Perineural pretreatment of bee venom attenuated the development of allodynia in the spinal nerve ligation injured neuropathic pain model; an experimental study

Abstract

Background: Diluted bee venom (BV) is known to have anti-nociceptive and anti-inflammatory effects. We therefore assessed whether perineural bee venom pretreatment could attenuate the development of neuropathic pain in the spinal nerve ligation injured animal model.

Methods: Neuropathic pain was surgically induced in 30 male Sprague Dawley rats by ligation of the L5 and L6 spinal nerves, with 10 rats each treated with saline and 0.05 and 0.1 mg BV. Behavioral testing for mechanical, cold, and thermal allodynia was conducted on postoperative days 3 to 29. Three rats in each group and 9 sham operated rats were sacrificed on day 9, and the expression of transient receptor potential vanilloid type 1 (TRPV1), ankyrin type 1 (TRPA1), and melastatin type 8 (TRPM8) receptors in the ipsilateral L5 dorsal root ganglion was analyzed.

Results: The perineural administration of BV to the spinal nerves attenuated the development of mechanical, thermal, and cold allodynia, and the BV pretreatment reduced the expression of TRPV1, TRPA1, TRPM8 and c - Fos in the ipsilateral dorsal root ganglion.

Conclusion: The current study demonstrates that the perineural pretreatment with diluted bee venom before the induction of spinal nerve ligation significantly suppresses the development of neuropathic pain. Furthermore, this bee venom induced suppression was strongly related with the involvement of transient receptor potential family members.

Figure 1

The results of behavioral testing after perineural diluted bee venom (DBV) pretreatment in time–course. A. Mechanical allodynia was suppressed in rats pretreated with diluted bee venom (DBV), relative to the control group. The paw withdrawal threshold in both DBV treated groups were significantly higher compared with the control group from observation day 3 to day 13. B. DBV effect on acetone stimulation test in rats. Cold allodynia was inhibited, beginning on day 3 in both groups of rats treated with DBV, compared with the control group and continued until the end of study. C. DBV effect on hot plate test in rats. Thermal allodynia was inhibited from day 3 until day 17 in 0.1 mg DBV group, and day 9 to day 17 in 0.05 mg DBV group. The PWL of control and both BV treated groups restored to normal values beginning on day 21. The preservation of PWL was dose related, being significantly higher in rats treated with 0.1 mg than with 0.05 mg DBV on days 3, 5, 7, 9, and 13. D. DBV effect on cold plate test in rats. The development of cold allodynia after SNL was inhibited in the rats pretreated with DBV, especially in the 0.1 mg group, beginning on day 5 until the end of study. The 0.1 mg DBV group presented significantly longer PWL compared with 0.05 mg group throughout the study period. Each point represents mean ± standard error of the mean (SEM). *p < 0.05 compared with the control group. †p < 0.05 compared with the BV 0.05 mg group. n = 7 animal per group.

Figure 2

The proportion of immunoreactive (IR) neurons observed at the ipsilateral L5 dorsal root ganglion (DRG). Histograms show the proportion of A. transient receptor potential vanilloid type 1 (TRPV1), B. ankyrin type 1 (TRPA1), C. melastatin type 8 (TRPM8) and D. c–Fos IR neurons. Compared with sham operated animals, spinal nerve ligation surgery (SNL) significantly increased the proportion of neurofilament (NF) 200 positive neurons presenting immunoreactivity for TRPV1, TRPA1, TRPM8 and c–Fos. In the NF200 negative neurons, SNL surgery significantly increased the proportion of TRPV1, TRPM8 and c–Fos IR neurons in the dorsal root ganglion. Perineural pretreatment with 0.05 mg or 0.1 mg diluted bee venom (DBV) significantly decreased the proportion of NF 200 positive and negative neurons expressing TRPV1, TRPA1, TRPM8 and c–Fos compared with the control group. Three rats in each group were analyzed. Each bar represents mean (%) ± standard deviation (SD). *p < 0.05 compared with the control group, † p < 0.05 compared with the BV 0.05 mg group.

Figure 3

Immunofluorescent images of dorsal root ganglion. A. Transient receptor potential vanilloid type 1 (TRPV1) immunoreactive (IR) neurons and merged images with neurofilament (NF) 200 and c–Fos observed through a confocal microscope. Immunofluorescent images showing neurons positive for TRPV1 (red), NF200 (green) and c–Fos (blue) in the dorsal root ganglion (DRG) 9 days after surgery in a) sham operated, b) spinal nerve ligation (SNL), and c) 0.1 mg diluted bee venom (DBV) treated SNL rats. Clockwise from upper left: c–Fos– NF200 –merged – TRPV1. B. Transient receptor potential ankyrin type 1 (TRPA1) IR neurons and merged images with NF200 and c–Fos. Neurons positive for TRPA1 (red), NF200 (green) and c–Fos (blue) in the DRG 9 days after surgery in a) sham operated, b) SNL, and c) 0.1 mg DBV treated SNL rats. Clockwise from upper left: c–Fos– NF200 –merged – TRPA1. C. Transient receptor potential melastatin type 8 (TRPM8) IR neurons and merged images with NF200 and c–Fos observed through a confocal microscope. Neurons positive for TRPM8 (red), NF200 (green) and c–Fos (blue) in the DRG 9 days after surgery in a) sham operated, b) SNL, and c) 0.1 mg DBV treated SNL rats. Clockwise from upper left: c–Fos– NF200 –merged – TRPA1. All images are magnified × 630, with scale bars = 40 μm. Three rats in each group were analyzed.