New approaches to second-degree burn healing: Polyvinyl alcohol membrane loaded to arnica combined to laser therapy.

Second-degree burns require greater care, as the damage is more extensive and worrisome and the use of a biomaterial can help in the cell repair process, with better planning, low cost, and better accessibility. Arnica has anti-inflammatory and analgesic properties in skin lesions treatments and laser therapy is another therapeutic alternative for burns. Evaluate the effects of arnica incorporated into PVA associated or not with low intensity laser on burns in rats. PVA and PVA with arnica (PVA+A) were obtained and characterized physicochemically. Through in vivo studies, the effects of PVA and PVA+A with or without the application of laser on the lesions allowed histological and immunohistochemical analyzes. PVA+A was biocompatible and with sustained release of the active, being a promising pharmacological tool and confirmed that laser therapy was effective in accelerating the healing process, due to its potential biomodulator, improving inflammatory aspects, promoting rapid healing in skin lesions.

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.

Cordia salicifolia and Lafoensia pacari plant extracts against the local effects of Bothrops jararacussu and Philodryas olfersii snake venoms.

Philodryas olfersii produces similar local effects to Bothrops jararacussu snakebite, which can induce misidentification and bothropic antivenom administration. Antivenom therapy is effective, but has its limitations regarding local damage. Since plants are used in folk medicine to treat snakebite victims, we evaluated the protective properties of Cordia salicifolia and Lafoensia pacari extracts against Philodryas olfersii and Bothrops jararacussu venoms. Preparations pretreated with both extracts inhibited > 90% the B. jararacussu venom-induced neuromuscular blockade, and 52% to 81% the P. olfersii venom-induced blockade. C. salicifolia inhibited the myonecrosis promoted by both venoms; however, L. pacari prevented only the myofilaments hypercontraction. Regarding haemorrhagic activity, C. salicifolia was more effective against B. jararacussu venom, while L. pacari was more effective against P. olfersii venom. On the other hand, for oedema-forming activity the results were the opposite. Considering that both extracts prevented (to different levels) the main manifestations of both snakebites (local symptoms), we endorse further studies involving these plants as coadjuvant in snakebite therapeutics.

Atividade antiofídica de Camellia sinensis contra a ação tóxica de venenos botrópicos em preparação neuromuscular de ave / Antiophidic activity of Camellia sinensis against the toxic action of bothropic venoms in bird neuromuscular preparations

Introdução: venenos de serpentes do gênero Bothrops são capazes de induzir bloqueio neuromuscular em preparações isoladas de mamífero e ave. O tratamento específico é a soroterapia, porém, nem sempre se mostra eficaz contra a toxicidade induzida pelo veneno. Por outro lado, compostos vegetais têm sido estudados com finalidade antiofídica, como é o caso da Camellia sinensis (chá verde). Objetivo: o objetivo foi de avaliar a atividade do extrato alcoólico de Camellia sinensis contra a ação de diferentes venenos botrópicos em preparações neuromusculares de ave. Métodos: foram utilizadas preparações neuromusculares de biventer cervicis de pintainho (BC) que foram incubadas com: venenos de diferentes serpentes do gênero Bothrops (B. jararaca, B. jararacussu, B. alternatus e B. neuwiedi, 50-100 µg/mL), extrato alcoólico de Camellia sinensis (Cs) 100 µg/ml ou pré-tratadas com Cs (30min) seguida da adição dos diferentes venenos botrópicos. Resultados: ao final do experimento, foi observado bloqueio da resposta contrátil em 69,7 ± 4,3% (B. jararaca), 83,1 ± 10% (B. jararacussu), 88,1 ± 4,5% (B. alternatus) e 92,4 ± 5,6% (B. neuwiedi). Quando as preparações foram pré incubadas com o extrato de Cs, as proteções foram de: 62%, 89,2%, 75,1% e 78,2%, respectivamente. Conclusão: conclui-se que o extrato de Camellia sinensis se mostrou eficaz contra a neurotoxicidade induzida por venenos botrópicos

Introduction: venoms from snakes of the Bothrops genus are capable of inducing neuromuscular blockade in preparations isolated from mammals and birds. The specific treatment is serum therapy, but it is not always effective against the toxicity induced by the venom. On the other hand, some plant compounds have been studied for antiophidic purposes, as is the case with Camellia sinensis (green tea). Objective: evaluate the activity of the alcoholic extract from Camellia sinensis against the action of various bothropic venoms in bird neuromuscular preparations. Methods: Chick biventer cervicis (BC) neuromuscular preparations were used which were incubated with venoms from various snakes of the Bothrops genus (B. jararaca, B. jararacussu, B. alternatus and B. neuwiedi, 50-100 µg/ml), Camellia sinensis (Cs) alcoholic extract, 100 µg/ml, or previously treated with Cs (30 min), followed by the addition of the various bothropic venoms. Results: blockade of the contractile response was observed in 69.7 ± 4.3% (B. jararaca), 83.1 ± 10% (B. jararacussu), 88.1 ± 4.5% (B. alternatus) and 92.4 ± 5.6% (B. neuwiedi). When the preparations were previously incubated with Cs extract, results were 62%, 89.2%, 75.1% and 78.2%, respectively. Conclusion: Camellia sinensis extract was found to be effective against neurotoxicity induced by bothropic venoms.

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.

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on µ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca(2+) by Sr(2+) and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.

Beneficial effect of crotamine in the treatment of myasthenic rats.

INTRODUCTION: Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats. METHODS: We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program. RESULTS: During the in-situ electromyographic recording, neostigmine (17 µg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine. CONCLUSIONS: These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom.

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 µl/ml and 118.9 ± 9.3% with 40 µl/ml in PND, and 120.7 ± 7.7% with 40 µl/ml and 114.5 ± 4.4% with 50 µl/ml in BC after 120 min; n = 4-6, mean ± SEM). In PND, the ethanol alone (40 µl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 µl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 µg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 µl/ml) for 30 min before incubation with venom (50 µg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 µg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

Biological characterization of Bothrops marajoensis snake venom.

This study describes the effects of Bothrops marajoensis venom (Marajó lancehead) on isolated neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND). At low concentrations (1µg/ml for CBC and 5µg/ml for PND), the venom exhibited a neuromuscular blocking without any damaging effect on the muscle integrity. At higher concentration (20µg/ml for PND), together with the neuromuscular blockade, there was a moderate myonecrosis. The results show differences between mammalian and avian preparations in response to venom concentration; the avian preparation was more sensitive to venom neurotoxic effect than the mammalian preparation. The possible presynaptic mechanism underlying the neuromuscular blocking effect was reinforced by the observed increase in MEPPs at the same time (at 15min) when the facilitation of twitch tension occurred. These results indicate that the B. marajoensis venom produced neuromuscular blockade, which appeared to be presynaptic at low concentrations with a postsynaptic component at high concentrations, leading to muscle oedema. These observations demand the fractionation of the crude venom and characterization of its active components for a better understanding of its biological dynamics.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu.

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes.

Structural characterization and neuromuscular activity of a new Lys49 phospholipase A(2) homologous (Bp-12) isolated from Bothrops pauloensis snake venom.

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on micro-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA(2) from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 +/- 6 min (1.4 microM) and 16 +/- 6 min (3.6 microM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca(2+) is replaced by Sr(2+), being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA(2) family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.

A study of the myotoxicity of bothropstoxin-i using manganese in mouse phrenic nerve-diaohragm and extensor digitorum longus preparations

Bothropstoxin-I (BthTX-I) from Bothrops jararacussu snake venom has a predominantly postsynaptic action that is responsible for this toxin´s myotoxicity. However, BthTX-I also has a presynaptic action that is counteracted by Mn2+, a reversible neuromuscular blocker that acts predominantly presynaptically. In this work, we used two nerve-muscle preparations (mouse phrenic nerve-diaphragm - PND and extensor digitorum longus - EDL) to investigate the ability of Mn2+ to protect against the myotoxicity of BthTX-I. The preparations were incubated with Tyrode solution (control), BthTX-I, or Mn2+ alone. BthTX-I (1.4 µM) produced irreversible blockade in both preparations, whereas the blockade by Mn2+ (0.9 mM) was total and reversible in PND but just partially reversible in EDL. Pretreating the preparations with Mn2+ resulted in 100% and 80% protection against BthTX-I-induced blockade, respectively. However, when Mn2+ (0.9 or 1.8 mM) and BthTX-I (1.4 µM) were co-incubated for 30 min before testing, the blockade was faster and sustained. Washing the preparations resulted in complete, sustained recovery in those exposed to 1.8 mM Mn2+ but not to 0.9 mM Mn2+. Morphological analysis showed that the extent of fiber damage by BthTXI (1.4 µM) was 82% (PND) and 68.5% (EDL), and that Mn2+ (0.9 mM) afforded 40% protection in both preparations and reduced the increase in muscle fiber cross-sectional area by 20% and 15%, respectively, compared to BthTX-I alone. Mn2+ (0.9 mM) significantly attenuated the release of creatine kinase by BthTXI. The low creatine kinase activity resulted from a protective action of Mn2+ on the sarcolemma and from direct inactivation of the released enzyme. These results show that Mn2+ prevents membrane disruption by BthTX-I and can protect against the myotoxicity and neurotoxicity caused by this toxin.

Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.