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Nociceptive and edematogenic responses elicited by a crude bristle extract of Lonomia obliqua caterpillars
Toxicon 43 (2004) 273–278 www.elsevier.com/locate/toxicon
Nociceptive and edematogenic responses elicited by a crude bristle extract of Lonomia obliqua caterpillars Lu´cia de Castro Bastosa, Ana Beatriz Gorini Veigab, Jorge A. Guimara˜esb, Carlos Roge´rio Tonussia,* a
b
Department of Pharmacology, Federal University of Santa Catarina, P.O. Box 476, Floriano´polis, SC 88040-900, Brazil Centro de Biotecnologia, Federal University of Rio Grande do Sul, Av. Bento Gonc¸alves, P.O. Box 15005, Porto Alegre, RS 91501-970, Brazil Received 2 October 2003; revised 10 December 2003; accepted 12 December 2003
Abstract Lyophilized Lonomia obliqua crude bristle extract (LOCBE) diluted in physiological saline (15, 35 and 50 mg of protein/paw) was injected in the plantar surface of the hind paw of the rat, causing a nociceptive response which lasted from 30 to a maximum of 50 min, peaking in the first 5 min. The animals also presented hematuria and nasal bleeding. Nociception was inhibited by indomethacin pretreatment (2.5 mg/kg, i.p., 60 min before), but not by guanethidine (30 mg/kg/day, s.c., for 3 days) or loratadine (5 mg/kg, p.o., 60 min before). LOCBE injection also produced paw edema peaking 1 h after injection and lasting for 6 h. Loratadine pretreatment, but neither guanethidine nor indomethacin, reduced edema. After the period of overt nociception, a nociceptive aftersensation response could be evoked up to 6 h after by immersing the paw into cold water (15 8C) for 10 s. Capsaicin (1.6 mg), formalin (0.5%) or prostaglandin E2 (500 ng) did not produce the same aftersensation phenomenon. These results suggest that LOCBE-induced nociception is largely facilitated by prostaglandin production, and edematogenic response seems to be facilitated by prostanoids and histamine. Finally, LOCBE induced a state of sensitization to cold, which seemed to be specific as it was not caused by other noxious chemicals. q 2004 Elsevier Ltd. All rights reserved. Keywords: Aftersensation; Allodynia; Inflammation; Pain; Hematuria
1. Introduction The species Lonomia obliqua (LO) is a saturniidae moth that occurs predominantly in Southern Brazil. When in the larval stage, also known as caterpillar, the animal is covered with sets of hard bristles, which constitute tiny injecting needles filled with a protein-concentrated hemolymph (Veiga et al., 2001). The contact of the human skin with these bristles produces an intense local burning pain sensation and disturbance in the blood clotting system. Within a few hours, hematomas and hematuria are observed in combination with intracerebral and intraperitoneal hemorrhage (in some cases accompanied by renal failure) * Corresponding author. Tel.: þ55-48-331-9491; fax: þ 55-48222-4164. E-mail address: [email protected] (C.R. Tonussi). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.12.003
(Da Silva et al., 1996; Arocha-Pinango et al., 2000). In LO crude bristles extract (LOCBE), three activities have been described: a prothrombin activator called LO prothrombin activator protease (LOPAP); a factor X activator; and a phospholipase A(2)-like activity called Lonomiatoxin (Donato et al., 1998; Reis et al., 1999; Arocha-Pinango et al., 2000). The only treatment for the human envenomation caused by skin contact with LO caterpillars has been the anti-lonomia serum (Da Silva et al., 1996; Rocha-Campos et al., 2001). The treatment of the local pain sensation, or even the evaluation of sensory sequela, is not conducted since all efforts are made to control the impairment of the blood coagulation, which may result in fatal hemorrhage. However, it is known that any source of acute nociception, depending on its duration, can give rise to sensitization of the nociceptive pathways, and may result in sensory sequela (Raja et al., 1999).
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In order to gather information which may improve the care of patients envenomed by LO, we characterized a model in rats for the study of the nociceptive response elicited by LOCBE, which may provide reliable data regarding nociceptive, inflammatory, and possibly the sensory sequela produced.
2. Materials and methods 2.1. Animals Experiments were performed on male Wistar rats (200 – 250 g) which were housed in temperature-controlled rooms (22 – 25 8C) under a 12 – 12 h light/dark cycle with free access to water and food. All experiments were conducted according to the NIH guide for the care and use of laboratory animals (NIH publications no. 8023, revised 1978), and the ethical guidelines of the International Association for the Study of Pain (IASP, 1983). 2.2. Preparation of Lonomia obliqua crude bristle extract (LOCBE) The caterpillar’s hard bristles were macerated and homogenized in deionized water (Milli-Qw) and centrifuged at 14,000 rpm for 20 min. The pellet was discarded and the supernatant was stored lyophilized. Samples (2 mg) of the lyophilized extract were resuspended in 1 ml of the physiological saline and kept frozen at 2 18 8C. This sample was thawed on the day of the experiment to remove the amount needed, and refrozen. 2.3. Nociception evaluation After the intraplantarly injection of either LOCBE solutions, capsaicin (1.6 mg), formalin (0.5%) or prostaglandin E2 (250 ng) into one hind paw, the animals were placed into a glass chamber ð29 £ 29 £ 35 cmÞ for observation of the nocifensive behavior. The number of shaking, lifting or licking movements of the injected paw, after LOCBE injection, was scored in clusters of 5 min of observation, during a total observation time of 60 min. One hour after the period of observation of spontaneous nocifensive behavior, i.e. 2 h after the paw injection, the animals receiving each of the noxious chemicals were assessed for aftersensation. This method consisted of immersing the injected hind paw into cold water (15 8C) for 10 s, after which the animals were returned to the glass chamber and observed for 10 min for the same nocifensive behavior. This procedure was repeated hourly until the sixth hour.
sulfate in water (v/v). The cuvette was fixed on the plate of an electronic balance, so that the immersion of the treated hind paw (at the level of the tibio-tarsal joint) was accompanied by an increase of the weight displayed. Since the weight in grams is directly correlated to the volume of the immersed paw (cuvette solution density ¼ 1 g/ml), the value displayed by the balance was readily assumed as the paw volume. Edema measurement was made just before, and hourly after the injection of LOCBE or other nociceptive chemicals (50 ml). 2.5. Evaluation of the hemorrhagic effect The hemorrhagic symptoms presented by animals receiving LOCBE were characterized by hematuria of variable intensity, and nasal or eye bleeding. Hematuria was visually detected by a red-colored urine. In order to match the occurrence of nociception with hemorrhage, we considered the percentage of animals presenting any of the above hemorrhagic symptoms, irrespective to their intensity. 2.6. Drugs and dilutions This study was conducted with carrageenan multi-type k/l (BDH chemicals, UK), capsaicin, guanethidine, and prostaglandin E2 (Sigma, St Louis, MO, USA), formalin (Merck AG, Darmstadt Germany), indomethacin (Prodome Quı´mica e Farmaceˆutica, Campinas, SP, Brazil), loratadine (EMS Sigma Pharma, Hortolaˆndia, SP, Brazil). Prostaglandin E2 was stored in absolute ethanol solution (500 mg/ml), and was freshly diluted in physiological saline just before its application. Indomethacin was diluted in a 1.29% sodium bicarbonate solution ðpH ¼ 7:4Þ; so that the final concentration of sodium was within the physiological range. The other substances were diluted in 0.9% saline. 2.7. Statistical analysis All statistical analyses were carried out using the GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). The one-way ANOVA for repeated measures was used for time-course curve comparison, and one-way ANOVA for unmatched measures was used for multiple comparisons of experimental groups at a specific time of observation. When a significance level of at least P , 0:05 was detected, analysis was followed by the Newman–Keuls test. Results are expressed as mean ^ s.e. mean of six animals.
3. Results
2.4. Edema measurement
3.1. Nociceptive and edematogenic dose-dependent effects produced by LOCBE
Edema formation was measured by immersing the injected paw into a cuvette (10 ml) filled with a solution of 2.5% lauryl
The lyophilized crude bristle extract of L. obliqua, diluted in physiological saline at the doses of 100, 50, 35 and
L. de Castro Bastos et al. / Toxicon 43 (2004) 273–278
Fig. 1. Dose-dependent nocifensive response elicited by Lonomia obliqua crude bristle extract (LOCBE). LOCBE (100, 50, 35 and 17.5 mg) diluted in 50 ml of the physiological saline was injected subcutaneously in the plantar side of the right hind paw of rats. Immediately after LOCBE injection, the animals were observed for the nocifensive behavior (flinches, lifting and licking of the paw) during 30 min. Each point represents the mean ^ s.e. of mean of six animals. * P , 0:05; * * P , 0:001 between indicated groups (Posthoc analysis following ANOVA for repeated measures).
17.5 mg/50 ml was applied as described in Section 2. Fig. 1 shows the number of nociceptive events grouped in 5-min intervals, during a 30-min period of observation. LOCBE produced a clear dose-dependent nociceptive effect with significant differences between doses of 100 and 50 mg ðP , 0:001Þ and 50 and 35 mg ðP , 0:05Þ: The edematogenic response, however, presented more restricted variation among doses, but this effect lasted longer.
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Fig. 3. Dissociation of the nociceptive and hemorrhagic effects induced by LOCBE subcutaneous injection. The figure parallels the mean þ s.e. of mean of the nocifensive behavior (N) and the percentage of animals presenting hematuria (H) relative to the aging of the LOCBE sample dilution. The nocifensive response induced by the 0-day-old LOCBE solution was not found to be different from that induced by the same sample 11 days after preparation (P . 0:05 ANOVA for unmatched measures).
The 50 mg-treated group differed from the 35 mg-treated group ðP , 0:001Þ: All groups presented intense edema for more than 6 h, and the group receiving 100 mg of LOCBE still presented some paw edema 24 h later (Fig. 2). We adopted a dose of 35 mg/paw for subsequent experiments since it was the dose producing a better nociceptive response with the fewest secondary hemorrhagic effects. 3.2. Hemorrhagic effect after subcutaneous LOCBE injection The injection of LOCBE produced hemorrhagic symptoms in several animals receiving the higher doses, and on the first few days after diluting a new sample of lyophilized extract. However, even in the cases of the most intense hematuria (intense red-colored urine), only two animals died in the subsequent 24 h. Indeed, animals surviving the initial 24 h recovered from the hemorrhagic signs and presented normal and active behavior. An important finding was that the hemorrhagic effect disappeared over the subsequent days after LOCBE sample re-suspension, although the nociceptive effect remained active with some degree of attenuation only (Fig. 3). This attenuation, however, was not found to be statistically significant when compared to the nociception elicited by a freshly diluted LOCBE sample.
Fig. 2. Edematogenic response elicited by LOCBE subcutaneous injection. LOCBE (100, 50, 35 and 17.5 mg) diluted in the physiological saline was injected subcutaneously in the plantar side of the right hind paw of rats. Control group (Sal) received only 50 ml of physiological saline instead of LOCBE. Paw volume was measured just before, 30 min after and in the subsequent 6 h after LOCBE injection. Twenty-four hours after LOCBE injection, only the group receiving 100 mg still presented some edema. Each point represents the mean ^ s.e. of mean of six animals. * P , 0:05 between indicated groups (Post-hoc analysis following ANOVA for repeated measures).
3.3. Effect of indomethacin, guanethidine and loratadine treatments on the nociceptive and edematogenic responses Indomethacin pretreatment was given intraperitoneally, at a dose of 2.5 mg/kg, 1 h before the LOCBE injection (35 mg) into the hind paw. This treatment was also given 1 h after the LOCBE injection, in another group, for evaluation of its effect on the edema response. Indomethacin pretreatment significantly inhibited the nocifensive response
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Fig. 4. Effect of indomethacin, guanethidine and loratadine pretreatment on the nocifensive response induced by LOCBE. Indomethacin (Indo; 2.5 mg/kg, i.p.) given 60 min before LOCBE injection (35 mg), produced a strong inhibition of the nocifensive response, while guanethidine (Gua; 3 £ 30 mg=kg=day; s.c.) and loratadine (Lora; 5 mg/kg, p.o.) did not cause antinociception. Inhibition of nocifensive response values were calculated by the difference between each treated group and their respective control groups, expressed as mean þ s.e. of mean ðn ¼ 6Þ: * P , 0:001 compared to saline (SAL) treated group (Post-hoc analysis following ANOVA for unmatched measures).
(P , 0:001; Fig. 4), although neither the pretreatment nor the post-treatment affected edema formation (Fig. 5A and B). Guanethidine (30 mg/kg, s.c., for 3 days) and loratadine (5 mg/kg, p.o., 1 h before) pretreatments did not affect the nociceptive response (Fig. 4), but loratadine inhibited edema formation (P , 0:001; Fig. 5D). Control groups received sodium bicarbonate solution (1.29%), instead of indomethacin, or saline instead of guanethidine or loratadine.
Fig. 5. Effect of indomethacin, guanethidine and loratadine treatments on the edematogenic response induced by LOCBE. T and U bars represent the treated (drugs) and untreated (saline) groups, respectively. S bars represent the edematogenic effect of saline injection (50 ml) instead of LOCBE (negative control). Volume measurements were made 1 and 6 h after paw injections. Indomethacin (2.5 mg/kg, i.p.) was given 60 min before (panel A), or 60 min after (panel B) LOCBE injection (35 mg). Guanethidine (panel C) pretreatment was given on three consecutive days (30 mg/kg/day, s.c.) and the experiment was performed in the same day after the third injection. Loratadine (5 mg/kg, p.o.) pretreatment was given 60 min before LOCBE injection (panel D). Each bar represents the mean þ s.e. of mean of six animals. * P , 0:05 compared to the respective point of the saline-treated group (Post-hoc analysis following ANOVA for unmatched measures).
3.4. Normalization of the nocifensive responses induced by three different noxious chemicals and LOCBE In order to investigate whether the secondary effects of LOCBE-induced nocifensive response were due to the nociceptive input only, we adjusted the doses of three different noxious chemicals to produce nearly equal nocifensive responses in the same nocifensive paradigm adopted here. For this purpose, formalin (0.5%), capsaicin (1.6 mg) and prostaglandin E2 (250 ng) were injected into the hind paw of the animals. The nocifensive responses induced by these stimuli and LOCBE (35 mg) are shown in Fig. 6. The control group received only an injection of physiological saline. Comparing the area under the timecourse of the edema curve induced by LOCBE ð1:92 ^ 0:21Þ with the other two potent nociceptive chemicals, formalin ð0:73 ^ 0:26Þ and capsaicin ð0:34 ^ 0:12Þ produced an edematogenic response significantly higher than that in saline ð0:31 ^ 0:19Þ-treated paws ðP , 0:001Þ: These data are not shown in the figure.
Fig. 6. Normalization of the nocifensive responses induced by three different noxious chemicals and LOCBE. The doses of capsaicin (C; 1.6 mg), formalin (F; 0.5%) and prostaglandin E2 (PG; 250 ng) were adjusted to produce a similar nocifensive response to that produced by LOCBE (L; 35 mg). The nocifensive response was recorded in the first 10 min after injection. Each bar represents the mean þ s.e. of mean of six animals. The control group received only a saline injection into the footpad. * P , 0:01; * * P , 0:001 compared to the saline-treated group (Post-hoc analysis following ANOVA for unmatched measures).
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Fig. 7. Nociceptive aftersensation induced by LOCBE to cold stimulation. Two hours after the chemical noxious stimulation with LOCBE (35 mg), capsaicin (1.6 mg), formalin (0.5%) and PGE2 (250 ng), the injected paw was further submitted to a 10-s immersion in cold water (15 8C), each hour, over 5 h. Immediately after each cold stimulation, the animals were observed for nocifensive responses. Each bar represents the mean þ s.e. of mean ðn ¼ 6Þ of the responses exhibited during 10 min. Only animals stimulated with LOCBE presented a significant aftersensation compared to the control (saline-treated) group ð* P , 0:01; Post-hoc analysis following ANOVA for repeated measures).
3.5. Aftersensation elicited by cold stimulation of paws pretreated with LOCBE We occasionally observed, beyond the time range of LOCBE-induced nocifensive behavior (. 60 min) that some animals receiving the LOCBE exhibited nocifensive behavior some minutes after having the paws immersed in water for edema measurement. In order to study this response, we submitted all the animals that received the 35 mg LOCBE injection to repeated cold water immersion ð15 ^ 1 8CÞ for 10 s, every hour. This procedure elicited consistent nocifensive responses 5 – 10 min after each cold stimulation. Fig. 7 shows the time-course curve for the responses elicited up to 6 h of observation. After this time, the aftersensation response subsided. For comparative purposes, animals that received capsaicin (1.6 mg), prostaglandin E2 (250 ng) or formalin (0.5%) were subsequently submitted to the cold stimulation. As shown in Fig. 7, these treatments did not lead to the aftersensation.
4. Discussion In the present study, the nociceptive and edematogenic responses produced by the L. obliqua crude bristles extract (LOCBE) were reproduced in rats. The intensity and duration of these responses were apparently similar to those described for humans (Arocha-Pinango et al., 2000). LOCBE produced a dose-related syndrome of shaking and lifting of the injected hind paw that appeared to be mainly
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mediated by prostaglandin release, since it was strongly inhibited by indomethacin pretreatment. High sensitivity to the cyclooxygenase inhibitor indomethacin, which suggests strong involvement of prostaglandin in this response, may be correlated to the phospholipase A2-like activity described in LOCBE (Arocha-Pinango et al., 2000; Seibert et al., 2003). This phospholipase activity could be producing large amounts of arachidonic acid that would be converted to prostaglandin by cyclooxygenase. The possible participation of catecholamines and histamine was also investigated because there were substantial data implicating the peripheral sympathetic system (Nakamura and Ferreira, 1987; Tonussi and Ferreira, 1992; Liu et al., 1996; Kinnman et al., 1997; Lorenzetti et al., 2002), or mast cells (Julius and Basbaum, 2001), in acute chemical nociception in rats. However, neither the peripheral depletion of the sympathetic catecholamines, by guanethidine, nor the H1 receptor blockade, by loratadine, caused any inhibition of the nociceptive effect of LOCBE. On the other hand, the edema response elicited was not significantly inhibited by indomethacin, but loratadine produced a significant inhibition. These pharmacological characteristics are compatible with a mild inflammatory condition. The rapid onset of the edematogenic response may also suggest a neurogenic driven response, i.e. an edema mainly produced by the vasodilator peptides released from nociceptors acutely activated by the noxious stimuli. However, formalin and capsaicin did not reproduce the LOCBE-induced edematogenic response. Since these noxious chemicals were producing similar nociceptive effects, thereby probably producing similar nociceptor activation, it is possible that LOCBE-induced edema is mediated by other factors in addition to those released by the activation of nociceptors. Another interesting observation regarding LOCBE is that the hemorrhagic factor, which is the main cause of morbidity in accidents with L. obliqua, did not seem to be responsible for nociception and edema, since it was observed here that the LOCBE samples gradually lost their hemorrhagic capability in the subsequent days after resuspension in saline, while the nociceptive and edematogenic capability persisted. This higher stability of the nociceptive factor may indicate that it is a small protein, or even a non-protein, molecule. In a second series of experiments, we observed that in addition to the nociceptive and edematogenic responses described above, LOCBE produced a clear nociceptive aftersensation due to cold stimulation. In our studies, this sensitivity to cold water stimulation lasted for about 6 h, but we did not have the opportunity to verify the sensitization time-course produced by higher concentrations of LOCBE injection. Thus, we do not know whether aftersensation could be present for a longer period. This aftersensation may be related to the afterdischarge phenomena, which occur in sensitized neurons in the dorsal horn of spinal cord, following repetitive nociceptor firing or peripheral damage (Woolf, 1992). However, it is difficult to reconcile the idea
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of afterdischarge, which persists only few seconds after the interruption of the eliciting stimuli, with the present case of aftersensation, which initiates minutes after the cold stimulation, and persists up to 10 min. In addition, our experiments have shown that formalin (1%), capsaicin (1.6 mg), or PGE2 (250 ng), all stimuli producing similar initial nociceptive responses, did not elicit this kind of aftersensation to cold. Thus, LOCBE-induced aftersensation to cold did not seem to occur as a function of the initial nociceptive activity, but may be due to a specific mechanism related to a factor in the extract. We may speculate that a factor present in LOCBE could be sensitizing cold-sensitive fibers specifically, or affecting sympathetic terminals that respond to the cooling. In conclusion, we demonstrated here that the nociceptive and inflammatory effects of the venom of L. obliqua may be studied in rats, with great sensitivity. The short-lived nociceptive response seemed to be not due to the substances causing the hemorrhagic syndrome. In addition to the pain and inflammatory effects, LOCBE also produced sensory sequela, characterized by the nociceptive aftersensation to cold stimuli. This finding suggests that human subjects must be evaluated for similar nociceptive disturbance, as allodynia to cold stimuli, even after the main nociceptive effect of LO venom has subsided. The specificity of LOCBE to produce this cold-induced aftersensation suggests that further fractionation of the venom will provide a new pharmacological tool for nociceptive studies. Additional investigations are in progress for the identification of the mechanisms involved in this aftersensation response.
Acknowledgements This work was conducted with the financial support of the Conselho Nacional de Pesquisa (CNPq) and Coordenac¸a˜o de Aperfeic¸oamento do Pessoal do Ensino Superior (CAPES), Brazil.
References Arocha-Pinango, C.L., Marval, E., Guerrero, B., 2000. Lonomia genus caterpillar toxins: biochemical aspects. Biochimie 82, 937–942. Da Silva, W.D., Campos, C.M., Goncalves, L.R., Sousa-e-Silva, M.C., Higashi, H.G., Yamagushi, I.K., Kelen, E.M., 1996. Development of an antivenom against toxins of Lonomia obliqua caterpillars. Toxicon 34, 1045–1049.
Donato, J.L., Moreno, R.A., Hyslop, S., Duarte, A., Antunes, E., Le Bonniec, B.F., Rendu, F., de Nucci, G., 1998. Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin. Thromb. Haemost. 79, 539–542. International Association for the Study of Pain, 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109–110. Julius, D., Basbaum, A.I., 2001. Molecular mechanisms of nociception. Nature 413, 203–210. Kinnman, E., Nygards, E.B., Hansson, P., 1997. Peripheral alphaadrenoceptors are involved in the development of capsaicininduced ongoing and stimulus evoked pain in humans. Pain 69, 79 –85. Liu, M., Parada, S., Rowan, J.S., Bennett, G.J., 1996. The sympathetic nervous system contributes to capsaicin-evoked mechanical allodynia but not pinprick hyperalgesia in humans. J. Neurosci. 16, 7331– 7335. Lorenzetti, B.B., Veiga, F.H., Canetti, C.A., Poole, S., Cunha, F.Q., Ferreira, S.H., 2002. CINC-1 mediates the sympathetic component of inflammatory mechanical hypersensitivity in rats. Eur. Cytokine Netw. 13, 456–461. Nakamura, M., Ferreira, S.H., 1987. A peripheral sympathetic component in inflammatory hyperalgesia. Eur. J. Pharmacol. 135, 145–153. Raja, S.N., Meyer, R.A., Ringkamp, M., Campbell, J.N., 1999. Peripheral neural mechanisms of nociception. In: Melzack, R., Wall, P.D. (Eds.), Textbook of Pain, Churchill Livingstone, London, UK, pp. 11 –57. Reis, C.V., Kelen, E.M., Farsky, S.H., Portaro, F.C., Sampaio, C.A., Fernandes, B.L., Camargo, A.C., Chudzinski-Tavassi, A.M., 1999. A Caþ þ activated serine protease (LOPAP) could be responsible for the haemorrhagic syndrome caused by the caterpillar Lonomia obliqua. Lancet 353, 1942. Rocha-Campos, A.C., Gonc¸alves, L.R., Higashi, H.G., Yamagushi, I.K., Fernandes, I., Oliveira, J.E., Ribela, M.T., Sousa-E-Silva, M.C., da Silva, W.D., 2001. Specific heterologous F(ab0 )2 antibodies revert blood incoagulability resulting from envenoming by Lonomia obliqua caterpillars. Am. J. Trop. Med. Hyg. 64, 283 –289. Seibert, C.S., Shinohara, E.M., Sano-Martins, I.S., 2003. In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes. Toxicon 41, 831–839. Tonussi, C.R., Ferreira, S.H., 1992. Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics. Pain 48, 421– 427. Veiga, A.B., Blochtein, B., Guimaraes, J.A., 2001. Structures involved in production, secretion and injection of the venom produced by the caterpillar Lonomia obliqua (Lepidoptera, Saturniidae). Toxicon 39, 1343–1351. Woolf, C.J., 1992. Excitability changes in the central neurons following peripheral damage: role of central sensitization in the pathogenesis of pain. In: Willis, W.D., (Ed.), Hyperalgesia and Allodynia, Raven Press, New York, pp. 221– 243.
Nociceptive and edematogenic responses elicited by a crude bristle extract of Lonomia obliqua caterpillars Lu´cia de Castro Bastosa, Ana Beatriz Gorini Veigab, Jorge A. Guimara˜esb, Carlos Roge´rio Tonussia,* a
b
Department of Pharmacology, Federal University of Santa Catarina, P.O. Box 476, Floriano´polis, SC 88040-900, Brazil Centro de Biotecnologia, Federal University of Rio Grande do Sul, Av. Bento Gonc¸alves, P.O. Box 15005, Porto Alegre, RS 91501-970, Brazil Received 2 October 2003; revised 10 December 2003; accepted 12 December 2003
Abstract Lyophilized Lonomia obliqua crude bristle extract (LOCBE) diluted in physiological saline (15, 35 and 50 mg of protein/paw) was injected in the plantar surface of the hind paw of the rat, causing a nociceptive response which lasted from 30 to a maximum of 50 min, peaking in the first 5 min. The animals also presented hematuria and nasal bleeding. Nociception was inhibited by indomethacin pretreatment (2.5 mg/kg, i.p., 60 min before), but not by guanethidine (30 mg/kg/day, s.c., for 3 days) or loratadine (5 mg/kg, p.o., 60 min before). LOCBE injection also produced paw edema peaking 1 h after injection and lasting for 6 h. Loratadine pretreatment, but neither guanethidine nor indomethacin, reduced edema. After the period of overt nociception, a nociceptive aftersensation response could be evoked up to 6 h after by immersing the paw into cold water (15 8C) for 10 s. Capsaicin (1.6 mg), formalin (0.5%) or prostaglandin E2 (500 ng) did not produce the same aftersensation phenomenon. These results suggest that LOCBE-induced nociception is largely facilitated by prostaglandin production, and edematogenic response seems to be facilitated by prostanoids and histamine. Finally, LOCBE induced a state of sensitization to cold, which seemed to be specific as it was not caused by other noxious chemicals. q 2004 Elsevier Ltd. All rights reserved. Keywords: Aftersensation; Allodynia; Inflammation; Pain; Hematuria
1. Introduction The species Lonomia obliqua (LO) is a saturniidae moth that occurs predominantly in Southern Brazil. When in the larval stage, also known as caterpillar, the animal is covered with sets of hard bristles, which constitute tiny injecting needles filled with a protein-concentrated hemolymph (Veiga et al., 2001). The contact of the human skin with these bristles produces an intense local burning pain sensation and disturbance in the blood clotting system. Within a few hours, hematomas and hematuria are observed in combination with intracerebral and intraperitoneal hemorrhage (in some cases accompanied by renal failure) * Corresponding author. Tel.: þ55-48-331-9491; fax: þ 55-48222-4164. E-mail address: [email protected] (C.R. Tonussi). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.12.003
(Da Silva et al., 1996; Arocha-Pinango et al., 2000). In LO crude bristles extract (LOCBE), three activities have been described: a prothrombin activator called LO prothrombin activator protease (LOPAP); a factor X activator; and a phospholipase A(2)-like activity called Lonomiatoxin (Donato et al., 1998; Reis et al., 1999; Arocha-Pinango et al., 2000). The only treatment for the human envenomation caused by skin contact with LO caterpillars has been the anti-lonomia serum (Da Silva et al., 1996; Rocha-Campos et al., 2001). The treatment of the local pain sensation, or even the evaluation of sensory sequela, is not conducted since all efforts are made to control the impairment of the blood coagulation, which may result in fatal hemorrhage. However, it is known that any source of acute nociception, depending on its duration, can give rise to sensitization of the nociceptive pathways, and may result in sensory sequela (Raja et al., 1999).
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In order to gather information which may improve the care of patients envenomed by LO, we characterized a model in rats for the study of the nociceptive response elicited by LOCBE, which may provide reliable data regarding nociceptive, inflammatory, and possibly the sensory sequela produced.
2. Materials and methods 2.1. Animals Experiments were performed on male Wistar rats (200 – 250 g) which were housed in temperature-controlled rooms (22 – 25 8C) under a 12 – 12 h light/dark cycle with free access to water and food. All experiments were conducted according to the NIH guide for the care and use of laboratory animals (NIH publications no. 8023, revised 1978), and the ethical guidelines of the International Association for the Study of Pain (IASP, 1983). 2.2. Preparation of Lonomia obliqua crude bristle extract (LOCBE) The caterpillar’s hard bristles were macerated and homogenized in deionized water (Milli-Qw) and centrifuged at 14,000 rpm for 20 min. The pellet was discarded and the supernatant was stored lyophilized. Samples (2 mg) of the lyophilized extract were resuspended in 1 ml of the physiological saline and kept frozen at 2 18 8C. This sample was thawed on the day of the experiment to remove the amount needed, and refrozen. 2.3. Nociception evaluation After the intraplantarly injection of either LOCBE solutions, capsaicin (1.6 mg), formalin (0.5%) or prostaglandin E2 (250 ng) into one hind paw, the animals were placed into a glass chamber ð29 £ 29 £ 35 cmÞ for observation of the nocifensive behavior. The number of shaking, lifting or licking movements of the injected paw, after LOCBE injection, was scored in clusters of 5 min of observation, during a total observation time of 60 min. One hour after the period of observation of spontaneous nocifensive behavior, i.e. 2 h after the paw injection, the animals receiving each of the noxious chemicals were assessed for aftersensation. This method consisted of immersing the injected hind paw into cold water (15 8C) for 10 s, after which the animals were returned to the glass chamber and observed for 10 min for the same nocifensive behavior. This procedure was repeated hourly until the sixth hour.
sulfate in water (v/v). The cuvette was fixed on the plate of an electronic balance, so that the immersion of the treated hind paw (at the level of the tibio-tarsal joint) was accompanied by an increase of the weight displayed. Since the weight in grams is directly correlated to the volume of the immersed paw (cuvette solution density ¼ 1 g/ml), the value displayed by the balance was readily assumed as the paw volume. Edema measurement was made just before, and hourly after the injection of LOCBE or other nociceptive chemicals (50 ml). 2.5. Evaluation of the hemorrhagic effect The hemorrhagic symptoms presented by animals receiving LOCBE were characterized by hematuria of variable intensity, and nasal or eye bleeding. Hematuria was visually detected by a red-colored urine. In order to match the occurrence of nociception with hemorrhage, we considered the percentage of animals presenting any of the above hemorrhagic symptoms, irrespective to their intensity. 2.6. Drugs and dilutions This study was conducted with carrageenan multi-type k/l (BDH chemicals, UK), capsaicin, guanethidine, and prostaglandin E2 (Sigma, St Louis, MO, USA), formalin (Merck AG, Darmstadt Germany), indomethacin (Prodome Quı´mica e Farmaceˆutica, Campinas, SP, Brazil), loratadine (EMS Sigma Pharma, Hortolaˆndia, SP, Brazil). Prostaglandin E2 was stored in absolute ethanol solution (500 mg/ml), and was freshly diluted in physiological saline just before its application. Indomethacin was diluted in a 1.29% sodium bicarbonate solution ðpH ¼ 7:4Þ; so that the final concentration of sodium was within the physiological range. The other substances were diluted in 0.9% saline. 2.7. Statistical analysis All statistical analyses were carried out using the GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). The one-way ANOVA for repeated measures was used for time-course curve comparison, and one-way ANOVA for unmatched measures was used for multiple comparisons of experimental groups at a specific time of observation. When a significance level of at least P , 0:05 was detected, analysis was followed by the Newman–Keuls test. Results are expressed as mean ^ s.e. mean of six animals.
3. Results
2.4. Edema measurement
3.1. Nociceptive and edematogenic dose-dependent effects produced by LOCBE
Edema formation was measured by immersing the injected paw into a cuvette (10 ml) filled with a solution of 2.5% lauryl
The lyophilized crude bristle extract of L. obliqua, diluted in physiological saline at the doses of 100, 50, 35 and
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Fig. 1. Dose-dependent nocifensive response elicited by Lonomia obliqua crude bristle extract (LOCBE). LOCBE (100, 50, 35 and 17.5 mg) diluted in 50 ml of the physiological saline was injected subcutaneously in the plantar side of the right hind paw of rats. Immediately after LOCBE injection, the animals were observed for the nocifensive behavior (flinches, lifting and licking of the paw) during 30 min. Each point represents the mean ^ s.e. of mean of six animals. * P , 0:05; * * P , 0:001 between indicated groups (Posthoc analysis following ANOVA for repeated measures).
17.5 mg/50 ml was applied as described in Section 2. Fig. 1 shows the number of nociceptive events grouped in 5-min intervals, during a 30-min period of observation. LOCBE produced a clear dose-dependent nociceptive effect with significant differences between doses of 100 and 50 mg ðP , 0:001Þ and 50 and 35 mg ðP , 0:05Þ: The edematogenic response, however, presented more restricted variation among doses, but this effect lasted longer.
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Fig. 3. Dissociation of the nociceptive and hemorrhagic effects induced by LOCBE subcutaneous injection. The figure parallels the mean þ s.e. of mean of the nocifensive behavior (N) and the percentage of animals presenting hematuria (H) relative to the aging of the LOCBE sample dilution. The nocifensive response induced by the 0-day-old LOCBE solution was not found to be different from that induced by the same sample 11 days after preparation (P . 0:05 ANOVA for unmatched measures).
The 50 mg-treated group differed from the 35 mg-treated group ðP , 0:001Þ: All groups presented intense edema for more than 6 h, and the group receiving 100 mg of LOCBE still presented some paw edema 24 h later (Fig. 2). We adopted a dose of 35 mg/paw for subsequent experiments since it was the dose producing a better nociceptive response with the fewest secondary hemorrhagic effects. 3.2. Hemorrhagic effect after subcutaneous LOCBE injection The injection of LOCBE produced hemorrhagic symptoms in several animals receiving the higher doses, and on the first few days after diluting a new sample of lyophilized extract. However, even in the cases of the most intense hematuria (intense red-colored urine), only two animals died in the subsequent 24 h. Indeed, animals surviving the initial 24 h recovered from the hemorrhagic signs and presented normal and active behavior. An important finding was that the hemorrhagic effect disappeared over the subsequent days after LOCBE sample re-suspension, although the nociceptive effect remained active with some degree of attenuation only (Fig. 3). This attenuation, however, was not found to be statistically significant when compared to the nociception elicited by a freshly diluted LOCBE sample.
Fig. 2. Edematogenic response elicited by LOCBE subcutaneous injection. LOCBE (100, 50, 35 and 17.5 mg) diluted in the physiological saline was injected subcutaneously in the plantar side of the right hind paw of rats. Control group (Sal) received only 50 ml of physiological saline instead of LOCBE. Paw volume was measured just before, 30 min after and in the subsequent 6 h after LOCBE injection. Twenty-four hours after LOCBE injection, only the group receiving 100 mg still presented some edema. Each point represents the mean ^ s.e. of mean of six animals. * P , 0:05 between indicated groups (Post-hoc analysis following ANOVA for repeated measures).
3.3. Effect of indomethacin, guanethidine and loratadine treatments on the nociceptive and edematogenic responses Indomethacin pretreatment was given intraperitoneally, at a dose of 2.5 mg/kg, 1 h before the LOCBE injection (35 mg) into the hind paw. This treatment was also given 1 h after the LOCBE injection, in another group, for evaluation of its effect on the edema response. Indomethacin pretreatment significantly inhibited the nocifensive response
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Fig. 4. Effect of indomethacin, guanethidine and loratadine pretreatment on the nocifensive response induced by LOCBE. Indomethacin (Indo; 2.5 mg/kg, i.p.) given 60 min before LOCBE injection (35 mg), produced a strong inhibition of the nocifensive response, while guanethidine (Gua; 3 £ 30 mg=kg=day; s.c.) and loratadine (Lora; 5 mg/kg, p.o.) did not cause antinociception. Inhibition of nocifensive response values were calculated by the difference between each treated group and their respective control groups, expressed as mean þ s.e. of mean ðn ¼ 6Þ: * P , 0:001 compared to saline (SAL) treated group (Post-hoc analysis following ANOVA for unmatched measures).
(P , 0:001; Fig. 4), although neither the pretreatment nor the post-treatment affected edema formation (Fig. 5A and B). Guanethidine (30 mg/kg, s.c., for 3 days) and loratadine (5 mg/kg, p.o., 1 h before) pretreatments did not affect the nociceptive response (Fig. 4), but loratadine inhibited edema formation (P , 0:001; Fig. 5D). Control groups received sodium bicarbonate solution (1.29%), instead of indomethacin, or saline instead of guanethidine or loratadine.
Fig. 5. Effect of indomethacin, guanethidine and loratadine treatments on the edematogenic response induced by LOCBE. T and U bars represent the treated (drugs) and untreated (saline) groups, respectively. S bars represent the edematogenic effect of saline injection (50 ml) instead of LOCBE (negative control). Volume measurements were made 1 and 6 h after paw injections. Indomethacin (2.5 mg/kg, i.p.) was given 60 min before (panel A), or 60 min after (panel B) LOCBE injection (35 mg). Guanethidine (panel C) pretreatment was given on three consecutive days (30 mg/kg/day, s.c.) and the experiment was performed in the same day after the third injection. Loratadine (5 mg/kg, p.o.) pretreatment was given 60 min before LOCBE injection (panel D). Each bar represents the mean þ s.e. of mean of six animals. * P , 0:05 compared to the respective point of the saline-treated group (Post-hoc analysis following ANOVA for unmatched measures).
3.4. Normalization of the nocifensive responses induced by three different noxious chemicals and LOCBE In order to investigate whether the secondary effects of LOCBE-induced nocifensive response were due to the nociceptive input only, we adjusted the doses of three different noxious chemicals to produce nearly equal nocifensive responses in the same nocifensive paradigm adopted here. For this purpose, formalin (0.5%), capsaicin (1.6 mg) and prostaglandin E2 (250 ng) were injected into the hind paw of the animals. The nocifensive responses induced by these stimuli and LOCBE (35 mg) are shown in Fig. 6. The control group received only an injection of physiological saline. Comparing the area under the timecourse of the edema curve induced by LOCBE ð1:92 ^ 0:21Þ with the other two potent nociceptive chemicals, formalin ð0:73 ^ 0:26Þ and capsaicin ð0:34 ^ 0:12Þ produced an edematogenic response significantly higher than that in saline ð0:31 ^ 0:19Þ-treated paws ðP , 0:001Þ: These data are not shown in the figure.
Fig. 6. Normalization of the nocifensive responses induced by three different noxious chemicals and LOCBE. The doses of capsaicin (C; 1.6 mg), formalin (F; 0.5%) and prostaglandin E2 (PG; 250 ng) were adjusted to produce a similar nocifensive response to that produced by LOCBE (L; 35 mg). The nocifensive response was recorded in the first 10 min after injection. Each bar represents the mean þ s.e. of mean of six animals. The control group received only a saline injection into the footpad. * P , 0:01; * * P , 0:001 compared to the saline-treated group (Post-hoc analysis following ANOVA for unmatched measures).
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Fig. 7. Nociceptive aftersensation induced by LOCBE to cold stimulation. Two hours after the chemical noxious stimulation with LOCBE (35 mg), capsaicin (1.6 mg), formalin (0.5%) and PGE2 (250 ng), the injected paw was further submitted to a 10-s immersion in cold water (15 8C), each hour, over 5 h. Immediately after each cold stimulation, the animals were observed for nocifensive responses. Each bar represents the mean þ s.e. of mean ðn ¼ 6Þ of the responses exhibited during 10 min. Only animals stimulated with LOCBE presented a significant aftersensation compared to the control (saline-treated) group ð* P , 0:01; Post-hoc analysis following ANOVA for repeated measures).
3.5. Aftersensation elicited by cold stimulation of paws pretreated with LOCBE We occasionally observed, beyond the time range of LOCBE-induced nocifensive behavior (. 60 min) that some animals receiving the LOCBE exhibited nocifensive behavior some minutes after having the paws immersed in water for edema measurement. In order to study this response, we submitted all the animals that received the 35 mg LOCBE injection to repeated cold water immersion ð15 ^ 1 8CÞ for 10 s, every hour. This procedure elicited consistent nocifensive responses 5 – 10 min after each cold stimulation. Fig. 7 shows the time-course curve for the responses elicited up to 6 h of observation. After this time, the aftersensation response subsided. For comparative purposes, animals that received capsaicin (1.6 mg), prostaglandin E2 (250 ng) or formalin (0.5%) were subsequently submitted to the cold stimulation. As shown in Fig. 7, these treatments did not lead to the aftersensation.
4. Discussion In the present study, the nociceptive and edematogenic responses produced by the L. obliqua crude bristles extract (LOCBE) were reproduced in rats. The intensity and duration of these responses were apparently similar to those described for humans (Arocha-Pinango et al., 2000). LOCBE produced a dose-related syndrome of shaking and lifting of the injected hind paw that appeared to be mainly
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mediated by prostaglandin release, since it was strongly inhibited by indomethacin pretreatment. High sensitivity to the cyclooxygenase inhibitor indomethacin, which suggests strong involvement of prostaglandin in this response, may be correlated to the phospholipase A2-like activity described in LOCBE (Arocha-Pinango et al., 2000; Seibert et al., 2003). This phospholipase activity could be producing large amounts of arachidonic acid that would be converted to prostaglandin by cyclooxygenase. The possible participation of catecholamines and histamine was also investigated because there were substantial data implicating the peripheral sympathetic system (Nakamura and Ferreira, 1987; Tonussi and Ferreira, 1992; Liu et al., 1996; Kinnman et al., 1997; Lorenzetti et al., 2002), or mast cells (Julius and Basbaum, 2001), in acute chemical nociception in rats. However, neither the peripheral depletion of the sympathetic catecholamines, by guanethidine, nor the H1 receptor blockade, by loratadine, caused any inhibition of the nociceptive effect of LOCBE. On the other hand, the edema response elicited was not significantly inhibited by indomethacin, but loratadine produced a significant inhibition. These pharmacological characteristics are compatible with a mild inflammatory condition. The rapid onset of the edematogenic response may also suggest a neurogenic driven response, i.e. an edema mainly produced by the vasodilator peptides released from nociceptors acutely activated by the noxious stimuli. However, formalin and capsaicin did not reproduce the LOCBE-induced edematogenic response. Since these noxious chemicals were producing similar nociceptive effects, thereby probably producing similar nociceptor activation, it is possible that LOCBE-induced edema is mediated by other factors in addition to those released by the activation of nociceptors. Another interesting observation regarding LOCBE is that the hemorrhagic factor, which is the main cause of morbidity in accidents with L. obliqua, did not seem to be responsible for nociception and edema, since it was observed here that the LOCBE samples gradually lost their hemorrhagic capability in the subsequent days after resuspension in saline, while the nociceptive and edematogenic capability persisted. This higher stability of the nociceptive factor may indicate that it is a small protein, or even a non-protein, molecule. In a second series of experiments, we observed that in addition to the nociceptive and edematogenic responses described above, LOCBE produced a clear nociceptive aftersensation due to cold stimulation. In our studies, this sensitivity to cold water stimulation lasted for about 6 h, but we did not have the opportunity to verify the sensitization time-course produced by higher concentrations of LOCBE injection. Thus, we do not know whether aftersensation could be present for a longer period. This aftersensation may be related to the afterdischarge phenomena, which occur in sensitized neurons in the dorsal horn of spinal cord, following repetitive nociceptor firing or peripheral damage (Woolf, 1992). However, it is difficult to reconcile the idea
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of afterdischarge, which persists only few seconds after the interruption of the eliciting stimuli, with the present case of aftersensation, which initiates minutes after the cold stimulation, and persists up to 10 min. In addition, our experiments have shown that formalin (1%), capsaicin (1.6 mg), or PGE2 (250 ng), all stimuli producing similar initial nociceptive responses, did not elicit this kind of aftersensation to cold. Thus, LOCBE-induced aftersensation to cold did not seem to occur as a function of the initial nociceptive activity, but may be due to a specific mechanism related to a factor in the extract. We may speculate that a factor present in LOCBE could be sensitizing cold-sensitive fibers specifically, or affecting sympathetic terminals that respond to the cooling. In conclusion, we demonstrated here that the nociceptive and inflammatory effects of the venom of L. obliqua may be studied in rats, with great sensitivity. The short-lived nociceptive response seemed to be not due to the substances causing the hemorrhagic syndrome. In addition to the pain and inflammatory effects, LOCBE also produced sensory sequela, characterized by the nociceptive aftersensation to cold stimuli. This finding suggests that human subjects must be evaluated for similar nociceptive disturbance, as allodynia to cold stimuli, even after the main nociceptive effect of LO venom has subsided. The specificity of LOCBE to produce this cold-induced aftersensation suggests that further fractionation of the venom will provide a new pharmacological tool for nociceptive studies. Additional investigations are in progress for the identification of the mechanisms involved in this aftersensation response.
Acknowledgements This work was conducted with the financial support of the Conselho Nacional de Pesquisa (CNPq) and Coordenac¸a˜o de Aperfeic¸oamento do Pessoal do Ensino Superior (CAPES), Brazil.
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