Snakebite – Snake Venom

PLOS Neglected Tropical Diseases.  Published: October 17, 2016

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

About the Authors

Nikele Nadur-Andrade
Contributed equally to this work with: Nikele Nadur-Andrade, Camila Squarzoni Dale

Affiliation Universidade Nove de Julho, São Paulo, São Paulo, Brazil

Camila Squarzoni Dale
Contributed equally to this work with: Nikele Nadur-Andrade, Camila Squarzoni Dale

Affiliation Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil

Victoria Regina da Silva Oliveira

Affiliation Universidade Nove de Julho, São Paulo, São Paulo, Brazil

Elaine Flamia Toniolo

Affiliation Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil

Regiane dos Santos Feliciano

Affiliation Universidade Nove de Julho, São Paulo, São Paulo, Brazil

José Antonio da Silva Jr.

Affiliation Universidade Nove de Julho, São Paulo, São Paulo, Brazil

Stella Regina Zamuner

Stella.rz@uninove.br

Affiliation Universidade Nove de Julho, São Paulo, São Paulo, Brazil

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

Conceived and designed the experiments: SRZ CSD JAdS. Performed the experiments: NNA VRdSO EFT RdSF. Analyzed the data: SRZ CSD VRdSO EFT. Contributed reagents/materials/analysis tools: SRZ CSD JAdS. Wrote the paper: SRZ CSD VRdSO EFT.

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 ug) 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-a, 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-a 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.

Author Summary

Envenoming caused by Bothrops snakes is characterized by drastic local tissue damage involving hemorrhage, blistering, myonecrosis, prominent inflammatory response and intense pain. The most effective treatment for Bothrops snakebites is antivenom therapy, which is very efficient in reversing systemic effects of envenomation but not the severe local effects. Thus, there exists a need to find novel complementary therapies that may further assist in the prevention or even counteract the severe local effects of bothrops snakebite. Several studies have shown the effectiveness of photobiomodulation in reducing local effects induced by Bothropic venoms, however its mechanisms still remain unknown. In this study, we analyzed the effectiveness of photobiomodulation in reducing BmV-induced mechanical allodynia and hyperalgesia as well as part of the mechanisms involved in such effect. Results demonstrate that photobiomodulation reduces venom-induced mechanical allodynia and hyperalgesia and this effect depends on a decrease of nociceptor activation at the spinal cord level and by a modulation of pro- and anti- inflammatory cytokines as well as kinin receptors at mRNA transcriptional levels. These findings make phtobiomodulation a promising candidate to be associated to antivenom therapy for the treatment of the local response induced by Bothrops venoms.

Introduction

Bothropic envenomation is characterized by severe local manifestation associated with oedema, myonecrosis, hemorrhage and intense pain [14] caused by the toxic action of venom components and aggravated by induced-inflammation. The local effects induced by bothropic venoms are the result of multifactorial and synergistic actions of toxins, which are still poorly understood. Bothrops moojeni is a venomous snake responsible for most of the snakebites in the Central region of Brazil [5]. Despite the medical importance, there are only a few studies related to the local inflammatory reaction caused by Bothrops moojeni venom (Bmv). In this sense, the literature shows that in the accidents caused by these snakes serious local complications occur, including a prominent edema formation, intense pain, swelling and pallor, which may develop into more severe outcomes such as muscle mass loss, neuropathy, and amputation [6, 7].

Currently, the most effective treatment for Bothrops snakebites accidents is the antivenom therapy (AV). However, although AV has proven to be effective in reversal the systemic response, its administration does not prevent local effects and resultant disabilities [3]. Consequently, there is a need to find therapeutic approaches associated with AV treatment that can be effective in reducing the local effects caused by Bothrops snakes envenoming in order to minimize or prevent the progression to a severe clinical status observed after Bothrops snakebites [8, 9].

Photobiomodulation is a form of light that triggers biochemical changes within cells, where the photons are absorbed by cellular photoreceptors and triggers chemical alterations [10]. The mechanisms of photobiomodulation essentially rely on particular visible red and infrared light waves in photoreceptors within sub-cellular components, particularly the respiratory chain within mitochondrial membranes due to the activation of various transcription factors by the immediate chemical signaling molecules produced from mitochondrial stimulation [11]. The most important of these signaling molecules are thought to be Adenosine Triphosphate (ATP), cyclic-AMP, nitric oxide (NO) and Reactive Oxygen Species (ROS) [12].

Many studies have demonstrated analgesic and anti-inflammatory effects provided by photobiomodulation in both experimental [13, 14] and clinical trials [15, 16]. Photobiomodulation has also proven to be an interesting and efficient complementary alternative for the treatment of local effects caused by bothropic venom through the ability of decreasing the observed local effects, such as myonecrosis [17, 18]; inflammation [1922] hemorrhage [21] and pain [20, 23]. In this context, we have recently demonstrated that photobiostimulation with LLLT and light emitting diode (LED) reverse edema formation, local hemorrhage and inflammatory hyperalgesia induced by Bohtrops moojeni venom (BmV) in mice [18, 24].

Although some studies have demonstrated the effectiveness of photobiomodulation in reducing hyperalgesia and allodynia induced by bothropic venom, the mechanism involved in this effect still remains unknown. In this context, the present experiments were designed to investigate the antinociceptive effect of photobiomodulation on BmV-induced allodynia and hyperalgesia and to explore possible underlying mechanisms.

Materials and Methods

Animals

Male Swiss mice weighing 20–25 g, age-matched, were used throughout this study. Animals were maintained under controlled light cycle (12/12 h) and temperature (21 ± 2°C) with free access to food and water.

Ethics Statement

All animal experimentation protocols received the approval by the Ethics Committee on the Use of Animals at of Hospital Sírio-Libanês (Protocol no. (CEUA 2010/01), in agreement with Brazilian federal law (11.794/2008, Decreto n° 6.899/2009). We followed institutional guidelines on animal manipulation, adhering to the “Principles of Laboratory Animal Care” (National Society for Medical Research, USA) and the “Guide for the Care and Use of Laboratory Animals” (National Academy of Sciences, USA).

Venom and Antivenom

Bothrops moojeni venom (Bmv) was supplied by the Serpentarium of the Center of Studies of Nature at UNIVAP. Bmv was lyophilized, kept refrigerated at 4°C and diluted in sterile saline solution (0.9%) immediately before use. Bmv was injected into the subplantar surface of the right hind paw at the concentration of 1.0 ug/50 uL. Equine antivenom (AV) used in the experiments was a polyvalent Bothrops AV (lot# 990504–18) raised against a pool of venom from B. alternatus, B. jararaca, B. jararacussu, B. cotiara, B. moojeni and B. neuwiedi obtained from the Butantan Institute (São Paulo, SP, Brazil). AV was injected through the intravenous route (0.2 uL of AV diluted in saline; final volume of 50 uL, considering that 1 mL of AV neutralizes 5 mg of Bothropic venom [25] 30 min after BmV injection.

Mechanical Hyperalgesia and Tactile Allodynia

Hyperalgesia and allodynia of the hind paw were assessed as described by Takasaki et al. [17]. Mice were placed individually in plastic cages with a wire bottom, which allowed access to their paws. To reduce stress, mice were habituated to the experimental environment one day before the first measurement. At the day of the test, the animals were placed in the cages 30 min before the beginning of each measurement and received an injection of 1.0 ug of crude Bmv diluted in 50 uL of sterile saline into the subplantar surface of the right hind paw. Control group animals received the same volume of sterile saline. Von Frey filaments with bending forces of 0.407 g (3.61 filament—allodynia stimulus), 0.692 g and 1.202 g (3.84 and 4.08 filaments—hyperalgesia stimulus) were pressed perpendicularly against the plantar skin and held for 5 s, at 1, 3, 6 and 24 h after venom injection. A stimulation of the same intensity was applied three times to each hind paw at intervals of 5 s. The responses to these stimuli were ranked as follows: 0, no response; 1, move away from von Frey filament and 2, immediate flinching or licking of the hind foot. The nociceptive score was calculated as follows:

 

Animals were returned to their home cages with free access to food and water between the 1 and 3 h, 3 and 6 h and 6 and 24 h measurements.

Light Source, Dose and Treatment

A low-level semiconductor Ga-As laser, Theralaser D.M.C. (São Carlos, SP, Brazil), operating with a wavelength of red 685 nm, was used through the experiments with a beam spot of 0,2 cm2 and an output power of 30 mW, energy density of 2.2 J/cm2 and exposure time of 15 s. Laser doses, low enough to avoid any thermal effect, were chosen on the basis of previous study from our laboratory [18]. Animals were gently manually restrained and the LLLT was applied to the same area as the injection of Bmv or saline solution. A control group was treated using the same experimental procedure but with the laser turned off. Animals were irradiated 30 min and 3 h after subplantar injection of either Bmv or saline and were immediately returned to their home cages with free access to food and water after each application.

Experiments were conducted in an environment with partial obscurity to not suffer interference from external light. The output power of the laser equipment was measured using the Laser Check1power meter (MM Optics, São Carlos, Brazil).

Immunohistochemistry

Six hours after the intraplantar (i.pl.) injection of Bmv or saline, mice were deeply anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg) and transcardially perfused with phosphate-buffered saline and 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The spinal cord (L4 and L5) was removed, left in the same fixative for 5–8 h and then cryoprotected overnight in 30% sucrose. Thirty ?m frozen sections were immunostained for Fos expression. The spinal cord sections were incubated free floating with a rabbit polyclonal antibody against the nuclear protein which is the product of the early response gene c-fos (Ab-5; Calbiochem, CA/USA), and diluted 1:1000 in PB containing 0.3% Triton X-100 plus 5% of normal goat serum. Incubation with the primary antibody was conducted overnight at 24°C. After three washes (10 min each) in PB, the sections were incubated with biotinylated goat anti-rabbit sera (Vector Labs, Burlingame, CA) diluted 1:200 in PB for 2 h at 24°C. The sections were washed again in PB and incubated with the avidin-biotin-peroxidase complex (ABC Elite; Vector Labs). After the reaction with 0.05% 3–3’ diaminobenzidine and a 0.01% solution of hydrogen peroxide in PB and intensification with 0.05% osmium tetroxide in water, the sections were mounted on gelatin- and chromoalumen-coated slides, dehydrated, cleared, and coversliped. The material was then analyzed on a light microscope, and digital images were collected. A quantitative analysis of the immunolabeled material was analyzed using a light microscope and the NIS Elements F3.0 Image analysis system (Nikon Instruments Inc., USA). A quantitative analysis was performed on the density of nuclei representative of thle immunoreactivity for Fos (Fos-IR) in: a) the dorsal horn of the spinal cord (DHSC; superficial laminae-I to IV according to the classification of Rexed. Measurements were taken from 10 different sections for each animal analyzed, including areas that were defined for each structure by using a 20 x objective for the DHSC. Measurements were performed with the program Image J and the operator was blinded to the animal treatment group.

Real-Time Quantitative Polymerase Chain Reaction (PCR)

Total RNA was isolated from subplantar muscles and spinal cord by TRIzol reagent (Gibco BRL, Gaithersburg, MD), according to the manufacturer’s protocol. RNA was subjected to DNase I digestion, followed by reverse transcription to cDNA, as previously described [26]. PCR was performed in a 7000 Sequence Detection System (ABI Prism, Applied Biosystems, Foster City, CA) using the SYBRGreen core reaction kit (Applied Biosystems). Primers used are described in Table 1.

Quantitative values for IL-6, IL-10, TNF-a, kinin B1 and B2 receptors, CAPDH and mRNA transcription were obtained from the threshold cycle number, where the increase in the signal associated with an exponential growth of PCR products begins to be detected. Melting curves were generated at the end of every run to ensure product uniformity. The relative target gene expression level was normalized on the basis of GADPH expression as endogenous RNA control [27]. Results are expressed as a ratio relative to the sum of GAPDH transcript levels as internal control.

Statistical Analysis

Results were expressed as the mean±SEM. Statistical analyses of data were generated by using GraphPad Prism, version 4.02 (GraphPad). A value of p<0.05 indicated a significant difference. Statistical comparison of more than two groups was performed using analysis of variance (ANOVA), followed by Bonferroni’s test. Statistical comparison for treatment over time was performed using two way ANOVA followed by Bonferroni’s test.

Results

Effect of Photobiomodulation on Bmv-Induced Mechanical Allodynia and Hyperalgesia

We initially investigated the effects of photobiomodulation on the allodynia and hyperalgesia induced by Bmv. We found that animals injected with Bmv showed significant mechanical allodynia and hyperalgesia when compared with baseline measurement taken before the test, as indicated by basal threshold in response to stimulation by von Frey filaments observed from 1st h after Bmv injection up to 24 h (Fig 1). Photobiomodulation treatment applied 30 min and 3 h after Bmv injection reversed mechanical allodynia of mice in all evaluated times (Fig 1A). Regarding hyperalgesia, LLL was able to interfere with mechanical sensitivity evaluated by 3.84 filament in all evaluated times (Fig 1B) however, for the 4.08 filament the reversion of hyperalgesia was observed only at the 3rd h of evaluation (Fig 1C). AV treatment itself did not interfere with mechanical sensitivity of mice (Fig 1).

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

Animals were injected intraplantar with Bmv (1 ?g; ?) 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).

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Effect of Photobiomodulation on Neuronal Activation

As demonstrated in Fig 2, intraplantar administration of Bmv induced a significant increase of Fos immunoreactivity observed in the dorsal horn of the spinal cord of animals injected with Bmv (42.75 ± 3.26) when compared to the saline group (10.65 ± 1.61). Photobiomodulation treatment significantly decreased Fos expression (26.58 ± 3.58; Fig 2).

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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.

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Effect of Photobiomodulation on IL-6, IL-10 and TNF-a mRNA Expression

Cytokine production was evaluated on samples obtained from either spinal cord or footpad of animals previously evaluated at the nociceptive tests. As shown in Fig 3, the mRNA concentrations of IL-6 and TNF-a increased significantly at 6 h after Bmv injection in the footpad of mice when compared with control group (Fig 3A and 3B). After laser treatment, a significant reduction of both IL-6 and TNF-a mRNA levels was found. Moreover, treatment with AV did not significantly interfere with either IL-6 or TNF-a mRNA levels. However, concomitant treatment of mice with AV and phtobiomodulation decreased both IL-6 and TNF-a mRNA levels (Fig 3A and 3B). Furthermore, no changes on IL-6 and TNF-a were observed in samples from spinal cord of mice (Fig 3D and 3E). IL-10 mRNA levels were decreased after Bmv injection on both footpad and spinal cord of mice. Photobiomodulation treatment increased IL-10 levels in both footpad and spinal cord samples (Fig 3C and 3F). AV treatment did not interfere with IL-10 levels, however it prevented the decrease of this cytokine on samples from spinal cord (Fig 3F).

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

mRNA levels of IL-6, TNF-a 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 ug) 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.

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Effect of Photobiomodulation on Kinin B1 and B2 Receptors mRNA Expression

A significant increase on mRNA expression of kinin B1 receptors was observed on Bmv-treated mice when compared to the control group (Fig 4A). LLLT, AV and the association of LLLT and AV induced a significant decrease of mRNA levels of kinin B1 receptors when compared with Bmv-treated animals (Fig 4A). Kinin B2 receptors mRNA expression was also significantly increased in envenomed mice paw when compared to control group (Fig 4B). Once again, LLLT or AV treatment decreased mRNA levels of B2 kinin receptors. More interestingly, the combination of LLLT and AV was more effective in decreasing B2 levels when compared with AV itself (Fig 4B).

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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 ug) 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.

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Discussion

The most effective treatment for venomous snakebites accidents is antivenom therapy. However, it is well known that such therapy is effective in neutralizing only the systemic effects of envenomation, without interfering with the severe local effects induced by these venoms [3]. Thus, given the importance framework triggered by local envenoming caused by bothropic venom and the incapacity of the antivenon to neutralize them, it is essential to investigate alternative therapies, with the greatest effectiveness in delaying progression and decreasing local symptoms of envenomed victims.

Among clinical symptoms induced by bothrops snakebites, local pain is a common and clinically relevant manifestation to the patient [28, 29]. Therefore, in this study, we investigated the capacity of photobiomodulation in reducing the nociceptive response caused by Bmv in mice footpad as well as the mechanisms involved. Herein, the intraplantar injection of Bmv induced mechanical allodynia and hyperalgesia. These results are in accordance with previous data demonstrating that Bmv induces potent mechanical allodynia and hyperalgesia in mice [24, 30]. Photobiomodulation applied 30 min and 3 h after Bmv reversed both mechanical allodynia and hyperalgesia. From these data, we confirmed that photobiomodulation, in fact, is effective in reducing Bmv-induced local pain. In our study, as in previous studies [24, 30], we observed that antinociception was not related to AV treatment, since it was not able to interfere with mechanical sensitivity of mice. Also, the association of LLLT and AV did not modify the effect of LLLT alone, reinforcing the therapeutic potential of LLL in treating local effects induced by bothrops venoms.

To better understand the capacity of photobiomodulation to decrease nociception, we evaluated the expression of Fos protein in the dorsal horn of the spinal cord of mice. The expression of proto-oncogenes from the c-fos, c-jun, and erg-1 family are extensively used as tools for the expression of enhanced activity of nociceptive neurons [20, 21]. Our results demonstrate that the intraplantar administration of Bmv induced a significant increase of Fos expression, observed in the dorsal horn of the spinal cord, which is characteristic of nociceptor activation. According to the results of this study, photobiomodulation not only significantly inhibited Bmv-induced mechanical allodynia and hyperalgesia, but also decreased nociceptor activation at the spinal level. More interestingly, we showed here that photobiomodulation is able to interfere with the transmission of Bmv-induced pain message to the central nervous system, reducing nociceptor activation at the central level. This result reveals sensory neurons as an important cellular target for photobiomodulation in the context of pain. In addition to nociceptor-mediated effects, other mechanism(s) may also take part in the antinociception observed in our experimental model. We hypothesized that photobiomodulation may reduce the inflammatory cytokines in the paw and spinal cord. Therefore, the next experiment was designed to further validate the proposed hypothesis.

It is commonly believed that proinflammatory cytokines such as TNF-a and IL-6 are involved in the pain process and that their peripheral and central levels are up-regulated in many pain models [31, 32]. In addition, as described in previous studies, Bothropic venom induces the accumulation of pro-inflammatory IL-6 and TNF-a cytokines in the local of venom injection, which contributes to the enhancement of local tissue damage [1, 33, 34]. Moreover, some studies suggest that the analgesic effect of LLLT may be due to the anti-inflammatory activity by the inhibition of inflammatory mediators [13, 35, 36]. Hence, to further analyze the mechanism by which photobiomodulation reduces nociception of mice induced by Bmv, the expression of pro-inflammatory IL-6 and TNF-a cytokines was evaluated on samples obtained from either footpad or spinal cord of animals. Our results showed that photobiomodulation was able to reduce IL-6 and TNF-a gene expression in the footpad of animals. Also, we showed that associated treatment of AV and LLLT induced the same decrease on IL-6 and TNF-a mRNA levels as the observed with LLLT alone. Moreover, no changes on IL-6 and TNF-a mRNA levels were observed in samples from spinal cord of mice, thus suggesting that inhibition of hyperalgesia depends on a peripheral inhibition of inflammatory cytokines. This result corroborates the study of Ferreira et al. (2005) [13] that proposed that the analgesic effect of LLLT involves the inhibition of hyperalgesic mediators.

Regarding IL-10, we observed that Bmv injection decreased IL-10 mRNA levels on both footpad and spinal cord samples. Also, LLLT increased IL-10 mRNA levels in both footpad and spinal cord. AV treatment did not interfere with IL-10 levels on samples from footpad of mice. However it prevented the decrease of this cytokine on samples from spinal cord. From these data, we confirmed that AV prevents systemic effects induced by Bmv however it did not protect against local hyperalgesia. IL-10 is considered a regulatory cytokine, related to the control of the inflammatory process due to its capacity of inhibiting the proinflammatory cytokine secretion [37]. Results presented herein suggest that laser irradiation was able to modulate the expression of this regulatory cytokine, both in the local of venom injection and in the spinal cord, and it appears likely that this modulation plays a role in the anti-nociception observed after bothropic venom in response to photobiomodulation.

To further analyze the mechanism by which photobiomodulation reduced Bmv-induced nociception, we evaluated the kinin receptors levels in the footpad of mice. Both kinin B1 and B2 receptors, evaluated here, play a central role in the pathophysiology of inflammation [38]. Kinin B2 receptors are broadly and constitutively expressed in most tissues, whereas B1 receptor is weakly expressed in most tissues under basal conditions but strongly upregulated following inflammation [39]. The involvement of bradykinin on Bmv-induced hyperalgesia and edema has been demonstrated [7, 40]. In addition, it was already demonstrated that the kinin B2 receptors are involved in hyperalgesic response induce by B. jararaca and B. aspervenoms [22, 41]. Our results demonstrate that both B1 and B2 kinin receptors are increased in the footpad of animals injected with Bmv. Among the treatments, we found that both LLLT and AV were able to reduce the expression of B1 and B2 kinin mRNA levels. However, the association of LLLT and AV showed greater effectiveness in reducing B2 kinin receptors. Considering that kinin receptors are important mediators on Bothrops-induced hyperalgesia [22,23] it is feasible to suggest that photobiostimulation reverses Bmv-induced hyperagesia, at least in part, by modulating bradikinin receptors involved in the process.

We conclude that photobiomodulation with low level laser is effective in decreasing nociceptor activation at the spinal level. Moreover LLL is effective in modulating pro- and anti-inflammatory cytokines as well as kinin receptors at mRNA transcriptional level. These effects, at least in part, contribute to the decrease of hyperalgesia observed after Bmv. Photobioestimulation with the parameters used herein should be considered as a potential therapeutic approach for the treatment of local effects of Bothrops snakebite.

Acknowledgments

The authors thank Dr José Carlos Cogo for providing the Bothrops moojeni venom. This study was performed in partial fulfillment of the requirements for the Ph.D. degree for N. Nadur-Andrade at Universidade Nove de Julho.

Author Contributions

  1. Conceived and designed the experiments: SRZ CSD JAdS.
  2. Performed the experiments: NNA VRdSO EFT RdSF.
  3. Analyzed the data: SRZ CSD VRdSO EFT.
  4. Contributed reagents/materials/analysis tools: SRZ CSD JAdS.
  5. Wrote the paper: SRZ CSD VRdSO EFT.

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PLoS One. 2016 Jul 8;11(7):e0158980. doi: 10.1371/journal.pone.0158980.

Low-Level Laser Therapy (904 nm) Counteracts Motor Deficit of Mice Hind Limb following Skeletal Muscle Injury Caused by Snakebite-Mimicking Intramuscular Venom Injection.

Vieira WF1,2, Kenzo-Kagawa B2, Cogo JC3, Baranauskas V1, Cruz-Höfling MA2.

Author information

  • 1Department of Semiconductors, Instruments and Photonics (DSIF)-Faculty of Electrical Engineering and Computation, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
  • 2Department of Biochemistry and Tissue Biology (DBBT)-Institute of Biology, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
  • 3Institute for Research and Development (IP&D)-University of Vale do Paraíba (UNIVAP), São José dos Campos, São Paulo, Brazil.

Abstract

Myotoxins present in Bothrops venom disrupt the sarcolemma of muscle fibers leading to the release of sarcoplasmic proteins and loss of muscle homeostasis. Myonecrosis and tissue anoxia induced by vascularization impairment can lead to amputation or motor functional deficit. The objective of this study was to investigate the dynamic behavior of motor function in mice subjected to injection of Bothrops jararacussu venom (Bjssu) and exposed to low-level laser therapy (LLLT). Male Swiss mice received Bjssu injection (830 ?g/kg) into the medial portion of the right gastrocnemius muscle. Three hours later the injected region was irradiated with diode semiconductor Gallium Arsenide (GaAs- 904 nm, 4 J/cm²) laser following by irradiation at 24, 48 and 72 hours. Saline injection (0.9% NaCl) was used as control. Gait analysis was performed 24 hours before Bjssu injection and at every period post-Bjssu using CatWalk method. Data from spatiotemporal parameters Stand, Maximum Intensity, Swing, Swing Speed, Stride Length and Step Cycle were considered. The period of 3 hours post venom-induced injury was considered critical for all parameters evaluated in the right hindlimb. Differences (p<0.05) were concentrated in venom and venom + placebo laser groups during the 3 hours post-injury period, in which the values of stand of most animals were null. After this period, the gait characteristics were re-established for all parameters. The venom + laser group kept the values at 3 hours post-Bjssu equal to that at 24 hours before Bjssu injection indicating that the GaAs laser therapy improved spatially and temporally gait parameters at the critical injury period caused by Bjssu. This is the first study to analyze with cutting edge technology the gait functional deficits caused by snake envenoming and gait gains produced by GaAs laser irradiation. In this sense, the study fills a gap on the field of motor function after laser treatment following snake envenoming.

PLoS One. 2016 Apr 8;11(4):e0152890. doi: 10.1371/journal.pone.0152890.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

Silva LM1, Silva CA1, Silva AD1, Vieira RP1, Mesquita-Ferrari RA2, Cogo JC3, Zamuner SR1.

Author information

  • 1Posgraduated Program in Medicine, Universidade Nove de Julho-UNINOVE, São Paulo, SP, Brazil.
  • 2Posgraduated Program in Biophotonics, Universidade Nove de Julho-UNINOVE, São Paulo, SP, Brazil.
  • 3Institute of Research and Development, Universidade do Vale do Paraíba, UNIVAP, São José dos Campos, SP, Brazil.

Abstract

BACKGROUND:

Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.

METHODOLOGY:

C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 ?g/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.

RESULTS:

In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.

CONCLUSION:

LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

 Photochem Photobiol Sci. 2014 Sep 18. [Epub ahead of print]

Photobiostimulation reduces edema formation induced in mice by Lys-49 phospholipases A2 isolated from Bothrops moojeni venom.

Nadur-Andrade N1, Dale CS, Santos AS, Soares AM, de Lima CJ, Zamuner SR.

Author information

  • 1Universidade Nove de Julho, São Paulo, SP, Brazil. Stella.rz@uninove.br.

Abstract

The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm-2, and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm-2, and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.

Photochem Photobiol Sci.  2013 Oct;12(10):1895-902. doi: 10.1039/c3pp50036e.

Effects of a low-level semiconductor gallium arsenide laser on local pathological alterations induced by Bothrops moojeni snake venom.

Aranha de Sousa E1, Bittencourt JA, Seabra de Oliveira NK, Correia Henriques SV, dos Santos Picanço LC, Lobato CP, Ribeiro JR, Pereira WL, Carvalho JC,da Silva JO.
  • 1Toxicology Laboratory, Pharmaceutical Science Course, Federal University of Amapá, Macapa, AP, Brazil. jocivania@unifap.br elziliam@ibest.com.br adolfo_bittencourt@yahoo.com.br nayanaseabra@yahoo.com.br shayanne_henriques@hotmail.com leide-caroline@hotmail.com camilalobato2011@hotmail.com jocivania@unifap.br.

Abstract

Antivenom therapy has been ineffective in neutralizing the tissue damage caused by snakebites. Among therapeutic strategies to minimize effects after envenoming, it was hypothesized that a low level laser would reduce complications and reduce the severity of local snake venom effects. In the current study, the effect of a low-level semiconductor gallium arsenide (GaAs) laser on the local pathological alterations induced by B. moojeni snake venom was investigated. The experimental groups consisted of five male mice, each administered either B. moojeni venom (VB), B. moojeni venom + antivenom (VAV), B. moojeni venom + laser (VL), B. moojeni venom + antivenom + laser (VAVL), or sterile saline solution (SSS) alone. Paw oedema was induced by intradermal administration of 0.05 mg kg(-1) of B. moojeni venom and was expressed in mm of directly induced oedema. Mice received by subcutaneous route 0.20 mg kg(-1) of venom for evaluating nociceptive activity and the time (in seconds) spent in licking and biting the injected paw was taken as an indicator of pain response. Inflammatory infiltration was determined by counting the number of leukocytes present in the gastrocnemius muscle after venom injection (0.10 mg kg(-1)). For histological examination of myonecrosis, venom (0.10 mg kg(-1)) was administered intramuscularly. The site of venom injection was irradiated by the GaAs laser and some animals received antivenom intraperitoneally. The results indicated that GaAs laser irradiation can help in reducing some local effects produced by the B. moojeni venom in mice, stimulating phagocytosis, proliferation of myoblasts and the regeneration of muscle fibers.

Lasers Med Sci.  2011 Apr 12. [Epub ahead of print]

Effects of photobiostimulation on edema and hemorrhage induced by Bothrops moojeni venom.

Nadur-Andrade N, Barbosa AM, Carlos FP, Lima CJ, Cogo JC, Zamuner SR.

Source

Laboratory of Physiology and Pharmacology, Institute of Research and Development, University of Vale do Paraíba, São José dos Campos, Brazil.

Abstract

Antivenom (AV) treatment has been ineffective in neutralizing the severe local fast-developing tissue damage following snake-bite envenoming. We studied the effectiveness of low-level laser (LLL) and light-emitting diode (LED) irradiation alone or in combination with AV in reducing local edema formation and hemorrhage induced by Bothrops moojeni venom (BmV) in mice. Edema formation was induced by injection of 1 ?g per paw of BmV into the right paw and was evaluated before and at several intervals after BmV intraplantar injection. Hemorrhagic activity was evaluated after intradermal injection of 20 ?g of BmV by measuring the diameter of the hemorrhagic area on the inner side of the skin. The site of BmV injection was irradiated by LLL or LED 30 min after BmV inoculation. AV was also administered intravenously 30 min after BmV injection. Irradiation with LLL at a wavelength of 685 nm and a dose of 2.2 J/cm(2) and with a red LED and an infrared LED at wavelengths of 635 nm and 945 nm, respectively, and a dose of 4 J/cm(2) reduced edema formation and hemorrhage induced by BmV (p?<?0.05). The combined AV and LLL or LED treatment showed the same reduction as LLL or LED irradiation separately. In conclusion, both LLL and LED irradiation reduced venom-induced local effects even though symptoms were already present. Thus, the effect of phototherapy in reducing local effects induced by BmV may be clinically relevant.

Photomed Laser Surg. 2009 Jun 16. [Epub ahead of print]

Effect of Low-Level Laser Therapy in the Myonecrosis Induced by Bothrops jararacussu Snake Venom.

Barbosa AM, Villaverde AB, Sousa LG, Munin E, Fernandez CM, Cogo JC, Zamuner SR.

1 Laboratory of Inflammation, Institute of Research and Development, University of Vale do Paraíba (UNIVAP) , São Jose dos Campos, SP, Brazil .

Abstract Objective: The aim of this work was to investigate the capacity of low-level laser therapy (LLLT) alone or in combination with antivenom (AV) to reduce myonecrosis induced by Bothrops jararacussu snake venom.

Background Data: Myonecrosis is the most pronounced local effect caused by B. jararacussu venom. AV therapy and other first-aid treatments do not reverse these local effects.

Material and Methods: Male Swiss mice were used. Myonecrosis was induced by injection of 0.6 mg/kg of B. jararacussu venom in the right gastrocnemius muscle and was evaluated at 3 or 24 h after venom injection. The site of venom administration was irradiated for 29 s with a low power semiconductor laser (685 nm) at a dose of 4.2 J/cm(2). Intravenous AV therapy (0.5 mL dose) was administered at different times: 30 min before venom injection or 0, 1, or 3 h afterward. Both AV therapy and LLLT treatments were duplicated in mice groups killed at 3 or 24 h.

Results: B. jararacussu venom caused a significant myonecrotic effect 3 and 24 h after venom injection. LLLT significantly reduced myonecrosis by 83.5% at 24 h (p < 0.05) but not at 3 h, and AV therapy alone was ineffective for reducing myonecrosis at 3 and 24 h.

Conclusion: Only LLLT significantly reduced myonecrosis of the envenomed muscle, suggesting that LLLT is a potentially therapeutic approach for treating the local effects of B. jararacussu venom.

Photochem Photobiol. 2009 Jan-Feb;85(1):63-9. Epub 2008 Jul 17.

The ability of low level laser therapy to prevent muscle tissue damage induced by snake venom.

Doin-Silva R, Baranauskas V, Rodrigues-Simioni L, da Cruz-Höfling MA.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.

Abstract

Antivenom therapy has been ineffective in neutralizing the severe local fast developing tissue damage following snakebite envenoming. Herein, some effects of in situ helium neon (HeNe) laser irradiation on rat nerve-muscle preparation injected with Bothrops jararacussu venom are described. The tibialis anterior muscle was injected with venom diluted in 0.9% saline solution (60 microg/0.02 mL) or saline solution alone. Sixty minutes after venom injection, laser (HeNe) treatment was administered at three incident energy densities: dose 1, a single exposure of 3.5 J cm(-2); dose 2, three exposures of 3.5 J cm(-2); dose 3, a single exposure of 10.5 J cm(-2). Muscle function was assessed through twitch tension recordings whereas muscle damage was evaluated through histopathologic analysis, morphometry of area of tissue affected and creatine kinase (CK) serum levels, and compared to unirradiated muscles. Laser application at the dose of 3.5 J cm(-2) reduced the area of injury by 64% (15.9 +/- 1.5%vs 44.2 +/- 5.7%), decreased the neuromuscular blockade (NMB) by 62% (11.5 +/- 2.5%vs 30.4 +/- 5.2%) and reduced CK levels by 58% (from 455 +/- 4.5% to 190.3 +/- 23.4%) when compared with unirradiated controls. Dose 2 showed a poorer benefit than dose 1, and dose 3 was ineffective in preventing the venom effects. Measurements of the absorbance of unirradiated and irradiated venom solution showed no difference in absorption spectra. In addition, no difference in the intensity of partial NMB in nerve-muscle preparation was shown by unirradiated and irradiated venom. The results indicate that the laser light did not alter venom toxicity. We conclude that HeNe laser irradiation at a dosage of 3.5 J cm(-2) effectively reduces myonecrosis and the neuromuscular transmission blocking effect caused by B. jararacussu snake venom. Thus, low level laser therapy may be a promising tool to minimize the severity of some of the local effects of snake envenoming.

Toxicon. 2008 Mar 10 [Epub ahead of print]

Effect of low-level laser therapy in the inflammatory response induced by Bothrops jararacussu snake venom.

Barbosa AM, Villaverde AB, Guimarães-Souza L, Ribeiro W, Cogo JC, Zamuner SR.

Laboratory of Inflammation, Institute of Research and Development, University of Vale do Paraíba, Av. Shishima Hifumi, 2911, Urbanova, CEP 12244-000, São José dos Campos, SP, Brazil.

This article reports the effect of low-level laser therapy (LLLT) on the edema formation and leukocyte influx caused by Bothrops jararacussu snake venom as an alternative treatment for Bothrops snakebites. The inflammatory reaction was induced by injection of 0.6mg/kg of B. jararacussu venom, in gastrocnemius muscle. Cell influx and edema were evaluated at 3 or 24h after venom injection. Mice were irradiated at the site of injury by a low-level laser (685nm) with a dose of 4.2J/cm(2). A therapy that combines LLLT and antivenom was also studied. B. jararacussu venom caused a significant edema formation 3 and 24h after its injection, and a prominent leukocyte infiltrate composed predominantly of neutrophils at 24h after venom inoculation. LLLT significantly reduced edema formation by 53% and 64% at 3 and 24h, respectively, and resulted in a reduction of neutrophils accumulation (P<0.05). The combined therapy showed to be more efficient than each therapy acting separately. In conclusion, LLLT significantly reduced the edema and leukocyte influx into the envenomed muscle, suggesting that LLLT should be considered as a potentially therapeutic approach for the treatment of the local effects of Bothrops species.

Lasers Surg Med. 2003;33(5):352-7.

Effects of the Ga-As laser irradiation on myonecrosis caused by Bothrops Moojeni snake venom.

Dourado DM, Fávero S, Baranauskas V, da Cruz-Höfling MA.

Departamento de Histologia e Embriologia, Instituto de Biologia, C.P. 6109, Universidade Estadual de Campinas (UNICAMP), 13 083-970 Campinas, Sao Paulo, Brazil.

Abstract

BACKGROUND AND OBJECTIVES: Viper snake envenoming induces in the victims systemic coagulopathy, and severe local tissue damage such as edema, hemorrhage, intense pain, and myonecrosis. Serumtherapy and other first-aid managements are ineffective in neutralizing these local effects. The effects of the gallium-arsenide (Ga-As) laser irradiation on mice gastrocnemius injected intramuscularly (i.m.) with Bothrops moojeni snake venom were investigated.

STUDY DESIGN/MATERIALS AND METHODS: Macroscopical, histopatological, and myonecrosis quantification through serum creatine kinase (CK) evaluation was done at 3, 12, and 24 hours (two, five, and eight irradiation sessions, 4 J/cm(2), 1 minute 32 seconds per period, respectively), were done after the venom or saline injection, and in venom-unirradiated mice.

RESULTS: In unirradiated gastrocnemius, the venom induced massive hemorrhage, vascular congestion, time-progressing myonecrosis, edema, abundant inflammatory infiltrate, and high CK serum levels. Ga-As irradiation significantly decreased the amount of myonecrosis in all the periods tested (P < 0.05).

CONCLUSIONS: The laser treatment significantly inhibited the ability of B. moojeni venom to rapidly disrupt the integrity of the plasma membrane.

LLLT ON DAMAGED MUSCLE CAUSED BY BOTHROPS MOOJENI SNAKE VENOM.

Dourado DM, Cruz-Höfling MA.

The venom of the bothrops moojeni snake was injected into the gastroscnemius of mice to mimic the effect of a snakebite. Traditional therapies for this snakebite have proven less effective. Three groups were tested: A=saline, B=venom and C=venom+ laser. Two sessions of HeNe laser at 4 J/cm2 during 1 m 32 s were administered and the animals were sacrificed at 24 h, 3 d and 7 d, respectively. The analysis showed myonecrosis with inflammation and an extensive area of degenerated fibres. In the laser group there was, by day 3, an incipient number of regenerating fibres. Laser accelerated the phagocytosis of fibre remnants and recovery of the tissue, decreasing the oedema and increasing regeneration.