Oxidative stress and changes in hematological and biochemical biomarkers in Crotalus durissus terrificus (South American rattlesnake) venom-induced acute kidney injury in rats / Estrés oxidativo y alteraciones en biomarcadores hematológicos y bioquímicos en lesión renal aguda inducida por el veneno de Crotalus durissus terrificus (cascabel de America del Sur) en ratones

Abstract Acute kidney injury (AKI) is the major cause of mortality following bites by the South American rattlesnake Crotalus durissus terrificus. We investigated the early onset of Crotalus durissus terrificus venom-induced AKI in rats within 2 h of venom injection and its attenuation by antivenom. Several biomarkers were used to monitor AKI in the absence or presence of antivenom. Male Wistar rats were divided into five groups (n=5 each): G1, rats injected with saline (control); G2, rats injected with venom (6 mg kg-1, intraperitoneally) and euthanized after 2 h to evaluate AKI; G3 and G4, rats injected with 0.9% sterile saline or antivenom 2 h after venom, respectively, and monitored until death or up to 24 h post-venom, and G5, rats injected with antivenom alone and monitored for 24 h. Blood, urine and renal tissue samples were collected immediately after death to assess oxidative stress, hematological and biochemical alterations, and renal histological damage. Venom caused AKI within 2 h (G2) that persisted for up to 8.2 ± 1.6 h (G3), as confirmed by increases in blood urea, creatinine, and renal proteinuria; these increases were attenuated by antivenom. There were no changes in blood protein concentrations in G2 and G3, whereas there were increases in blood reduced glutathione, glutathione peroxidase, and plasma TBARS (but not in catalase) that were attenuated to varying extents by antivenom. There were no marked changes in platelets or leukocytes, but an increase in erythrocytes after 8.2 h with venom alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom groups alone was attenuated by antivenom. Renal glomerular and tubular damage was greatest after 2 h post-venom and declined thereafter. Venom caused early-onset AKI, with variable effects on lipid peroxidation and oxidative stress. Antivenom attenuated the AKI, as shown by the decrease in blood urea and the normalization of proteinuria, without protecting against lipid peroxidation.

Resumen La injuria o lesión renal aguda (LRA) es la mayor causa de mortalidad debido a las mordeduras por cascabeles Crotalus durissus terrificus. Se estudió la instalación precoz de LRA, en ratas, inducida por el veneno de Crotalus durissus terrificus después de 2 h de su inoculación y la atenuación por el antiveneno. Se utilizaron diversos biomarcadores para monitorear LRA en ausencia o presencia del antiveneno. Ratas Wistar machos fueron divididos en 5 grupos (n=5 por grupo): G1, ratas inoculadas con solución salina (control); G2, ratas inoculadas con veneno (6 mg kg-1 dosis, vía intraperitoneal), y sacrificadas después de 2 h para evaluar LRA; G3 y G4, ratas inoculadas con 0.9% de solución salina esterilizada o antiveneno luego de 2 h después de inoculado el veneno, respectivamente, y monitoreadas hasta su muerte o hasta 24 h después de inoculado el veneno; y G5, ratas inoculadas con antiveneno solo y monitoreadas durante 24 h. Las muestras de sangre, orina, y tejido renal fueron colectadas inmediatamente después de la muerte de los animales para evaluar estrés oxidativo, alteraciones hematológicas y bioquímicas, y daño histológico renal. El veneno causó LRA dentro de las 2 h (G2) persistiendo durante más de 8,2 ± 1,6 h (G3), estando esto confirmado por el incremento de urea sanguínea, creatinina, y proteinuria renal; estos aumentos disminuyeron con la aplicación del antiveneno. No se observaron alteraciones en las concentraciones de proteínas sanguíneas en G2 y G3, mientras que se encontraron incrementos en glutatión reducido sanguíneo, glutatión peroxidasa y TBARS plasmática (pero no en catalasa), que disminuyeron con la aplicación del antiveneno aunque en diferente grado. No ocurrieron alteraciones marcadas de plaquetas o leucocitos, mientras que el aumento de glóbulos rojos observado luego de 8,2 h de la inoculación con veneno, disminuyó con el antiveneno. El daño renal glomerular y tubular fue más importante luego de 2 h de la inoculación con veneno y posteriormente disminuyó. El veneno causó LRA precoz a las 2 h, con efectos variables sobre la peroxidación lipídica y el estrés oxidativo. El antiveneno redujo el daño renal, conforme lo demostrado por la disminución en la urea sanguínea y por la normalización de la proteinuria, aunque no se observó protección contra la peroxidación lipídica.

Evaluation of Protection by Caffeic Acid, Chlorogenic Acid, Quercetin and Tannic Acid against the In Vitro Neurotoxicity and In Vivo Lethality of Crotalus durissus terrificus (South American Rattlesnake) Venom.

Systemic envenomation by Crotalus durissus terrificus (South American rattlesnake) can cause coagulopathy, rabdomyolysis, acute kidney injury, and peripheral neuromuscular blockade, the latter resulting in flaccid paralysis. Previous studies have shown that plant products such as tannic acid and theaflavin can protect against the neuromuscular blockade caused by C. d. terrificus venom in vitro. In this work, we used mouse-isolated phrenic nerve-diaphragm preparations to examine the ability of caffeic acid, chlorogenic acid, and quercetin to protect against C. d. terrificus venom-induced neuromuscular blockade in vitro. In addition, the ability of tannic acid to protect against the systemic effects of severe envenomation was assessed in rats. Preincubation of venom with caffeic acid (0.5 mg/mL), chlorogenic acid (1 mg/mL), or quercetin (0.5 mg/mL) failed to protect against venom (10 µg/mL)-induced neuromuscular blockade. In rats, venom (6 mg kg-1, i.p.) caused death in ~8 h, which was prevented by preincubation of venom with tannic acid or the administration of antivenom 2 h post-venom, whereas tannic acid given 2 h post-venom prolonged survival (~18.5 h) but did not prevent death. Tannic acid (in preincubation protocols or given 2 h post-venom) had a variable effect on blood creatinine and urea and blood/urine protein levels and prevented venom-induced leukocytosis. Tannic acid attenuated the histological lesions associated with renal damage in a manner similar to antivenom. The protective effect of tannic acid appeared to be mediated by interaction with venom proteins, as assessed by SDS-PAGE. These findings suggest that tannic acid could be a potentially useful ancillary treatment for envenomation by C. d. terrificus.

The Influence of Silver Nanoparticles Against Toxic Effects of Philodryas olfersii Venom.

PURPOSE: A silver nanoparticle obtained by reducing salts with solid dispersion of curcumin (130 nm, 0.081 mg mL-1) was used to counteract against the toxic - edematogenic, myotoxic, and neurotoxic - effects of Philodryas olfersii venom. METHODS: The edematogenic effect was evaluated by plasma extravasation in rat dorsal skin after injection of 50 µg per site of venom alone or preincubated with 1, 10, and 100 µL of AgNPs; the myotoxicity was evaluated by measuring the creatine kinase released into the organ-bath before the treatment and at the end of each experiment; and neurotoxicity was evaluated in chick biventer cervicis using the conventional myographic technique, face to the exogenous acetylcholine (ACh) and potassium chloride (KCl) added into the bath before the treatment and after each experiment. Preliminarily, a concentration-response curve of AgNPs was carried out to select the concentration to be used for neutralizing assays, which consists of neutralizing the venom-induced neuromuscular paralysis and edema by preincubating AgNPs with venom for 30 min. RESULTS: The P. olfersii venom-induced edema (n=6) and a complete neuromuscular blockade (n=4) that includes the total and unrecovered block of ACh and KCl contractures. AgNPs produced a concentration-dependent decrease the venom-induced edema (n=6) from 223.3% to 134.4% and to 100.5% after 10 and 100 µL AgNPs-preincubation, respectively. The preincubation of venom with AgNPs (1 µL; n=6) was able to maintain 46.5 ± 10.9% of neuromuscular response under indirect stimuli, 39.2 ± 9.7% of extrinsic nicotinic receptors functioning in absence of electrical stimulus and 28.3 ± 8.1% of responsiveness to potassium on the sarcolemmal membrane. The CK release was not affected by any experimental protocol which was like control. CONCLUSION: AgNPs interact with constituents of P. olfersii venom responsible for the edema-forming activity and neuromuscular blockade, but not on the sarcolemma membrane-acting constituents. The protective effect of the studied AgNPs on avian preparation points out to molecular targets as intrinsic and extrinsic nicotinic receptors.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom.

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 A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) 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 PLA2-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.

Experimental model for removal of snake venom via hemoperfusion in rats.

OBJECTIVE: To examine the efficiency of hemoperfusion in removing South American rattlesnake (Crotalus durissus terrificus) venom from rats compared with neutralization by antivenom. DESIGN: An exploratory experimental investigation in rats involving the injection of snake venom with or without subsequent hemoperfusion or antivenom administration. SETTING: Basic animal research laboratory in a private university. ANIMALS: Normal, healthy male Wistar rats (0.29-0.40 kg, 3-6 months old) from a commercial breeder. INTERVENTIONS: Four experimental groups of randomly allocated rats (n = 3/group) were studied: Group 1: rats were injected with a single dose of venom (5 mg/kg, IM, in the right thigh) with no other treatment; blood samples were collected minutes before death to determine leukocyte, platelet, and erythrocyte counts; Group 2 (Control): rats underwent hemoperfusion alone for 60 min using a hemoperfusion cartridge designed for protein adsorption (by granulated charcoal) and protein precipitation (by tannic acid); Group 3 (Venom + antivenom): rats were injected with venom (5 mg/kg, IM) and, 10 min later, were treated with antivenom at the venom:antivenom ratio recommended by the manufacturer; Group 4 (Venom + hemoperfusion): Rats were injected with venom (5 mg/kg, IM) and, 10 min later, were hemoperfused for 60 min. In groups 2-4, blood samples were collected for leukocyte, platelet, and erythrocyte counts 24 h after venom. MEASUREMENTS AND MAIN RESULTS: Rats injected with venom alone (Group 1) developed signs of neurotoxicity and ataxia and died in 9.0 ± 0.43 h but showed no changes in leukocyte or erythrocyte counts. In contrast, there were no deaths in groups 2-4. The lack of deaths in Groups 3 and 4 indicated that antivenom and hemoperfusion, respectively, protected against the lethal effects of the venom. CONCLUSIONS: Hemoperfusion with a double-action hemoperfusion cartridge capable of protein adsorption and precipitation protected rats against C. d. terrificus venom.

Acute kidney injury caused by the intraperitoneal injection of Bothrops jararaca venom in rats.

We examined the ability of Bothrops jararaca venom (12.5 mg/kg) injected intraperitoneally (i.p.) to cause acute kidney injury (AKI) in rats. Blood urea and creatinine (AKI biomarkers, in g dL-1) were elevated after 2 h in venom-treated rats (urea: from 0.41 ± 0.1 to 0.7 ± 0.03; creatinine from 46.7 ± 3.1 to 85 ± 6.7; p < 0.05; n = 3 each), with no change in circulating reduced glutathione. Venom-treated rats survived for ∼6 h, at which point platelets were reduced (×103 µL-1; from 763.8 ± 30.2 to 52.5 ± 18.2) whereas leukocytes and erythrocytes were slightly increased (from 4.7 ± 0.3 to 6.6 ± 0.1 × 103 µL-1 and from 8.38 ± 0.1 to 9.2 ± 0.09 × 106 µL-1, respectively; p < 0.05); blood protein (5.2 ± 0.4 g dL-1) and albumin (2.7 ± 0.1 g dL-1) were normal, whereas blood and urinary urea and creatinine were increased. All parameters returned to normal with antivenom given 2 h post-envenomation. The i.p. injection of venom caused AKI similar to that seen with other routes of administration.

Photobiomodulation reduces cell death and cytokine production in C2C12 cells exposed to Bothrops venoms.

Snakebites caused by the genus Bothrops are often associated with severe and complex local manifestations such as edema, pain, hemorrhage, and myonecrosis. Conventional treatment minimizes the systemic effects of venom; however, their local action is not neutralized. The purpose of this study was to evaluate the effect of photobiomodulation (PBM) on C2C12 muscle cells exposed to B. jararaca, B. jararacussu, and B. moojeni venoms on events involved in cell death and the release of inflammatory mediators. Cells were exposed to venoms and immediately irradiated with low-level laser (LLL) application in continuous wave at the wavelength of 660 nm, energy density of 4.4 J/cm2, power of 10 mW, area of 0.045 cm2, and time of 20 s. Cell integrity was analyzed by phase contrast microscope and cell death was performed by flow cytometry. In addition, interleukin IL1-ß, IL-6, and IL-10 levels were measured in the supernatant. Our results showed that the application of PBM increases cell viability and decreases cell death by apoptosis and necrosis. Moreover, the release of pro-inflammatory interleukins was also reduced. The data reported here indicate that PBM resulted in cytoprotection on myoblast C2C12 cells after venom exposure. This protection involves the modulation of cell death mechanism and decreased pro-inflammatory cytokine release.

Pharmacological and transcriptomic characterization of the nitric oxide pathway in aortic rings isolated from the tortoise Chelonoidis carbonaria.

In this study the nitric oxide (NO)-soluble guanylate cyclase (sGC) and phosphodiesterase-5 (PDE-5) pathway was characterized in tortoise Chelonoidis carbonaria aorta. Concentration response curves (CCR) to ATP, ADP, AMP, adenosine and histamine were performed in the presence and absence of L-NAME in aorta pre-contracted with ACh (3 µM). CCR to SNP, BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator) and tadalafil (PDE-5 inhibitor) were constructed in the presence and absence of ODQ (10 µM). ATP (pEC50 6.1 ±â€¯0.1), ADP (pEC50 6.0 ±â€¯0.2), AMP (pEC50 6.8 ±â€¯0.1) and histamine (pEC50 6.8 ±â€¯0.12) relaxed Chelonoidis aorta and the addition of L-NAME reduced their efficacy (p < .05). Adenosine effects (pEC50 6.6 ±â€¯0.1) were not changed in the presence of L-NAME. SNP (pEC50 7.5 ±â€¯0.7; Emax 102.2 ±â€¯2.5%), BAY 41-2272 (pEC50 7.3 ±â€¯0.2; Emax 130.3 ±â€¯10.2%), BAY 60-2770 (pEC50 11.4 ±â€¯0.1; Emax 130.3 ±â€¯6.5%) and tadalafil (pEC50 6.7 ±â€¯0.3; Emax 121.3 ±â€¯15.3%) relaxed Chelonoidis aorta. The addition of ODQ reduced the SNP and tadalafil maximum response (p < .05) and promoted 63 fold right shift on BAY 41-2272 curve. In contrast, no alteration was observed on BAY 60-2770 response. Transcriptomic analysis for eNOS and sGC were found in aorta and brain libraries with high homology when compared with human transcripts. The NO-sGC-PDE-5 is functionally present in Chelonoidis aorta with a functional and genomic similarity to mammalian vessels. Unlike most of mammalian vessels, ACh did not cause endothelium-dependent relaxation in Chelonoidis carbonaria aortic rings.

Prospective study of a Bothrops jararacussu venom batch (Bj2015) - phospholipase A2 activity, immunogenicity, neurotoxicity, and myotoxicity parameters.

Bothrops jararacussu venom's (Bj2015) batch was biomonitored quarterly for one year to assess phospholipase A2 (PLA2) activity, immunogenicity, neurotoxicity, and myotoxicity. In silico models were applied to evaluate losses using decay model and recoveries by predictive trend analysis. Mice were immunized with Bj2015. Antibodies were detected by double-immunodiffusion and total protein and albumin were measured. Neuromuscular blockade-induced by 40 µg mL-1 venom solution was carried out using mouse nerve phrenic-diaphragm preparation. Resulting muscles were submitted to light microscopy to evaluate the myotoxicity. PLA2 activity of 0.1 mg mL-1 Bj2015 was measured using 4-nitro-3-(octanoyloxy)benzoic acid as substrate. Over time, greater losses occurred in neurotoxicity than PLA2, but not in myotoxicity and immunogenicity. Concluding, the neurotoxicity decrease can be related to enzymatic losses, including PLA2. Depending on the purpose of use, the collected venom responds on a long time, avoiding unnecessary new collections, improving life quality of animals in captivity and increasing their longevity.

Activity of silver nanoparticles on prokaryotic cells and Bothrops jararacussu snake venom.

Nanoparticle-conjugated venom-toxins of venomous animals and its therapeutic efficacy against emerging or neglecting diseases is a promising strategy. In this study, silver nanoparticles (AgNPs ∼50 nm, 0.081 mg mL-1) were studied against the neuromuscular blockade, myotoxic effects induced by Bothrops jararacussu venom (60 µg mL-1) and also against prokaryotic cells. The neurotoxicity was evaluated on ex vivo mouse phrenic nerve-diaphragm using traditional myographic technique, able to obtain functional contractile responses and to check the neurotransmission. The myotoxicity on mammalian cells was evaluated in muscles resulting from pharmacological assays using routine histological techniques and light microscopy. The toxicity to prokaryotic cells was evaluated on Salmonella typhimurium TA100 without metabolic activation. The in vitro preincubation model between AgNPs and venom was enough to abolish toxic effects of B. jararacussu venom, but mammalian cells were highly sensitive to AgNPs more than prokaryotic cells, by acting as dose-independently and dose-dependently parameters, respectively. These results allowed us to conclude that AgNPs showed promising activity as antivenom agent but for its safer use, the toxicity should be evaluated on experimental animals.

Electrical field-induced contractions on Crotalus durissus terrificus and Bothrops jararaca aortae are caused by endothelium-derived catecholamine.

Endothelium is the main source of catecholamine release in the electrical-field stimulation (EFS)-induced aortic contractions of the non- venomous snake Panterophis guttatus. However, adrenergic vasomotor control in venomous snakes such as Crotalus durissus terrificus and Bothrops jararaca has not yet been investigated. Crotalus and Bothrops aortic rings were mounted in an organ bath system. EFS-induced aortae contractions were performed in the presence and absence of guanethidine (30 µM), phentolamine (10 µM) or tetrodotoxin (1 µM). Frequency-induced contractions were also performed in aortae with endothelium removed. Immunohistochemical localization of both tyrosine hydroxylase (TH) and S-100 protein in snake aortic rings and brains, as well as in human tissue (paraganglioma tumour) were carried out. EFS (4 to 16 Hz) induced frequency-dependent aortic contractions in both Crotalus and Bothrops. The EFS-induced contractions were significantly reduced in the presence of either guanethidine or phentolamine in both snakes (p<0.05), whereas tetrodotoxin had no effect in either. Removal of the endothelium abolished the EFS-induced contractions in both snakes aortae (p<0.05). Immunohistochemistry revealed TH localization in endothelium of both snake aortae and human vessels. Nerve fibers were not observed in either snake aortae. In contrast, both TH and S100 protein were observed in snake brains and human tissue. Vascular endothelium is the main source of catecholamine release in EFS-induced contractions in Crotalus and Bothrops aortae. Human endothelial cells also expressed TH, indicating that endothelium- derived catecholamines possibly occur in mammalian vessels.

Electrical field stimulation-induced contractions on Pantherophis guttatus corpora cavernosa and aortae.

A tetrodotoxin (TTX)-resistant mechanism is responsible for the electrical field stimulation (EFS)-induced contractions and relaxations of Crotalus durissus terrificus corpora cavernosa. Here it was investigated whether this mechanism also occurs in corpora cavernosa and aortae of the non-venomous snake Pantherophis guttatus corpora cavernosa and aortae. Corpora cavernosa and aortic rings isolated from Pantherophis guttatus snake were mounted in organ bath system for isometric tension recording. EFS-induced contractions in both tissues were performed in the presence and absence of guanethidine (30 µM), phentolamine (10 µM) and tetrodotoxin (1 µM). In another set of experiments, the endothelium was removed from aortic rings and EFS-induced contractions were performed in the denuded rings. Electrical field stimulation-induced contractions were frequency-dependent in Pantherophis guttatus corpora cavernosa and aortic rings. The contractions were significantly reduced in the presence of guanethidine (30 µM) or phentolamine (10 µM). Pre-treatment with tetrodotoxin had no effect on the EFS-induced contractions of either corpora cavernosa or aortic rings. Surprisingly, the EFS-induced contractions of aortic rings denuded of endothelium were almost abolished. These results indicate that the TTX-resistant mechanism is present in EFS-induced contractions of Pantherophis guttatus corpora cavernosa and aortae. The experiments performed in the aorta indicate that the endothelium is the main source for the release of catecholamines induced by EFS.

Photobiomodulation of local alterations induced by BthTX-I, a phospholipase A2 myotoxin from Bothrops jararacussu snake venom: In vivo and in vitro evaluation.

This report describes the effect of photobiomodulation (PBM) on edema formation, leukocyte influx, prostaglandin E2 (PGE2) biosynthesis and cytotoxicity caused by bothropstoxin-I (BthTX-I), a phospholipase A2 (PLA2) homologue isolated from Bothrops jararacussu snake venom. Swiss mice or C2C12 cells were irradiated with low-level laser (LLL) at 685nm wavelength, an energy density of 4.6J/cm2 and an irradiation time of 13s. To evaluate the effect on edema formation and leukocyte influx, LLL was applied to the site of inoculation 30min and 3h post-injection. C2C12 cells were exposed to BthTX-I and immediately irradiated. PBM significantly reduced paw edema formation, peritoneal leukocyte influx and PGE2 synthesis, but increased the viability of C2C12 muscle cells after BthTX-I incubation. These findings demonstrate that PBM attenuated the inflammatory events induced by BthTX-I. The attenuation of PGE2 synthesis could be an important factor in the reduced inflammatory response caused by laser irradiation. The ability of LLL irradiation to protect muscle cells against the deleterious effects of BthTX-I may indicate preservation of the plasma membrane.

Tetrodotoxin-insensitive electrical field stimulation-induced contractions on Crotalus durissus terrificus corpus cavernosum.

Reptiles are the first amniotes to develop an intromitent penis, however until now the mechanisms involved in the electrical field stimulation-induced contraction on corpora cavernosa isolated from Crotalus durissus terrificus were not investigated. Crotalus and rabbit corpora cavernosa were mounted in 10 mL organ baths for isometric tension recording. Electrical field stimulation (EFS)-induced contractions were performed in presence/absence of phentolamine (10 µM), guanethidine (30 µM), tetrodotoxin (1 µM and 1mM), A-803467 (10 µM), 3-iodo-L-Tyrosine (1 mM), salsolinol (3 µM) and a modified Krebs solution (equimolar substitution of NaCl by N-methyl-D-glucamine). Immuno-histochemistry for tyrosine hydroxylase was also performed. Electrical field stimulation (EFS; 8 Hz and 16 Hz) caused contractions in both Crotalus and rabbit corpora cavernosa. The contractions were abolished by previous incubation with either phentolamine or guanethidine. Tetrodotoxin (1 µM) also abolished the EFS-induced contractions of rabbit CC, but did not affect EFS-induced contractions of Crotalus CC. Addition of A-803467 (10 µM) did not change the EFS-induced contractions of Crotalus CC but abolished rabbit CC contractions. 3-iodo-L-Tyrosine and salsolinol had no effect on EFS-induced contractions of Crotalus CC and Rabbit CC. Replacement of NaCl by N- Methyl-D-glucamine (NMDG) abolished EFS-induced contractions of rabbit CC, but did not affect Crotalus CC. The presence of tyrosine hydroxylase was identified in endothelial cells only of Crotalus CC. Since the EFS-induced contractions of Crotalus CC is dependent on catecholamine release, insensitive to TTX, insensitive to A803467 and to NaCl replacement, it indicates that the source of cathecolamine is unlikely to be from adrenergic terminals. The finding that tyrosine hydroxylase is present in endothelial cells suggests that these cells can modulate Crotalus CC tone.

Neutralising ability of Terminalia fagifolia extract (Combretaceae) against the in vitro neuromuscular effects of Bothrops jararacussu venom.

The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 µg/mL and 500 µg/mL were tested against 40 and 200 µg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.

Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom.

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA2) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA2 activity inhibition: 24 °C bath temperature, Ca2+ - Sr2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca2+ or Sr2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 µg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA2, proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca2+ - Sr2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca2+ Tyrode solution at 37 °C (usual conditions); on Sr2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca2+ Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA2 (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA2 activity; Ca2+ - Sr2+ replacement reduced only the PLA2 activity (to 15.3%). We observed a strict relation between the inhibition of PLA2 activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA2 or other venom components, such as metalloproteinases.

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

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.

Low-intensity laser therapy improves tetanic contractions in mouse anterior tibialis muscle injected with Bothrops jararaca snake venom

Abstract Introduction Envenomation by Bothrops snakes can produce local pain, edema, hemorrhage and myonecrosis. However, standard antivenom therapy is generally ineffective in neutralizing these effects so that alternative methods of treatment have been investigated. In experimental animals, low-level laser therapy (LLLT) attenuates the local effects of Bothrops venoms, but the benefits of LLLT on muscle function after envenomation are unclear. In this study, we examined the influence of LLLT on the contractile activity of mouse skeletal muscle injected with venom from Bothrops jararaca, the principal cause of snakebite in southeastern Brazil. Methods Twenty-seven male mice were used. Mice were injected with venom (40 μg in 50 μl) in the right anterior tibialis muscle, after which the muscle tendon was exposed, connected to an isometric transducer and subjected to a resting tension of 1 g. A bipolar electrode was attached to the tibial nerve for electrical stimulation. The mice were randomly allocated to five groups: A – Control (n = 3), B – Venom 3 h (n = 6), C – Venom 9 h (n = 6), D – Venom + Laser 3 h (n = 6), E – Venom + Laser 9 h (n = 6). Results The two groups that received LLLT post-venom showed improved muscle contraction and contracture in relation to muscle treated with venom alone. Conclusion These results indicate that LLLT can improve muscle function after damage induced by B. jararaca venom.

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

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 µg/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.