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.

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.

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.

Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom.

BACKGROUND: Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian "cerrado" (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). METHODS: The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf's ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode's solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 µg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. RESULTS: TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis. CONCLUSIONS: The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria.

"Low-intensity laser therapy effect on the recovery of traumatic spinal cord injury".

Scientific advances have been made to optimize the healing process in spinal cord injury. Studies have been developed to obtain effective treatments in controlling the secondary injury that occurs after spinal cord injury, which substantially changes the prognosis. Low-intensity laser therapy (LILT) has been applied in neuroscience due to its anti-inflammatory effects on biological tissue in the repairing process. Few studies have been made associating LILT to the spinal cord injury. The objective of this study was to investigate the effect of the LILT (GaAlAs laser-780 nm) on the locomotor functional recovery, histomorphometric, and histopathological changes of the spinal cord after moderate traumatic injury in rats (spinal cord injury at T9 and T10). Thirty-one adult Wistar rats were used, which were divided into seven groups: control without surgery (n = 3), control surgery (n = 3), laser 6 h after surgery (n = 5), laser 48 h after surgery (n = 5), medullar lesion (n = 5) without phototherapy, medullar lesion + laser 6 h after surgery (n = 5), and medullar lesion + laser 48 h after surgery (n = 5). The assessment of the motor function was performed using Basso, Beattie, and Bresnahan (BBB) scale and adapted Sciatic Functional Index (aSFI). The assessment of urinary dysfunction was clinically performed. After 21 days postoperative, the animals were euthanized for histological and histomorphometric analysis of the spinal cord. The results showed faster motor evolution in rats with spinal contusion treated with LILT, maintenance of the effectiveness of the urinary system, and preservation of nerve tissue in the lesion area, with a notorious inflammation control and increased number of nerve cells and connections. In conclusion, positive effects on spinal cord recovery after moderate traumatic spinal cord injury were shown after LILT.

Photobiomodulation mitigates Bothrops jararacussu venom-induced damage in myoblast cells by enhancing myogenic factors and reducing cytokine production.

BACKGROUND: Photobiomodulation has exhibited promise in mitigating the local effects induced by Bothrops snakebite envenoming; however, the mechanisms underlying this protection are not yet fully understood. Herein, the effectiveness of photobiomodulation effects on regenerative response of C2C12 myoblast cells following exposure to Bothrops jararacussu venom (BjsuV), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: C2C12 myoblast cells were exposed to BjsuV (12.5 µg/mL) and irradiated once for 10 seconds with laser light of 660 nm (14.08 mW; 0.04 cm2; 352 mW/cm2) or 780 nm (17.6 mW; 0.04 cm2; 440 mW/ cm2) to provide energy densities of 3.52 and 4.4 J/cm2, and total energies of 0.1408 and 0.176 J, respectively. Cell migration was assessed through a wound-healing assay. The expression of MAPK p38-α, NF-Кß, Myf5, Pax-7, MyoD, and myogenin proteins were assessed by western blotting analysis. In addition, interleukin IL1-ß, IL-6, TNF-alfa and IL-10 levels were measured in the supernatant by ELISA. The PBM applied to C2C12 cells exposed to BjsuV promoted cell migration, increase the expression of myogenic factors (Pax7, MyF5, MyoD and myogenin), reduced the levels of proinflammatory cytokines, IL1-ß, IL-6, TNF-alfa, and increased the levels of anti-inflammatory cytokine IL-10. In addition, PBM downregulates the expression of NF-kB, and had no effect on p38 MAKP. CONCLUSION/SIGNIFICANCE: These data demonstrated that protection of the muscle cell by PBM seems to be related to the increase of myogenic factors as well as the modulation of inflammatory mediators. PBM therapy may offer a new therapeutic strategy to address the local effects of snakebite envenoming by promoting muscle regeneration and reducing the inflammatory process.

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

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 µg 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 µg 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.

Role of a novel tetrodotoxin-resistant sodium channel in the nitrergic relaxation of corpus cavernosum from the South American rattlesnake Crotalus durissus terrificus.

INTRODUCTION: Coitus in snakes may last up to 28 hours; however, the mechanisms involved are unknown. AIM: To evaluate the relevance of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-phosphodiesterase type 5 (PDE5) system in snake corpus cavernosum reactivity. METHODS: Hemipenes were removed from anesthetized South American rattlesnakes (Crotalus durissus terrificus) and studied by light and scanning electronic microscopy. Isolated Crotalus corpora cavernosa (CCC) were dissected from the non-spiny region of the hemipenises, and tissue reactivity was assessed in organ baths. MAIN OUTCOME MEASURES: Cumulative concentration-response curves were constructed for acetylcholine (ACh), sodium nitroprusside (SNP), 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272), and tadalafil in CCC precontracted with phenylephrine. Relaxation induced by electrical field stimulation (EFS) was also done in the absence and presence of N(ω) nitro-L-arginine methyl ester (L-NAME; 100 µM), 1H-[1, 2, 4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) and tetrodotoxin (TTX; 1 µM). RESULTS: The hemipenes consisted of two functionally concentric corpora cavernosa, one of them containing radiating bundles of smooth muscle fibers (confirmed by α-actin immunostaining). Endothelial and neural nitric oxide synthases were present in the endothelium and neural structures, respectively; whereas soluble guanylate cyclase and PDE5 were expressed in trabecular smooth muscle. ACh and SNP relaxed isolated CCC, with the relaxations being markedly reduced by L-NAME and ODQ, respectively. BAY 41-2272 and tadalafil caused sustained relaxations with potency (pEC(50) ) values of 5.84 ± 0.17 and 5.10 ± 0.08 (N=3-4), respectively. In precontracted CCC, EFS caused frequency-dependent relaxations that lasted three times longer than those in mammalian CC. Although these relaxations were almost abolished by either L-NAME or ODQ, they were unaffected by TTX. In contrast, EFS-induced relaxations in marmoset CC were abolished by TTX. CONCLUSIONS: Rattlesnake CC relaxation is mediated by the NO-cGMP-PDE5 pathway in a manner similar to mammals. The novel TTX-resistant Na channel identified here may be responsible for the slow response of smooth muscle following nerve stimulation and could explain the extraordinary duration of snake coitus.

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.

In vitro antiophidian properties of Dipteryx alata Vogel bark extracts.

Extracts from Dipteryx alata bark obtained with different solvents (hexane, dichloromethane, ethyl acetate and methanol) were mixed in vitro with Bothrops jararacussu (Bjssu, 40 µg/mL) and Crotalus durissus terrificus (Cdt, 15 µg/mL) snake venoms, and applied to a mouse phrenic nerve-diaphragm preparation to evaluate the possible neutralization of venom effects. Cdt venom neurotoxic effect was not inhibited by any of the extracts, while the neurotoxic and myotoxic actions of Bjssu venom were decreased by the methanolic extract. This inhibition appears to be augmented by tannins. Dichloromethane bark extract inhibited ~40% of Bjssu venom effects and delayed blockade induced by Cdt. The methodology used to determine which extract was active allows inferring that: (i) phenolic acids and flavonoids contained in the methanolic extract plus tannins were responsible mostly for neutralization of Bjssu effects; (ii) terpenoids from the dichloromethane extract may participate in the anti-Cdt and anti-Bjssu venom effects; (iii) a given extract could not inhibit venoms from different species even if those belong to the same family, so it is improper to generalize a certain plant as antiophidian; (iv) different polarity extracts do not present the same inhibitory capability, thus demonstrating the need for characterizing both venom pharmacology and the phytochemistry of medicinal plant compounds.

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.

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.

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.

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

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 0.6 mg/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 (685 nm) 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.

Pulmonary mechanic and lung histology injury induced by Crotalus durissus terrificus snake venom.

In the present work we investigated the effects of Crotalus durissus terrificus venom (CdtV) on the pulmonary mechanic events [static and dynamic elastance, resistive (DeltaP1) and viscoelastic pressures (DeltaP2)] and histology after intramuscular injection of saline solution (control) or venom (0.6 microg/g). The static and dynamic elastance values were increased significantly after 3 h of venom inoculation, but were reduced at control values in the other periods studied. The DeltaP1 values that correspond to the resistive properties of lung tissue presented a significant increase after 6h of CdtV injection, reducing to basal levels 12h after the venom injection. In DeltaP2 analysis, correspondent to viscoelastic components, an increase occurred 12 h after the venom injection, returning to control values at 24 h. CdtV also caused an increase of leukocytes recruitment (3-24 h) to the airways wall as well as to the lung parenchyma. In conclusion, C. durissus terrificus rattlesnake venom leads to lung injury which is reverted, after 24 h of inoculation.

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.

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.