Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition, Cytokines and Kinin Receptors Modulation

Abstract

Background: Envenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated.

Methodology/principal findings: Bmv (1 μg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT.

Conclusion/significance: These data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.

Fig 1. Effect of photobiomodulation on mechanical allodynia (A) or hyperalgesia (B and C) of mice.

Animals were injected intraplantar with Bmv (1 μg; ■) and after 30 min and 3 h were treated with LLL at 685 nm (Bmv + LLLT; ●) or a combination of laser and antivenom (Bmv + LLLT + AV; ▼). Allodynia was measured by the mechanical response to tactile stimulation assessed by a von Frey filament of 0.407 g (3.61 filament; A). Hyperalgesia was measured by the mechanical response to nociceptive stimulation assessed by von Frey filaments of 0.692 g (3.84 filament; B) and 1.202 g (4.08 filament; C). Pain threshold was determined before (baseline values, time 0) and 1, 3, 6, 12 and 24 h after BmV injection. Animals injected only with Bmv or with a combination of Bmv+AV (□) were submitted to the same protocol. Each point represents the mean ± SEM of five to seven animals per group. (*p<0.05, ≈ p<0.01 or #p<0.001 vs Bmv group are indicated).

Fig 2. Fos immunolabel in the in the dorsal horn of the spinal cord (DHSC) of mice.

Photomicrographs illustrating Fos immunostaining (arrows) in the dorsal horn of the spinal cord (DHSC) of mice injected intraplantar with saline (A), Bmv (B) or Bmv and laser at 685 nm (C). (D) Mean of the density of nuclei labeled for Fos protein in the DHSC of mice. Data shows the proto-oncogene immunoreactivity on the right side, ipsilateral to the paw irradiated. Values represent the mean ± SEM of 5 animals for group. Statistically significant differences vs. saline (^#p<0.05) or vs. BmV (*p<0.05) are indicated.

Fig 3. Effect of LLLT on IL-6 (A and D), TNF-α (B and E) or IL-10 (C and F) mRNA expression.

mRNA levels of IL-6, TNF-α or IL-10 were evaluated by RT-PCR on samples from foot pad (A, B and C) or spinal cord (D, E and F) of mice injected intraplantar with saline or Bmv (1 μg) and treated or not with LLLT at 685 nm or antivenom (AV) or a combination of LLLT and AV were collected 6 h after treatments. Experiments were performed in triplicates. Data are expressed as means ± SEM of 5 animals from each group. Statistically significant differences vs. saline (^#p<0.05) or vs. Bmv (*p<0.05) are indicated.

Fig 4. Effect of LLLT on kinin receptor mRNA expression.

mRNA levels of B1 (A) or B2 (B) kinin receptors were evaluated by RT-PCR on footpad samples of the mice injected intraplantar with saline or Bmv (1 μg) and treated or not with LLLT 685 nm at or antivenom (AV) or a combination of LLLT and AV. Samples were collected 6 h after saline or Bmv injection. Experiments were performed in triplicates. Data are expressed as means ± SEM of 5 animals from each group. Statistically significant differences vs. saline (^#p<0.05) or vs. Bmv (*p<0.05) or vs Bmv + AV (^&p<0.05) are indicated.