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Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A 2 myotoxins, BthTx-I (Lys-49 PLA 2 ) and BthTx-II (Asp-49 PLA 2 ) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 μl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 μl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 μl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA 2 -homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The pathogenesis of myonecrosis caused by myotoxins from bothropic venom is associated with local extracellular matrix (ECM) disintegration, hemorrhage, and inflammation. Search for alternative methods associated with serum therapy is mandatory to neutralize the fast development of local damage following snakebites. The experimental use of photobiomodulation therapy (PBMT) in murine models has shown promising results relative to structural and functional recovery from bothropic snakebite-induced myonecrosis. This study pioneered in using Raman and Fourier transform infrared (FTIR) spectroscopies to characterize biochemical alterations in the gastrocnemius that had been injected with Bothrops jararacussu venom and exposed to local PBMT. Results show that vibrational spectra from lyophilized and diluted venom (1307 cm -1 ) was also found in the envenomed gastrocnemius indicating venom presence in the unirradiated muscle 48 h post-injection; but any longer visible after PBMT at this time exposure or 72 h post-injection regardless irradiated or not. Raman and FTIR analyses indicated that the bands with higher area and intensity were 1657 and 1547 cm -1 and 1667 and 1452 cm -1 , respectively; all are assignments for proteins, especially collagen, and are higher in the PBMT-exposed gastrocnemius. The infrared spectra suggest that laser treatment was able to change protein in tissue and that such change indicates collagen as the main target. We hypothesize that the findings reflect remodeling of ECM with key participation of collagen and faster tissue recovery for an anabolic condition.

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 μg/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.