Schematic representation of the sucrose splash test.

<p>Schematic representation of the sucrose splash test.</p

Effect of crotamine on contraction force of isolated diaphragm of mice.

<p>The contraction of isolated mouse diaphragms was induced by transmural electrical stimulation (0.1 Hz, 2 ms duration, under optimum voltage and supramaximal voltage) and the isometric contractions were determined by plotting the average value of contraction force of each 5 min. Crotamine (from 3 to 30 nM) increased by about 17 to 46% the amplitude of twitch-contraction of isolated muscle induced by the direct transmural electrical stimuli. The time of crotamine addition was considered as zero (vertical dashed line), and the baseline contraction amplitude was considered as 100% (horizontal dashed line). The results (in percentage) were expressed as mean ± SEM (N = 6 independent experiments). The value <i>p</i> < 0.05 for ANOVA statistical analysis compared to the control was considered significant as indicated by *, ^# and ^<i>a</i>, for 30, 10 and 3 nM of crotamine, respectively.</p

Sucrose splash test.

<p>The period of grooming denotes the time spent by the animal in self cleaning activity, and the grooming was quantified for periods of 5 min each, statistically significant differences between animals receiving crotamine (7.5 μg/animal or 0.3 mg/kg BW) by intraperitoneal (<i>ip</i>) route compared to vehicle receiving control animals were observed only after the second splash (A). The animals (N = 9) were continuously monitored this experiment during 1.5 h, and two splashes were performed in this interval, as indicated by the vertical dashed lines. The significant decreased activity was observed in the animals that received crotamine (average 754 s) compared to control animals (average 1498 s) was observed (B). The results (mean ± SE) were expressed as grooming period (s). * <i>p</i> ≤ 0.05 for unpaired two-tailed t-test was used to compare total grooming observed in each group during the splash test.</p

Mean distance traveled by animals after the administration of crotamine and the voltage-dependent K⁺ channels blocker 4-AP.

<p>The distance travelled by the animal treated with crotamine and 4-AP was quantified in timeframe periods of 10 min each (<b>A</b>), as well as the total distance travelled during 60 min was detemined (<b>B</b>), and the time of paralysis was also recorded (<b>C</b>). The values were considered significant for <i>p</i> < 0.05 for ANOVA statistical analysis. *, ^# and ^<i>a</i> in panels A and B refer to comparisons with control, 4-AP (30 μg/animal, which corresponds to 1.2 mg/kg BW) and crotamine (7.5 μg/animal or 0.3 mg/kg BW), respectively, while ^<i>b</i> refers to comparisons with animals receiving only crotamine.</p

Evaluation of paralysis time and animal behavior in the open field.

<p>Time of paralysis (<b>A</b>), number of times that the animal crossed the central area of the open field (<b>B</b>) and the external area of the open field (<b>C</b>), and amount of stools deposited by the animal in the field (<b>D</b>) were recorded and expressed in minutes (min) for time of paralysis onset (in A) and in numbers of events (N) for B, C and D. Different doses of crotamine (7.5, 15 or 30 μg/animal which correspond to 0.3, 0.6 and 1.2 mg/kg BW, respectively) was administered by intraperitoneal (<i>ip</i>) injection. The last bar in panel A, indicated by the 24 h in the open box, refers to the <i>ip</i> administration of same dose of crotamine (7.5 μg/animal or 0.3 mg/kg BW) in the same animal 24 h after first crotamine administration, showing a significant decrease in the time for paralysis onset compared to the first administration of crotamine in naïve animal, a day before. *<i>p</i> < 0.05 for ANOVA statistical analysis.</p

Paralysis of mice hind limbs after intraperitoneal route administration of crotamine and/or ion channel active compounds.

<p>Paralysis of mice hind limbs after intraperitoneal route administration of crotamine and/or ion channel active compounds.</p

Mean distance traveled by the animals after the administration of different doses of crotamine.

<p>The dose-response effect of crotamine was confirmed by the differences in the mean distance traveled by the animals after receiving 30, 15 or 7.5 μg/animal (which correspond to 0.3, 0.6 and 1.2 mg/kg BW, respectively) by intraperitoneal (<i>ip</i>) injection, quantified during 60 min <b>(A)</b> or measured in periods of each 10 min <b>(B)</b>. Total locomotor activity was continuously monitored during 60 min, and each bar in the graphic represent the average traveled distance (in meters) of at least 6–7 animals/group. The value of <i>p</i> < 0.05 for ANOVA statistical analysis was considered significant. *, ^# and ^<i>a</i> represent the comparison with control, 7.5 μg and 15 μg of crotamine per animal, respectively.</p

Effect of K⁺ channel blockers on the effect of crotamine on the contraction force of isolated diaphragm of mice.

<p>The increases of muscle contraction force induced by crotamine (30 nM) were evaluated in the presence of the voltage-dependent K⁺ channel blocker, namely 4-aminopyridine (4-AP, 1 μM) (panels A and B), and Ca²⁺-dependent K⁺ channel blockers, namely apamin (APA, 50 nM) (panels C and D) and charibdotoxin (CBTx, 10 nM) (panels E and F), which were added into the preparation after (panels A, C and E) or before (panels B, D and F) the addition of crotamine. Crotamine or channel blockers were applied at time zero or 35 min after the first compound addition, as indicated by the vertical dashed lines. The baseline contraction amplitude was considered as 100% (horizontal dashed line). The results (mean ± SEM, N = 6 independent experiments) were expressed as a percentage. *<i>p</i> < 0.05 for ANOVA statistical analysis compared to their respective control.</p

Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both <i>ex vivo</i> and <i>in vivo</i> assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles

<div><p>The high medical importance of <i>Crotalus</i> snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the <i>Bothrops</i> genus, the <i>Crotalus</i> genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all <i>Crotalus</i> snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10–25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed <i>in vitro</i> and <i>in vivo</i> assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, <i>ex vivo</i> assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine <i>in vivo</i>. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (<i>ip</i>) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.</p></div

Schematic representation of the <i>ex vivo</i> assays.

<p>Schematic representation of the <i>ex vivo</i> assays.</p