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Asian scorpion BmK venom induces plasma extravasation and thermal hyperalgesia in the rat
Toxicon 40 (2002) 527±533
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Asian scorpion BmK venom induces plasma extravasation and thermal hyperalgesia in the rat Bing Chen 1, Xiaopan Zhuo 1, Congying Wang, Yonghua Ji* Institute of Physiology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China Received 4 April 2001; accepted 4 October 2001
Abstract In the present study, the effects of scorpion Buthus martensi Karsch (BmK) venom on plasma extravasation and paw withdrawal latency (PWL) to radiant heat have been investigated in rats. BmK venom (20±200 mg/kg) by subcutaneous injection under the surface of the rat hindpaw causes dose-dependant increased plasma extravasation that could be partially inhibited by intraperitoneal (i.p.) injected morphine (6 mg/kg). Peak plasma extravasation was reached at 10 min and persisted for 60 min at a dose of 200 mg/kg. BmK venom induced cutaneous hyperalgesia as indicated by decreased PWL to radiant heat in the ipsilateral paw following subcutaneous injection of 20 mg/kg BmK venom without effect on PWL of the contralateral hindpaw. Meanwhile, it was found that i.p. morphine injection could inhibit this decreased ipsilateral PWL. The results thus suggest that BmK venom could induce peripheral in¯ammation in rat by subcutaneous injection, and may prove a valuable animal model for investigating the pathophysiology of a number of in¯ammatory diseases and identifying potential antiin¯ammatory and analgesic drugs. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Scorpion Buthus martensi Karsch venom; Plasma extravasion; Peripheral in¯ammation; Morphine; Paw withdrawal latency; Thermal hyperalgesia
1. Introduction The Asian scorpion Buthus martensi Karsch is a species belonging to the Buthidae family, widely distributed from northwestern China to Mongolia and Korea. Although this species is not dangerously venomous for mammals, stings from scorpions are followed by severe pain, swelling, edema, burning sensation and redness (Balozet, 1971). These symptoms may last for several hours. Several bioactive agents like histamine and 5-HT have been identi®ed in the venom of other scorpion species (Basu et al., 1990), which are partly responsible for the immediate in¯ammatory pathophysiological changes. In¯ammation is often associated with either tissue damage or nerve injury, and considered to be a critical reaction to irritation and infection, and is characterized by * Corresponding author. Tel.: 186-21-6474-8057; fax: 186-216433-2445. E-mail address: [email protected] (Y. Ji). 1 Both authors contribute equally in this study.
redness, heat, swelling, loss of function and primary hyperalgesia (Levine and Reichling, 1999). Redness and heat are the results of increase in blood ¯ow; swelling is caused by an increase in vascular permeability. The changes result from the concerted action of a number of local mediators. Increased vascular permeability leads to the site of tissue injury being invaded by a host of immune cells, which in turn stimulate the release of other active substances. Associated with the series of events is hyperalgesia, due to the stimulation of nociceptors, the sensory neurons convey `painful' afferent information to the spinal cord. Primary hyperalgesia is characterized by a lowering of response threshold and/or an increased response to suprathreshold stimulus at the site of injury. Meanwhile, primary hyperalgesia can result in sensitization of primary nociceptors to mechanical and thermal stimulus at the in¯ammation site. The pain is due to increasing activation, and sensitization of primary afferent nerve ®bers (Andrew and Greenspan, 1999). The process of in¯ammation and hyperalgesia may lead to the removal of injured tissue and repair of the site. All these sensitization reactions ordinarily
0041-0101/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00248-3
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depend on the release, synthesis or attraction of in¯ammatory mediators to the site of injury. Although it has been identi®ed that scorpion stings can induce edema (Deshpande et al., 1999), any detailed and quantitative data analysis on edema responses induced by scorpion venom has not been attempted till date. In the present study, we determined plasma extravasation and paw withdrawal latency (PWL) to radiant heat of rats injected with scorpion BmK venom, and also determined the inhibitory effect of morphine on the ensuring in¯ammatory responses. Possible mechanisms are discussed. 2. Materials and methods Adult male Sprague±Dawley rats (Shanghai experimental animals center, Shanghai, China) weighing 220± 250 g were used. The animals were individually housed in cages with free access to water and food ad libition, and maintained in a room temperature of 21±238C with a 12 h light/dark cycle. Each animal was tested only once. Guidelines on ethical standards for investigation of experimental pain conscious animals were performed strictly according to principles described by Zimmermann (1983), and every effort was made to minimize animal suffering. Crude venom of scorpion B. martensi Karsch (BmK), collected by electrical stimulation, was purchased from an individual scorpion culture farm in Zhengzhou, Henan Province, China. Rats were classi®ed into three groups for extravasation measurement. The ®rst group was used to determine the time course of plasma extravasation of 200 mg/kg BmK venom (dissolved in 50 ml saline) following plantar injection. The second group of rats were used to determine the amount of plasma extravasation at different doses: 20, 40, 200, 400 mg/kg BmK venom (dissolved in 50 ml saline) under the surface of one hind paw. Control animals received an equivalent volume of saline. The third group of rats were used for the investigation of morphine's effect (10 mg/ml morphine hydrochloride, Shenyang First Pharmaceutical Factory, China) on plasma extravasation induced by BmK venom. Animals were pretreated with 6 mg/kg morphine (i.p.) before 40 mg/kg BmK venom injection. Control rats were pretreated with saline (i.p.) instead of morphine. Another four group of rats were employed to measure PWL to radiant heat. The ®rst group of rats were injected with 20 mg/kg BmK venom n 18: The second control group of rats were injected with 50 ml saline n 18: The third group of rats received 6 mg/kg morphine (i.p.) 10 min before 20 mg/kg BmK venom injection n 18: The fourth group of rats were pretreated with an equivalent volume of saline instead of morphine 10 min before BmK venom injection n 18: 2.1. Determination of rat plasma extravasation Plasma extravasation was measured according to
Humphrey (1993) and John et al. (1998). The rats were intravenously injected with 40 mg/kg Evans Blue while anesthetized with 30 mg/kg sodium pentobarbital (i.p.), 0, 30 or 50 min, later the anesthetized animals were injected with 50 ml BmK venom or saline. One hour after Evans Blue injection, the rats were cardiac punctured, a 1 ml blood sample was obtained and the animals were then intracardially perfused with 300 ml of 0.9% saline. The blood sample was centrifuged at 3000 rpm for 5 min, and the plasma supernatant was kept at 2208C prior to determination of the Evans Blue concentration. The contralateral and ipsilateral hindpaws were removed at the ankle joint for determination of plasma extravasation induced by BmK venom. The hindpaws were frozen at 2208C for several hours. The tissue was then weighed, minced, placed into a 10 ml mixture of acetone and 0.5% sodium sulfate (7:3, v/v; ACS) and homogenized for 30 s using a tissuetearer (XHF-1 High velocity tearer, Shanghai Xin-hua Electric Apparatus Factory, China). The homogenate sat overnight at room temperature and was centrifuged at 2000g for 15 min. The supernatant was decanted into another centrifuge tube and was centrifuged at 10 000g for 10 min. The frozen plasma supernatant was diluted 1:50 with 5 ml ACS and centrifuged at 10 000g for 10 min. The concentration of Evans Blue in the plasma and the tissue supernatants was measured spectrophotometrically from the light absorbance at 625 nm with a Beckman DU-650 spectrophotometer (USA) using glass cuvettes with light path lengths of 10 mm. The ACS mixture served as the blank. To determine plasma extravasation induced by BmK venom, the Evans Blue content in the tissue was normalized to paw weight. The data was expressed as the volume of exudate (ml exudate/g tissue). It was calculated by the formula, V F £ Atissue = 50 £ Aplasma ; where A is the absorbance at 625 nm, F is the total volume of ACS in homogenized tissue. The dilution of the plasma by 1:50 for Evans Blue measurement was accounted for by multiplying the Aplasma by 50 in the calculation of the exudate (V). 2.2. Determination of rat's paw withdrawal latency Paw withdrawal latency (PWL) to radiant heat was examined for thermal hyperalgesia of animals according to method described by Hargreaves et al. (1988a). Rats were placed individually in clear plastic chambers on a glass plate and the temperature of the chamber was maintained at 308C. Animals were allowed to adapt for 30 min before the experiment. Radiant heat was applied to the plantar surface (RTY-1 type, Thermal Pain Test Instrument, the Fourth Military Medical University, Xi-an, China) of the hind paw until the rat lifted its paw. The time from the onset of heat application to paw lifting was considered to be the PWL. Both hindpaws were tested independently with a 10 min interval between trials. The determination of PWL
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Fig. 1. Time-course of plasma extravasation induced by BmK venom. Plasma extravasation of the ipsilateral and contralateral paws were induced with a dose of 200 mg/kg BmK venom at 10, 30 and 60 min. Ordinate: Plasma extravasation expressed as ml exudate/g paw weight. The data was shown as the mean ^ SEM of ®ve rats. There was no signi®cant difference among these three time points (P . 0.05). But there was signi®cant difference between contralateral and ipsilateral injection sites (*P , 0.05).
was performed to the nearest 0.1 s. To prevent tissue damage, the cut-off latency was set at 20.0 s. While anesthetized with ether, the rat was subcutaneously injected with BmK venom or saline under the plantar surface of either hind paw (random choice between left and right paw). The rat recovered in 1 or 2 min after anesthesia. 2.3. Data analysis Data were presented as mean ^ SEM. The difference between groups was determined by a one-way or two-way analysis of variance (ANOVA) for repeated measures, and *P , 0.05, **P , 0.01, ***P 0.005 was considered as a signi®cant difference. The software used in anglicizing data is SigmaStat 2.0 (Copyright 1992±1995, Jandel Corporation.) 3. Results BmK venom injection into the plantar surface of the rat hind paw induces edema and hyperalgesia, which was limited to the ipsilateral hind paw. Other side effects such as respiratory changes and dyskinesia (Freire-Maia et al., 1970; Knaus et al., 1994) were not apparent. 3.1. Time course of plasma extravasation induced by BmK venom BmK venom injection with a dose of 200 mg/kg rapidly produce conspicuous redness and swelling on the ipsilateral hindpaw, and there were no signi®cant changes in the contralateral hindpaw. The plasma exudate of the ipsilateral hindpaw was determined to be 106 ^ 17.4 n 5;
135 ^ 19.0 n 5 and 144 ^ 21.8 ml plasma/g tissue n 5 while the exudate of the contralateral hind paw was 21 ^ 7.7 n 5; 18 ^ 4.7 n 5; 19 ^ 4.0 ml plasma/g tissue n 5 at 10, 30 and 60 min, respectively (Fig. 1). Comparison of the increases in plasma extravasation, there was no signi®cant difference among these three time points (P . 0.05), But there was signi®cant difference between contralateral and ipsilateral injection sites (*P , 0.05). Results indicated that plasma extravasation induced by BmK venom intra-plantar injection could be achieved peak at 10 min and persisted for at least 60 min. 3.2. Plasma extravasation induced by different doses of BmK venom From the earlier experiments we chose 30 min as the optimal time-point to assess the plasma exudates at different doses. A small amount of Evans Blue was recovered from the right and left hind paws of rats that were not injected with either saline or venom. This corresponds to the baseline plasma extravasation of about 15 ^ 2.3 ml plasma/g tissue n 4: Subcutaneous injection of saline produced a small increase in plasma extravasation in the ipsilateral hindpaw, the exudates was determined as 20 ^ 2.5 ml plasma/g tissue n 4; and the exudates of the contralateral hindpaw was 13 ^ 1.6 ml plasma/g tissue n 4: The difference between exudates of the contralateral and the ipsilateral hindpaw may be induced by the mechanical stimulation of the needle. But this will not in¯uence the overall response, and in this study, we use the saline injected animals as control to reduce the systematic error induced by mechanical factor. BmK venom injection could induce a concentration-dependent increase of plasma extravasation in the ipsilateral hindpaw. Thirty minutes after BmK venom
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Fig. 2. Plasma extravasation induced by BmK venom plasma extravasation was determined 30 min after BmK venom was injected into the rat plantar region. Ordinate: plasma extravasation expressed as ml exudate/g paw weight. The data was shown as the mean ^ SEM of four rats. There was a signi®cant effect of dose of scorpion venom (***P , 0.005) between each two groups except 200 and 400 mg/kg (P . 0.05).
injection with four doses of 20, 40, 200 and 400 mg/kg, the amount of the ipsilateral paw's plasma extravasation was determined to be 44 ^ 8.5 n 4; 109 ^ 8.8 n 4; 142 ^ 13.9 n 4 and 147 ^ 14.6 ml plasma/g tissue n 4, respectively, and the contralateral paw's plasma extravasation was 15 ^ 2.8 n 4; 17 ^ 1.3 n 4; 19 ^ 5.1 n 4 and 17 ^ 3.2 ml plasma/g tissue n 4:
Compared with control animals, a signi®cant plasma extravasation of rat's ipsilateral paw increase could be induced at the dose of 20 mg/kg BmK venom (***P , 0.005) and there was a signi®cant effect of dose of scorpion venom (***P , 0.005) between the two groups except 200 and 400 mg/kg (P . 0.05). It suggested that the maximal plasma extravasation reached when dose of BmK venom
Fig. 3. Inhibitory effect of morphine on plasma extravasation induced by BmK venom. Plasma extravasation induced by BmK venom intraplantar injection at a dose of 40 mg/kg was inhibited by morphine (6 mg/kg, intra-peritoneal). Ordinate: plasma extravasation expressed as ml exudate/g tissue. The data was shown as the mean ^ SEM of ®ve rats. Asterisks indicate a signi®cant different from saline injection (**P , 0.01).
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Fig. 4. Time course for thermal hyperalgesia induced by BmK venom and modulation by morphine. Effects of BmK venom 40 mg/kg on: (a) ipsilateral PWL and (b) contralateral PWL. Abscissa: time after BmK venom injection, ordinate: paw withdrawal latency. The data was shown as the mean ^ SEM of 18 rats. Asterisks indicate a signi®cant different (**P , 0.01, ***P , 0.005).
rose to 200 mg/kg and higher concentrations of BmK venom did not produce any further plasma extravasation. (Fig. 2). However, no concentration-dependent increase of plasma extravasation was observed in the contralateral hind paw of these rats, and there is no signi®cant difference between the plasma extravasations of the contralateral paw and the base line measurements (P . 0.05) (Fig. 2). 3.3. Effects of morphine on plasma extravasation induced by BmK venom Plasma extravasation of the ipsilateral hindpaw induced by BmK venom was decreased when rats were pretreated with 6 mg/kg morphine (i.p.) as compared to the control group. The exudate was reduced by 23% as 75 ^ 8.9 ml plasma/g n 5 morphine pretreatment from 95 ^ 2.3 ml plasma/g n 5 saline pretreatment (**P , 0.01). On the other hand, it was found that morphine did not alter the amount of exudate in the contralateral hindpaw (Fig. 3) (data not shown).
group) to 5.2 ^ 0.2 s (BmK venom treated group) (***P , 0.005). While there was no signi®cant difference in the contralateral PWLs of the saline and BmK venom treated groups (16.0 ^ 0.5 and 13.9 ^ 0.4) (P . 0.05) as shown in Fig. 4. Antinociception induced by morphine was investigated. The PWLs of both hindpaws rapidly increased after intraperitoneal morphine injection. The PWLs of the ipsilateral and contralateral hindpaw were 8.7 ^ 0.3 s n 18 and 18.0 ^ 0.8 s n 18, respectively (***P , 0.005). Compared with those of the group treated with BmK venom without morphine injection, there was signi®cant difference between the ipsilateral PWLs (**P , 0.01), but no signi®cant changes in the contralateral PWLs (P . 0.05). In the control group, saline pretreatment could not prolong detectable PWL in rat (P . 0.05) (Fig. 4). This indicates that morphine could partially inhibit the decrease in the PWL induced by BmK venom.
4. Discussion 3.4. Thermal hyperalgesia induced by BmK venom and modulation of morphine The concentration-dependent increase in plasma extravasation was paralleled in behavior pain score, but not in contralateral hind paw of these rats. Redness and swelling were noticeable in the rat's ipsilateral hindpaw after BmK venom injection. A signi®cant reduction in the response of PWL to heat also indicated the development of cutaneous hyperalgesia, which lasted for more than 1 h. Unilateral BmK venom injection was found to have no effect on PWL in the contralateral paw. PWL was decreased from 15.0 ^ 0.5 s (saline injection
Scorpion venom contains a vast number of substances with different bioactivities. However, it seems to be still obscure as to what component induces in¯ammation. Leme et al. (1978) found that the venom of the scorpion Tityus serrulatus, and its active principle tityustoxin (TsTX), applied to the peripheral cut end of the sciatic or saphenous nerve of the rat, induced in¯ammatory reactions in the areas. The reactions were attributed to the release of neurogenic permeability factors by tityustoxin. Nowadays, it is well known that neurogenic in¯ammation is mediated via sensory peptides released from the peripheral terminal of sensory nerves. The local release of pro-in¯ammatory
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neuropeptides such as substance P and calcitonin generelated peptide (CGRP) in the periphery have been associated with the development of neurogenic in¯ammation (Helyse et al., 1997; Holzer, 1998). In this study, BmK venom was found to cause a concentration-dependent increase in plasma extravasation in rat ipsilateral hind paw at doses from 20 to 200 mg/kg. Rat plasma extravasation reached a maximum at a dose of 200 mg/kg and lasted from 10 to 60 min. It was also noted that 20 mg/kg BmK venom could induce a signi®cant reduction in the PWL to heat in the ipsilateral hindpaw, but the PWL of the contralateral hindpaw did not change signi®cantly. In addition, pretreatment with morphine could inhibit rat plasma extravasation and decrease PWL to heat induced by BmK venom. These results suggest that BmK venom can induce peripheral in¯ammation in rats following subcutaneous injection. It has been demonstrated that carragenan-induced rat paw swelling and plasma extravasation can be inhibited in a dose-dependent manner by m- and k-opioid receptor agonists given systemically, and partially antagonized by opioid antagonists (Hargreaves et al., 1988b). In addition, morphine can attenuate the in¯ammation of arthritis when administered either systemically or centrally (Walker et al., 1996; Levine et al., 1985). It clearly indicates that morphine is a potent antagonist of peripheral in¯ammation. Thus, action could be due to several possible effects of opioids: (a) primary afferent neurons contain opioid receptor (Coggeshall et al., 1997) which appear to be physiologically active, since opiates administered into in¯amed tissue, suppress the release of substance P (Yaksh, 1988; Yonehara et al., 1988), reduce spontaneous ®ring of small-diameter afferents (Russell et al., 1987) and produce analgesia (Joris et al., 1987; Stein et al., 1988); (b) peripheral in¯ammation may be related to the presence of opioid binding sites on immune cells (Bryant et al., 1987); (c) opioids can exert anti-in¯ammatory actions including inhibition of calciumdependent release of pro-in¯ammatory peptides such as CGRP from peripheral nerve endings (Li et al., 1998; Malcangio and Bowery, 1996; Taddese et al., 1995); (d) opioids can inhibit local blood ¯ow which has been shown to depress formalin-induced plasma extravasation (Taylor et al., 2000). The results obtained in this study show that morphine can inhibit plasma extravasation induced by BmK venom possible via one or many of the mechanisms mentioned earlier. Histamine, 5-HT, histamine-releasing factor and hyaluronidase are considered to be partly involved in the pathophysiological changes induced by scorpion venom (Basu et al., 1990). It had been demonstrated that T. serrulatus crude venom injection could induce a signi®cant release of in¯ammatory mediators in rats such as bradykinin, platelet activating factor, prostaglandins, leukotrienes, amines, purines, cytokines and chemokines (Amaral and Rezende, 1997). Even several long-chain BmK neurotoxic polypeptides, which interact with sodium channels in excitable
membranes, and few short-chain BmK peptides, which were deemed to be potassium channel-selective ligands have been demonstrated extensively (Ji et al., 1994a,b, 1996, 1999a,b; Jia et al., 1999, 2000; Li et al., 2000; Tong et al., 2000). Therefore, it cannot be excluded that rat plasma extravasation induced by BmK venom might operate through a pathway of modulating K 1 channel activity in vascular smooth muscle cells, which resulted to alter the vascular tone and permeability. The neurotoxins or substances in the venom play an important role in the in¯ammatory response requires further investigation. In addition, peripheral in¯ammation induced by BmK venom subcutaneous injection, might prove a valuable animal model for investigating pathophysiological and molecular mechanisms of a number of in¯ammatory diseases, and identifying potential anti-in¯ammatory and analgesic drugs. Acknowledgements This study was supported by National Basic Research Program of China (1999054001), National Nature Sciences Foundation of China (39625010), and partially by Chinese Academy of Sciences. References Amaral, C.F.S., Rezende, N.A., 1997. Both cardiogenic and non-cardiogenic factors are involved in the patholgensis of pulmonary edema after scorpion envenoming. Toxicon 35, 997±998. Andrew, D., Greenspan, J.D., 1999. Mechanical and heat sensitization of cutaneous nociceptors after peripheral in¯ammation in the rat. J. Neurophysiol. 82, 2649±2656. Balozet, L., 1971. Scorpionism in the old world. In: Bucherl, W., Buckley, E.E. (Eds.). Venomous animal and their venoms, vol. 3. Academic Press, New York, pp. 349±371. Basu, A., Gomes, A., Dasgupta, S.C., Lahiri, S.C., 1990. Histamine, 5-HT and hyaluronidase in the venom of scorpion laevifrons (Pock). Indian J. Med. Res. 92, 371±373. Bryant, H.U., Bernton, E.W., Holaday, J.W., 1987. Immunosuppressive effects of chronic morphine treatment in mice. Life Sci. 41, 1731±1738. Coggeshall, R.E., Zhou, S., Carlton, S.M., 1997. Opioid receptors on peripheral sensory axons. Brain Res. 764, 126±132. Deshpande, S.B., Bagchi, S., Rain, O.P., Aryya, N.C., 1999. Pulmonary oedema produced by scorpion venom augments a phenyldiguanide-induced re¯ex response in anaesthetized rats. J. Physiol. 521, 537±544 pt 2. Freire-Maia, L., Ribeiro, R.M., Beraldo, W.T., 1970. Effects of puri®ed scorpion toxin on respiratory movements in the rat. Toxicon 8, 307±310. Hargreaves, K.M., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77±88. Hargreaves, K.M., Dubner, R., Joris, J.L., 1988. Peripheral actions of opiates in the blockade of carrageenan-induced in¯ammation. In: Dubner, R., Gebhart, G.F., Bond, M.R. (Eds.). Proceedings
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Asian scorpion BmK venom induces plasma extravasation and thermal hyperalgesia in the rat Bing Chen 1, Xiaopan Zhuo 1, Congying Wang, Yonghua Ji* Institute of Physiology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China Received 4 April 2001; accepted 4 October 2001
Abstract In the present study, the effects of scorpion Buthus martensi Karsch (BmK) venom on plasma extravasation and paw withdrawal latency (PWL) to radiant heat have been investigated in rats. BmK venom (20±200 mg/kg) by subcutaneous injection under the surface of the rat hindpaw causes dose-dependant increased plasma extravasation that could be partially inhibited by intraperitoneal (i.p.) injected morphine (6 mg/kg). Peak plasma extravasation was reached at 10 min and persisted for 60 min at a dose of 200 mg/kg. BmK venom induced cutaneous hyperalgesia as indicated by decreased PWL to radiant heat in the ipsilateral paw following subcutaneous injection of 20 mg/kg BmK venom without effect on PWL of the contralateral hindpaw. Meanwhile, it was found that i.p. morphine injection could inhibit this decreased ipsilateral PWL. The results thus suggest that BmK venom could induce peripheral in¯ammation in rat by subcutaneous injection, and may prove a valuable animal model for investigating the pathophysiology of a number of in¯ammatory diseases and identifying potential antiin¯ammatory and analgesic drugs. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Scorpion Buthus martensi Karsch venom; Plasma extravasion; Peripheral in¯ammation; Morphine; Paw withdrawal latency; Thermal hyperalgesia
1. Introduction The Asian scorpion Buthus martensi Karsch is a species belonging to the Buthidae family, widely distributed from northwestern China to Mongolia and Korea. Although this species is not dangerously venomous for mammals, stings from scorpions are followed by severe pain, swelling, edema, burning sensation and redness (Balozet, 1971). These symptoms may last for several hours. Several bioactive agents like histamine and 5-HT have been identi®ed in the venom of other scorpion species (Basu et al., 1990), which are partly responsible for the immediate in¯ammatory pathophysiological changes. In¯ammation is often associated with either tissue damage or nerve injury, and considered to be a critical reaction to irritation and infection, and is characterized by * Corresponding author. Tel.: 186-21-6474-8057; fax: 186-216433-2445. E-mail address: [email protected] (Y. Ji). 1 Both authors contribute equally in this study.
redness, heat, swelling, loss of function and primary hyperalgesia (Levine and Reichling, 1999). Redness and heat are the results of increase in blood ¯ow; swelling is caused by an increase in vascular permeability. The changes result from the concerted action of a number of local mediators. Increased vascular permeability leads to the site of tissue injury being invaded by a host of immune cells, which in turn stimulate the release of other active substances. Associated with the series of events is hyperalgesia, due to the stimulation of nociceptors, the sensory neurons convey `painful' afferent information to the spinal cord. Primary hyperalgesia is characterized by a lowering of response threshold and/or an increased response to suprathreshold stimulus at the site of injury. Meanwhile, primary hyperalgesia can result in sensitization of primary nociceptors to mechanical and thermal stimulus at the in¯ammation site. The pain is due to increasing activation, and sensitization of primary afferent nerve ®bers (Andrew and Greenspan, 1999). The process of in¯ammation and hyperalgesia may lead to the removal of injured tissue and repair of the site. All these sensitization reactions ordinarily
0041-0101/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00248-3
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depend on the release, synthesis or attraction of in¯ammatory mediators to the site of injury. Although it has been identi®ed that scorpion stings can induce edema (Deshpande et al., 1999), any detailed and quantitative data analysis on edema responses induced by scorpion venom has not been attempted till date. In the present study, we determined plasma extravasation and paw withdrawal latency (PWL) to radiant heat of rats injected with scorpion BmK venom, and also determined the inhibitory effect of morphine on the ensuring in¯ammatory responses. Possible mechanisms are discussed. 2. Materials and methods Adult male Sprague±Dawley rats (Shanghai experimental animals center, Shanghai, China) weighing 220± 250 g were used. The animals were individually housed in cages with free access to water and food ad libition, and maintained in a room temperature of 21±238C with a 12 h light/dark cycle. Each animal was tested only once. Guidelines on ethical standards for investigation of experimental pain conscious animals were performed strictly according to principles described by Zimmermann (1983), and every effort was made to minimize animal suffering. Crude venom of scorpion B. martensi Karsch (BmK), collected by electrical stimulation, was purchased from an individual scorpion culture farm in Zhengzhou, Henan Province, China. Rats were classi®ed into three groups for extravasation measurement. The ®rst group was used to determine the time course of plasma extravasation of 200 mg/kg BmK venom (dissolved in 50 ml saline) following plantar injection. The second group of rats were used to determine the amount of plasma extravasation at different doses: 20, 40, 200, 400 mg/kg BmK venom (dissolved in 50 ml saline) under the surface of one hind paw. Control animals received an equivalent volume of saline. The third group of rats were used for the investigation of morphine's effect (10 mg/ml morphine hydrochloride, Shenyang First Pharmaceutical Factory, China) on plasma extravasation induced by BmK venom. Animals were pretreated with 6 mg/kg morphine (i.p.) before 40 mg/kg BmK venom injection. Control rats were pretreated with saline (i.p.) instead of morphine. Another four group of rats were employed to measure PWL to radiant heat. The ®rst group of rats were injected with 20 mg/kg BmK venom n 18: The second control group of rats were injected with 50 ml saline n 18: The third group of rats received 6 mg/kg morphine (i.p.) 10 min before 20 mg/kg BmK venom injection n 18: The fourth group of rats were pretreated with an equivalent volume of saline instead of morphine 10 min before BmK venom injection n 18: 2.1. Determination of rat plasma extravasation Plasma extravasation was measured according to
Humphrey (1993) and John et al. (1998). The rats were intravenously injected with 40 mg/kg Evans Blue while anesthetized with 30 mg/kg sodium pentobarbital (i.p.), 0, 30 or 50 min, later the anesthetized animals were injected with 50 ml BmK venom or saline. One hour after Evans Blue injection, the rats were cardiac punctured, a 1 ml blood sample was obtained and the animals were then intracardially perfused with 300 ml of 0.9% saline. The blood sample was centrifuged at 3000 rpm for 5 min, and the plasma supernatant was kept at 2208C prior to determination of the Evans Blue concentration. The contralateral and ipsilateral hindpaws were removed at the ankle joint for determination of plasma extravasation induced by BmK venom. The hindpaws were frozen at 2208C for several hours. The tissue was then weighed, minced, placed into a 10 ml mixture of acetone and 0.5% sodium sulfate (7:3, v/v; ACS) and homogenized for 30 s using a tissuetearer (XHF-1 High velocity tearer, Shanghai Xin-hua Electric Apparatus Factory, China). The homogenate sat overnight at room temperature and was centrifuged at 2000g for 15 min. The supernatant was decanted into another centrifuge tube and was centrifuged at 10 000g for 10 min. The frozen plasma supernatant was diluted 1:50 with 5 ml ACS and centrifuged at 10 000g for 10 min. The concentration of Evans Blue in the plasma and the tissue supernatants was measured spectrophotometrically from the light absorbance at 625 nm with a Beckman DU-650 spectrophotometer (USA) using glass cuvettes with light path lengths of 10 mm. The ACS mixture served as the blank. To determine plasma extravasation induced by BmK venom, the Evans Blue content in the tissue was normalized to paw weight. The data was expressed as the volume of exudate (ml exudate/g tissue). It was calculated by the formula, V F £ Atissue = 50 £ Aplasma ; where A is the absorbance at 625 nm, F is the total volume of ACS in homogenized tissue. The dilution of the plasma by 1:50 for Evans Blue measurement was accounted for by multiplying the Aplasma by 50 in the calculation of the exudate (V). 2.2. Determination of rat's paw withdrawal latency Paw withdrawal latency (PWL) to radiant heat was examined for thermal hyperalgesia of animals according to method described by Hargreaves et al. (1988a). Rats were placed individually in clear plastic chambers on a glass plate and the temperature of the chamber was maintained at 308C. Animals were allowed to adapt for 30 min before the experiment. Radiant heat was applied to the plantar surface (RTY-1 type, Thermal Pain Test Instrument, the Fourth Military Medical University, Xi-an, China) of the hind paw until the rat lifted its paw. The time from the onset of heat application to paw lifting was considered to be the PWL. Both hindpaws were tested independently with a 10 min interval between trials. The determination of PWL
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Fig. 1. Time-course of plasma extravasation induced by BmK venom. Plasma extravasation of the ipsilateral and contralateral paws were induced with a dose of 200 mg/kg BmK venom at 10, 30 and 60 min. Ordinate: Plasma extravasation expressed as ml exudate/g paw weight. The data was shown as the mean ^ SEM of ®ve rats. There was no signi®cant difference among these three time points (P . 0.05). But there was signi®cant difference between contralateral and ipsilateral injection sites (*P , 0.05).
was performed to the nearest 0.1 s. To prevent tissue damage, the cut-off latency was set at 20.0 s. While anesthetized with ether, the rat was subcutaneously injected with BmK venom or saline under the plantar surface of either hind paw (random choice between left and right paw). The rat recovered in 1 or 2 min after anesthesia. 2.3. Data analysis Data were presented as mean ^ SEM. The difference between groups was determined by a one-way or two-way analysis of variance (ANOVA) for repeated measures, and *P , 0.05, **P , 0.01, ***P 0.005 was considered as a signi®cant difference. The software used in anglicizing data is SigmaStat 2.0 (Copyright 1992±1995, Jandel Corporation.) 3. Results BmK venom injection into the plantar surface of the rat hind paw induces edema and hyperalgesia, which was limited to the ipsilateral hind paw. Other side effects such as respiratory changes and dyskinesia (Freire-Maia et al., 1970; Knaus et al., 1994) were not apparent. 3.1. Time course of plasma extravasation induced by BmK venom BmK venom injection with a dose of 200 mg/kg rapidly produce conspicuous redness and swelling on the ipsilateral hindpaw, and there were no signi®cant changes in the contralateral hindpaw. The plasma exudate of the ipsilateral hindpaw was determined to be 106 ^ 17.4 n 5;
135 ^ 19.0 n 5 and 144 ^ 21.8 ml plasma/g tissue n 5 while the exudate of the contralateral hind paw was 21 ^ 7.7 n 5; 18 ^ 4.7 n 5; 19 ^ 4.0 ml plasma/g tissue n 5 at 10, 30 and 60 min, respectively (Fig. 1). Comparison of the increases in plasma extravasation, there was no signi®cant difference among these three time points (P . 0.05), But there was signi®cant difference between contralateral and ipsilateral injection sites (*P , 0.05). Results indicated that plasma extravasation induced by BmK venom intra-plantar injection could be achieved peak at 10 min and persisted for at least 60 min. 3.2. Plasma extravasation induced by different doses of BmK venom From the earlier experiments we chose 30 min as the optimal time-point to assess the plasma exudates at different doses. A small amount of Evans Blue was recovered from the right and left hind paws of rats that were not injected with either saline or venom. This corresponds to the baseline plasma extravasation of about 15 ^ 2.3 ml plasma/g tissue n 4: Subcutaneous injection of saline produced a small increase in plasma extravasation in the ipsilateral hindpaw, the exudates was determined as 20 ^ 2.5 ml plasma/g tissue n 4; and the exudates of the contralateral hindpaw was 13 ^ 1.6 ml plasma/g tissue n 4: The difference between exudates of the contralateral and the ipsilateral hindpaw may be induced by the mechanical stimulation of the needle. But this will not in¯uence the overall response, and in this study, we use the saline injected animals as control to reduce the systematic error induced by mechanical factor. BmK venom injection could induce a concentration-dependent increase of plasma extravasation in the ipsilateral hindpaw. Thirty minutes after BmK venom
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Fig. 2. Plasma extravasation induced by BmK venom plasma extravasation was determined 30 min after BmK venom was injected into the rat plantar region. Ordinate: plasma extravasation expressed as ml exudate/g paw weight. The data was shown as the mean ^ SEM of four rats. There was a signi®cant effect of dose of scorpion venom (***P , 0.005) between each two groups except 200 and 400 mg/kg (P . 0.05).
injection with four doses of 20, 40, 200 and 400 mg/kg, the amount of the ipsilateral paw's plasma extravasation was determined to be 44 ^ 8.5 n 4; 109 ^ 8.8 n 4; 142 ^ 13.9 n 4 and 147 ^ 14.6 ml plasma/g tissue n 4, respectively, and the contralateral paw's plasma extravasation was 15 ^ 2.8 n 4; 17 ^ 1.3 n 4; 19 ^ 5.1 n 4 and 17 ^ 3.2 ml plasma/g tissue n 4:
Compared with control animals, a signi®cant plasma extravasation of rat's ipsilateral paw increase could be induced at the dose of 20 mg/kg BmK venom (***P , 0.005) and there was a signi®cant effect of dose of scorpion venom (***P , 0.005) between the two groups except 200 and 400 mg/kg (P . 0.05). It suggested that the maximal plasma extravasation reached when dose of BmK venom
Fig. 3. Inhibitory effect of morphine on plasma extravasation induced by BmK venom. Plasma extravasation induced by BmK venom intraplantar injection at a dose of 40 mg/kg was inhibited by morphine (6 mg/kg, intra-peritoneal). Ordinate: plasma extravasation expressed as ml exudate/g tissue. The data was shown as the mean ^ SEM of ®ve rats. Asterisks indicate a signi®cant different from saline injection (**P , 0.01).
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Fig. 4. Time course for thermal hyperalgesia induced by BmK venom and modulation by morphine. Effects of BmK venom 40 mg/kg on: (a) ipsilateral PWL and (b) contralateral PWL. Abscissa: time after BmK venom injection, ordinate: paw withdrawal latency. The data was shown as the mean ^ SEM of 18 rats. Asterisks indicate a signi®cant different (**P , 0.01, ***P , 0.005).
rose to 200 mg/kg and higher concentrations of BmK venom did not produce any further plasma extravasation. (Fig. 2). However, no concentration-dependent increase of plasma extravasation was observed in the contralateral hind paw of these rats, and there is no signi®cant difference between the plasma extravasations of the contralateral paw and the base line measurements (P . 0.05) (Fig. 2). 3.3. Effects of morphine on plasma extravasation induced by BmK venom Plasma extravasation of the ipsilateral hindpaw induced by BmK venom was decreased when rats were pretreated with 6 mg/kg morphine (i.p.) as compared to the control group. The exudate was reduced by 23% as 75 ^ 8.9 ml plasma/g n 5 morphine pretreatment from 95 ^ 2.3 ml plasma/g n 5 saline pretreatment (**P , 0.01). On the other hand, it was found that morphine did not alter the amount of exudate in the contralateral hindpaw (Fig. 3) (data not shown).
group) to 5.2 ^ 0.2 s (BmK venom treated group) (***P , 0.005). While there was no signi®cant difference in the contralateral PWLs of the saline and BmK venom treated groups (16.0 ^ 0.5 and 13.9 ^ 0.4) (P . 0.05) as shown in Fig. 4. Antinociception induced by morphine was investigated. The PWLs of both hindpaws rapidly increased after intraperitoneal morphine injection. The PWLs of the ipsilateral and contralateral hindpaw were 8.7 ^ 0.3 s n 18 and 18.0 ^ 0.8 s n 18, respectively (***P , 0.005). Compared with those of the group treated with BmK venom without morphine injection, there was signi®cant difference between the ipsilateral PWLs (**P , 0.01), but no signi®cant changes in the contralateral PWLs (P . 0.05). In the control group, saline pretreatment could not prolong detectable PWL in rat (P . 0.05) (Fig. 4). This indicates that morphine could partially inhibit the decrease in the PWL induced by BmK venom.
4. Discussion 3.4. Thermal hyperalgesia induced by BmK venom and modulation of morphine The concentration-dependent increase in plasma extravasation was paralleled in behavior pain score, but not in contralateral hind paw of these rats. Redness and swelling were noticeable in the rat's ipsilateral hindpaw after BmK venom injection. A signi®cant reduction in the response of PWL to heat also indicated the development of cutaneous hyperalgesia, which lasted for more than 1 h. Unilateral BmK venom injection was found to have no effect on PWL in the contralateral paw. PWL was decreased from 15.0 ^ 0.5 s (saline injection
Scorpion venom contains a vast number of substances with different bioactivities. However, it seems to be still obscure as to what component induces in¯ammation. Leme et al. (1978) found that the venom of the scorpion Tityus serrulatus, and its active principle tityustoxin (TsTX), applied to the peripheral cut end of the sciatic or saphenous nerve of the rat, induced in¯ammatory reactions in the areas. The reactions were attributed to the release of neurogenic permeability factors by tityustoxin. Nowadays, it is well known that neurogenic in¯ammation is mediated via sensory peptides released from the peripheral terminal of sensory nerves. The local release of pro-in¯ammatory
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neuropeptides such as substance P and calcitonin generelated peptide (CGRP) in the periphery have been associated with the development of neurogenic in¯ammation (Helyse et al., 1997; Holzer, 1998). In this study, BmK venom was found to cause a concentration-dependent increase in plasma extravasation in rat ipsilateral hind paw at doses from 20 to 200 mg/kg. Rat plasma extravasation reached a maximum at a dose of 200 mg/kg and lasted from 10 to 60 min. It was also noted that 20 mg/kg BmK venom could induce a signi®cant reduction in the PWL to heat in the ipsilateral hindpaw, but the PWL of the contralateral hindpaw did not change signi®cantly. In addition, pretreatment with morphine could inhibit rat plasma extravasation and decrease PWL to heat induced by BmK venom. These results suggest that BmK venom can induce peripheral in¯ammation in rats following subcutaneous injection. It has been demonstrated that carragenan-induced rat paw swelling and plasma extravasation can be inhibited in a dose-dependent manner by m- and k-opioid receptor agonists given systemically, and partially antagonized by opioid antagonists (Hargreaves et al., 1988b). In addition, morphine can attenuate the in¯ammation of arthritis when administered either systemically or centrally (Walker et al., 1996; Levine et al., 1985). It clearly indicates that morphine is a potent antagonist of peripheral in¯ammation. Thus, action could be due to several possible effects of opioids: (a) primary afferent neurons contain opioid receptor (Coggeshall et al., 1997) which appear to be physiologically active, since opiates administered into in¯amed tissue, suppress the release of substance P (Yaksh, 1988; Yonehara et al., 1988), reduce spontaneous ®ring of small-diameter afferents (Russell et al., 1987) and produce analgesia (Joris et al., 1987; Stein et al., 1988); (b) peripheral in¯ammation may be related to the presence of opioid binding sites on immune cells (Bryant et al., 1987); (c) opioids can exert anti-in¯ammatory actions including inhibition of calciumdependent release of pro-in¯ammatory peptides such as CGRP from peripheral nerve endings (Li et al., 1998; Malcangio and Bowery, 1996; Taddese et al., 1995); (d) opioids can inhibit local blood ¯ow which has been shown to depress formalin-induced plasma extravasation (Taylor et al., 2000). The results obtained in this study show that morphine can inhibit plasma extravasation induced by BmK venom possible via one or many of the mechanisms mentioned earlier. Histamine, 5-HT, histamine-releasing factor and hyaluronidase are considered to be partly involved in the pathophysiological changes induced by scorpion venom (Basu et al., 1990). It had been demonstrated that T. serrulatus crude venom injection could induce a signi®cant release of in¯ammatory mediators in rats such as bradykinin, platelet activating factor, prostaglandins, leukotrienes, amines, purines, cytokines and chemokines (Amaral and Rezende, 1997). Even several long-chain BmK neurotoxic polypeptides, which interact with sodium channels in excitable
membranes, and few short-chain BmK peptides, which were deemed to be potassium channel-selective ligands have been demonstrated extensively (Ji et al., 1994a,b, 1996, 1999a,b; Jia et al., 1999, 2000; Li et al., 2000; Tong et al., 2000). Therefore, it cannot be excluded that rat plasma extravasation induced by BmK venom might operate through a pathway of modulating K 1 channel activity in vascular smooth muscle cells, which resulted to alter the vascular tone and permeability. The neurotoxins or substances in the venom play an important role in the in¯ammatory response requires further investigation. In addition, peripheral in¯ammation induced by BmK venom subcutaneous injection, might prove a valuable animal model for investigating pathophysiological and molecular mechanisms of a number of in¯ammatory diseases, and identifying potential anti-in¯ammatory and analgesic drugs. Acknowledgements This study was supported by National Basic Research Program of China (1999054001), National Nature Sciences Foundation of China (39625010), and partially by Chinese Academy of Sciences. References Amaral, C.F.S., Rezende, N.A., 1997. Both cardiogenic and non-cardiogenic factors are involved in the patholgensis of pulmonary edema after scorpion envenoming. Toxicon 35, 997±998. Andrew, D., Greenspan, J.D., 1999. Mechanical and heat sensitization of cutaneous nociceptors after peripheral in¯ammation in the rat. J. Neurophysiol. 82, 2649±2656. Balozet, L., 1971. Scorpionism in the old world. In: Bucherl, W., Buckley, E.E. (Eds.). Venomous animal and their venoms, vol. 3. Academic Press, New York, pp. 349±371. Basu, A., Gomes, A., Dasgupta, S.C., Lahiri, S.C., 1990. Histamine, 5-HT and hyaluronidase in the venom of scorpion laevifrons (Pock). Indian J. Med. Res. 92, 371±373. Bryant, H.U., Bernton, E.W., Holaday, J.W., 1987. Immunosuppressive effects of chronic morphine treatment in mice. Life Sci. 41, 1731±1738. Coggeshall, R.E., Zhou, S., Carlton, S.M., 1997. Opioid receptors on peripheral sensory axons. Brain Res. 764, 126±132. Deshpande, S.B., Bagchi, S., Rain, O.P., Aryya, N.C., 1999. Pulmonary oedema produced by scorpion venom augments a phenyldiguanide-induced re¯ex response in anaesthetized rats. J. Physiol. 521, 537±544 pt 2. Freire-Maia, L., Ribeiro, R.M., Beraldo, W.T., 1970. Effects of puri®ed scorpion toxin on respiratory movements in the rat. Toxicon 8, 307±310. Hargreaves, K.M., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77±88. Hargreaves, K.M., Dubner, R., Joris, J.L., 1988. Peripheral actions of opiates in the blockade of carrageenan-induced in¯ammation. In: Dubner, R., Gebhart, G.F., Bond, M.R. (Eds.). Proceedings
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