DRGs in the S-O group [Figure 3].

Figure 3

(a) Pathological changes of DRGs light microscopically on week 4 in sham-operated group (b) Pathological changes of DRGs light microscopically on week 4 in saline group (c) Pathological changes of DRGs light microscopically on week 4 in saline + VEGF-antibody group (d) Pathological changes of DRGs light microscopically on week 4 in Buflomedil group (e) Pathological changes of DRGs light microscopically on week 4 in Buflomedil + VEGF-antibody group

Discussion

Relevant research indicated that the expression of VEGF could be upregulated by anoxia.[13-15] The results of this experiment showed that with Buflomedil treatment, VEGF mRNA as well as VEGF-positive DRG neurons increased at 3 and 6 h (P < 0.01 at 6 h) after crushing, and both of them decreased to baseline on day 7 in this experiment. The findings demonstrated that the vasoactive agent, Buflomedil treatment in case of sciatic nerve injury would result in a momentary effect in the expression of VEGF and VEGF receptor in DRGs of rats after crush injury to sciatic nerves. It was interesting especially that the number of the VEGF plus VEGF receptor-positive DRG neurons increased with Buflomedil treatment, but decreased when the VEGF antibody was used. When VEGF-Ab was applied, they would combine with VEGF, and block the combination of VEGF with VEGF receptors. It is well-known that VEGF-Ab by itself would not influence the expression of VEGF. Therefore, VEGF mRNA remained unchanged in S and S+VEGF-Ab, as well as B and B+VEGF-Ab. However, VEGF-positive DRG neurons were different in S, or B group compared with S+VEGF-Ab, or B+VEGF-Ab, respectively, and this is due to some of the VEGF is occupied by VEGF-Ab. It is known that neuroprotection is one of the many important functions of VEGF in the course of injury-repair. The VEGF-positive DRG neurons in S+VEGF-Ab, or B+VEGF-Ab were decreased significantly compared with that in S, or B, respectively, in this experiment. The results suggested that this might be due to the biological activity of VEGF was inactivated or decreased when combined with the VEGF antibody. Taken together, the results, above, suggested that Buflomedil could upregulate VEGF and increase the combined expression of VEGF and VEGF receptors in DRGs after crush injury to sciatic nerves. It has been shown that VEGF was a key mediator of the angiogenesis in ischemic lesions in nervous tissues.[16-18] Uesaka demonstrated that angiogenesis is essential for the enlargement of any solid tumor, and VEGF is considered to be a major regulator.[19] Aramoto found that VEGF could induce mild vasodilation and obvious increases in microvascular permeability.[20] Recent evidence suggested that VEGF was protective against the effects of lesions in neurons or nerves in brain ischemia,[9-1116] spinal cord disorders[1121] and even impairment of peripheral nerves.[1621-24]

Li[25] found that when neurolemma was dissected during facial nerve decompression, the edematous nerves would immediately bulge, the blood vessels of the epineurium became dilate, the color of the nerves turned from pale to red, and facial movement would recover dramatically. The results of pathology in this experiment indicated that Buflomedil could reduce the pathological injury and improve the functional recovery after crushing injury to sciatic nerves.

Conclusions

The outcomes of this study indicate that the vasoactive agent Buflomedil may decrease the pathological lesion of peripheral nerves and improve the rehabilitation of the neural function, which may relate to upregulation of the expression of VEGF, following crush injury to peripheral nerves.