Heparin and commercial bothropic antivenom against the paralyzing effect of Bothrops jararacussu snake venom

The crude venom of Bothrops jararacussu (Bjssu) is known to induce muscular paralysis in vitro. Many studies have shown that various substances, including heparin, neutralize the damage caused by snake venom. In the present study, the ability of heparin (Hep) and commercial bothropic antivenom (CBA) to neutralize neuromuscular effects of Bjssu venom, at different time-points, was analyzed. Mouse phrenic nerve-diaphragm preparation was used through a conventional myographic technique, following five different protocols: Group 1 was incubated with Bjssu (40 μg/mL) without any other treatment; Groups 2 and 3 were pretreated with heparin (1 μL/mL) and CBA (120 μL/mL), respectively, for 15 minutes before venom addition; Group 4 after 50% neuromuscular blockade induced by Bjssu crude venom received 1 μL/mL of heparin while Group 5 received a mixture of Hep:CBA:Bjssu. Control preparations (Tyrode) were treated with Hep and CBA (mean ± SEM; n = 3-6). After 120 minutes of venom incubation, Group 1 preparations presented twitch-tension of 12 ± 2%. However, in Groups 2 and 3, the neutralizations were 92 ± 1.9% and 81 ± 6%, respectively. The heparin addition, after 50% neuromuscular blockade by Bjssu, produced 40 ± 6% muscular response after 120 minutes of incubation. Hep:CBA:Bjssu mixture displayed a protective effect of 84 ± 10% against venom action. In conclusion, heparin and commercial bothropic antivenom efficiently neutralized the neurotoxic effects caused by B. jararacussu crude venom, even at different incubation time-points.


INTRODUCTION
In Brazil venomous snakebite accidents represent an important public health problem.About 90% of them are caused by Bothrops snakes and these cases are distributed among different areas of the country (1,2).
Bothrops jararacussu is one of the largest species, in relation to size, of crotalid snakes found in southern Brazil, northern Argentina and eastern Paraguay.Its venom has been studied since the early 1900s (3), and produces envenomation signs and symptoms similar to other Bothrops species (4).
Serum therapy has been used worldwide for decades to treat patients after snakebites (10).However, in the last few years, many other strategies have been used to minimize the damages caused by these toxins (11)(12)(13).For example heparin, a natural anticoagulant molecule that, due to its acidity, can neutralize basic phospholipases A 2 , is under intense investigation as an alternative treatment (14).
In the present study, different types of heparin treatment were examined to prevent the neurotoxic activity of B. jararacussu crude venom.Commercial bothropic antivenom was also used on mouse neuromuscular preparation to enable further comparative analysis.

Mouse Phrenic Nerve-Diaphragm Preparation
Mice were deeply anesthetized with halothane (Cristália, Brazil) and killed by exsanguination to enable obtainment of phrenic nerve-diaphragm preparations (PND) (15).The diaphragm was removed, mounted under a tension of 5 g in a 5 mL organ bath containing Tyrode solution (NaCl 137 mM, KCl 2.7 mM; CaCl 2 1.8 mM; MgCl 2 0.49 mM; NaH 2 PO 4 0.42 mM; NaHCO 3 11.9 mM and glucose 11.1 mM) (pH 7.4, 37 C) and aerated with 95% O 2 and 5% CO 2 .Supramaximal stimuli (4 x thresholds, 0.1 Hz, 0.2 ms) delivered from a Grass S48® stimulator (Astro-Med Inc., USA) were applied to the nerve through bipolar electrodes.Isometric muscle tension was recorded by a force displacement transducer (Load Cell BG-10 GM®, Kulite Semiconductor Products Inc., USA) coupled to a physiograph (Gould, model RS 3400) via a Gould universal amplifier (both from Gould Inc., Recording Systems Division, USA).The preparations were allowed to stabilize for at least 20 minutes before addition of either treatment.

Statistical analysis
All data were presented as mean ± SEM of the number of experiments done (three to eight repetitions each protocol).Statistical comparisons were accomplished using ANOVA (repeated measures) with p < 0.05 indicating significance.

Submitted to Different Treatments
Figure 1 shows the results after submitting the nerve-diaphragm preparations to Tyrode solution, heparin (1 µL/mL), antivenom (120 µL/mL) and B. jaracussu crude venom (40 μg/mL).Note that the preparation treated with commercial antivenom displayed facilitative effect (increase of twitch-tension amplitude).Significant differences between treated and control results were observed (p < 0.05).

Contractile Response of Pretreated PND after Bjssu Venom Addition at Different Time-Points
At the end of 120 minutes of observation, pretreated preparations (heparin or antivenom for 15 minutes), before venom addition (Figure 2 -A and B), displayed respective twitch-tensions of 92 ± 1.9% (n = 3) and 81 ± 6% (n = 3).In the second protocol the heparin addition after the venom-induced 50% blockade (Figure 2 -C   against the Bjssu venom.Nevertheless, in C, when the Bjssu effect was deflagrated and the heparin was added earlier, the heparin efficacy was reduced (W, washing).

DISCUSSION
Envenomations caused by B. jararacussu frequently produce local and systemic effects (16).These effects are induced by a variety of venom compounds, including myotoxic phospholipases A 2 and metalloproteinases, among other proteins and peptides (17).B. jararacussu venom is not a potent neuromuscular blocker in vivo, and is less nocive in vitro than the venoms of certain neurotoxic snakes, which contain α and β neurotoxins that induce neuromuscular blockade at concentrations lower than 5 µg/mL (18).However, this venom also causes total neuromuscular blockade in isolated mouse preparation at doses above 50 µg/mL (5,6).
Heparin is a highly acidic sulfated glycosaminoglycan with a high sulfate concentration, which is able to form inactive acid-base complexes with basic myotoxins from Bothrops venoms (19)(20)(21)(22)(23). Due to its polyanionic nature, heparin can interact with many molecules that have cationic sites, including proteins from the extracellular matrix (fibronectin, laminae, vitronectin) (5,19,(22)(23)(24).Heparin can also interact with phospholipases A 2 present in many snake venoms that may or may not affect the enzymatic activity of these proteins (25,26) (28).The antivenom neutralizes the neuromuscular blockade caused by these venoms, evidencing the formation of an antigen-antibody complex.In this study, the antivenom (120 µL/mL) addition promoted a facilitative effect in isolated preparation, corroborating the findings of Oshima-Franco et al. (29), who argued that this effect is caused by the presence of a thermostable substance or may simply be a non-specific protein-concentration (excess) effect.The correct explanation for this effect remains unclear, but involves the antivenom production.Antivenom fractionation could help elucidate this phenomenon.Melo et al. (21) described the need for a pre-incubation with heparin and venom to form an acid-base complex.However, this procedure is unnecessary as demonstrated in the present study, since the incubation of heparin, before or after the venom addition, displayed an efficient venom-neutralizing effect.Oshima-Franco et al. (29) showed that the pre-incubation of heparin (60 µL/mL) with bothropstoxin-I, the principal toxin of B. jararacussu venom, for 30 minutes, induced 100% protection in the contractile response in mouse neuromuscular preparations.
In our experimental conditions, when examining pretreatment with heparin (1 µL/mL) or antivenom (120 µL/mL), for 15 minutes before venom addition, we observed a protective ability via the twitch-tension of mouse phrenic nerve-diaphragm.Under a different protocol, heparin addition after 50% blockade (about 50 minutes after incubation) induced by the venom itself, produced a 40% contractile response after 120 minutes, demonstrating that heparin was efficient in the heparin-venom complex, even when added in the presence of neuromuscular blockade, which indicates the use of different incubation time-points.
Boechat et al. (30) found the association between heparin and antivenom to be more effective in neutralizing the lethal activity of Bothrops envenomation.In this study the mixture (heparin:antivenom) was also effective in neutralizing its neurotoxic venom effects, but no significant difference was observed when compared to heparin:venom and antivenom:venom incubations.
As examples of venom or toxin neutralization, many other substances have been assayed, ranging from manganese salt, which provides significant protection against Bjssu venom and its toxin neurotoxicity, to medicinal plants, as shown by hydroalcoholic extract of Casearia sylvestris against the neuromuscular paralyzing effect of BThTX-I (31)(32)(33).Therefore, new therapies in parallel with the antivenom treatment would be fundamental in minimizing local effects induced by snake venoms, specially those from the Bothrops genus (30).
In conclusion, this study shows that heparin and commercial bothropic antivenom were capable of neutralizing the neurotoxic effects induced by whole B. jararacussu venom, even when the treatment is examined at different time-points.Heparin showed a more efficient protection than antivenom, considering the lower amount required (1 µL/mL) in relation to antivenom (120 µL/mL).

Figure 1 .
Figure 1.Neuromuscular effects of Tyrode solution, heparin, antivenom and Bjssu crude venom on the indirectly stimulated mouse phrenic nerve-diaphragm preparations.The points represent the mean ± SEM of experiments indicated in parentheses.*p < 0.05 ) maintained the neuromuscular response in 40 ± 6% (n = 4).The Bjssu:Hep:CBA mixtures produced (Figure 2 -D) a twitch-tension of 84 ± 10% (n = 3) against the venom action.In all protocols the protective effect of heparin against the toxic action of B. jararacussu venom was observed.

Figure 2 .
Figure 2. Record of twitch-tension in the PND after heparin and antivenom addition.