Local Haemorrhage Induced by Bothrops Jararaca Venom

Research Paper

Mediators of Inflammation, 9, 101–107 (2000)

WE in vestigated m orphological alterations in duced Local haemorrhage induced by by s.c. in jection of 2.5 m g of Both ro p s jararac a venom in rats. In tense disorganisation of collagen fibres w as Bothrops jararaca venom: observed 1 m in after the venom in jection, particularly at regions near vessels and nerves. Mast cells were relationship to neurogenic degranulated, and erythrocytes w ere seen leavin g inflammation venules throughout the endothelial junctions. At this tim e, dam aged endothelial cells were not observed. In rats envenom ed as above, but im m ediately after 1,CA 2 cardiorespiratory failure induced by deep ether Luis Roberto C. Gonçalves and Mario Mariano anaesthesia, alterations in the connective tissue struc- tures, as previously described, were not observed. Th e m ediation of this haem orrhage w as investigated 1Laboratorio de Fisiopatologia, Instituto Butantan, by injecting the venom in to the foot pad of m ice and Av. Vital Brazil, 1500, 05503–900; and 2Disciplina de com pared to th e m ediation of oedem a. Local haem or- Imunologia, Universidade Federal de Sao˜ Paulo, rhage w as significantly reduced in m ice pre-treated w ith capsaicin or guaneth idine or subm itted to a Escola Paulista de Medicina, Sao˜ Paulo-SP, Brazil surgical section of sciatic and saphenous nerves. In these anim als, oedem a w as not affected. Groups treated w ith m ethysergide or m orphin e showed both CA Corresponding Author haem orrhage and oedem a significantly reduced. Tel: 55 11 813–7222 ex t. 2164 In dom ethacin or dex am ethasone pre-treatm ents sig- Fax: 55 11 813–7222 ext. 2162 nificantly reduced the oedem a, but not the haem orrhage. Moreover, in anim als treated w ith prom- Email: [email protected] ethazin e or m epyram ine, oedem a and haem orrhage w ere not affected. Th ese data suggest that local haem orrhage induced by Both ro p s jararaca venom is partially controlled by serotonin and neurohu- m oral m ediators. Furtherm ore, results in dicate that haem orrhage and oedem a are m ediated by different pharm acological system s.

Key w ords: Snake venom, Bo th ro ps ja ra raca , Local haemorrhage, Oedema, Neurogenic inflammation

Introduction meters of the inflammatory response 25 – 29 induced by viperid venoms. Nevertheless, all this information is Haemorrhage and oedema, besides blood coagulation not sufficient to draw a comprehensive mechanism to disturbances, are the main signs of envenoming by explain the local lesions observed in this type of Bo thro ps snake venoms.1,2 Despite serum therapy envenoming. being an effective treatment for the control of In the present study it is show n that oedema and systemic symptoms in this type of envenoming, it haemorrhage induced by B. ja ra ra ca venom have does not control the oedema and local haemorrhagic distinct pharmacological mediation and our results and necrotic lesions w hich develop at the site of suggest that haemorrhage is partially controlled by venom inoculation.1,3 – 5 serotonin and neurogenic mediators. Among the toxins responsible for these local effects, haemorrhagic factors w ere isolated from various viperid venoms.6 Even though some of these haemorrhagic factors are devoid of proteolytic activ- Methods itie s,7–11 the y are characterised as metalloprotei- Venoms nases.6 Some of these factors digest proteins from the extracellular matrix, and this property has been A pool of lyophilised venom obtained from adult related to the pathogeny of haemorrhagic specimens of B. ja ra ra ca snakes, supplied by the lesions.6,1 2 –17 Laboratory of Herpetology, Instituto Butantan, w as On the other hand, efforts have also been made to used throughout this investigation. The venom was characterise the pharmacological mediators involved kept at –20°C, and venom solutions w ere prepared in oedema formation,18 – 23 pain24 and other para- (w /v ) w ith sterile saline just before use.

Animals haemoglobin; 44= 44 mmol–1 /cm= molecular absorp- tion of haemoglobin; d=1= cuvette thickness; 1000= Male Wistar rats (220–250 g ) or male Sw iss mice converting factor (litre to millilitre ); 4= volume of von (18–22 g ) w ere used. The animals w ere maintained Kampen–Zijlstra reagent. w ith w ater and food a d libitum in appropriate In order to eliminate the influence of oedema, the environment conditions and used under ethical condi- concentration of haemoglobin obtained in the paw tions, according to guidelines of the International injected w ith venom w as divided by the w eight of the Society of Toxinology.30 paw injected w ith saline. The difference between haemoglobin content of the paw injected w ith venom Morphology of venom-induced lesions and of that injected w ith saline w as the estimated haemorrhage, expressed as mg Hb /g of tissue. The Rats, maintained under ether anaesthesia, w ere injec- minimal haemorrhagic dose (MHD) w as defined as ted into the subcutaneous tissue of the scrotal bag the minimal concentration of venom able to induce w ith 2.5 m g of venom (50 m l) in saline. Control rats an increase of three times the haemoglobin concen- received the same volume of saline. One minute after tration in relation to that of a control tissue. Finally, the injection, animals w ere killed and samples of oedema, expressed in mg, w as evaluated by the tissue from injected areas w ere obtained. In order to difference in w eight betw een one paw injected w ith evaluate the role played by blood circulation in the venom and the other injected w ith saline.32 evolution of the venom-induced lesions, the same procedure as above w as performed in rats w hose heartbeats had just stopped due to prolonged ether anaesthesia. Tissue samples from the sites of venom Treatment of animals injection were collected 1 min after the injection and Groups of 6 to 10 mice w ere injected s.c. into the fix ed in 2.5% buffered glutaraldehyde. Routine double foot pad w ith 5 MHDs of the venom (5 m g in 50 m l) fixation, embedding in Araldite and staining w ith after the follow ing treatments: (1) dexamethasone uranyl acetate and lead citrate w ere employed. The (corticosteroid, phospholipase A2 inhibitor: Deca- processed samples w ere analysed in a Phillips (EM– dron®, Promade, Brazil), 1 and 0.4 mg/kg, i.p., 24 201) electron microscope. and 1 h before the venom; (2) indomethacin (inhibitor of the prostaglandin-forming cyclooxigenase: Sigma), 30 mg/kg, s.c., 30 min before the venom; (3) Evaluation of haemorrhage and oedema methysergide (5HT receptor antagonist: Sigma), Local haemorrhage and oedema w ere simultaneously 0.8 mg/kg, s.c., 30 min before the venom; (4) prom- evaluated using a modification of the methods descri- ethazine (H1 receptor antagonist: Rhodia Pharma, bed by Ow nby et al.31 and by Yamakaw a et al.32 Mice Brazil), 10 mg/kg, i.p., 30 min before the venom; (5) w ere injected s.c. in the right foot pad w ith 50 m l of mepyramine (H1 receptor antagonist: Sigma), 5 mg/ the venom solution. The contra-lateral foot pad kg, i.p., 30 min before the venom; (6) morphine received the same volume of sterile saline. Three (opioid: Merck), 30 mg/kg, s.c., 30 min before the hours later (time determined by a time-course experi- venom; (7) guanethidine (peripheral post-ganglionic ment for haemorrhage and oedema), the animals w ere adrenergic neurone inhibitor; sympathetic blockade: sacrificed in an ether chamber. The paw s w ere Ciba Geigy), three doses of 30 mg/kg, s.c., w ith 24 h removed at the level of the tibio-tarsic junction, intervals, before the venom injection; (8) chronic w eighted, fragmented and put in tubes containing denervation: mice w ere anaesthetised w ith a Keta- 4 ml of von Kampen–Zijlstra reagent modified by lar® (Park–Davis ) – Rumpum® (Bayer) solution 33 Matsubara et al. (200 mg K3[Fe(CN)6], 50 mg KCN, (1:2 v /v, diluted 1:4 v/v in saline ), i.p., 0.1 ml/10 g 120 mg KH2PO4 , 50 mg NaCl and 1 ml non-ionic body w eight, and the sciatic and saphenous nerves detergent per litre ). The tissue w as homogenised and of the right leg w ere surgically sectioned and the the tubes were centrifuged at 3000 g for 30 min. The venom injected into the right foot pad 7 days later; supernatant w as filtered in Millipore membranes (9) capsaicin (substance P releaser from sensory (0.8 m pores ), and the absorbance at 540 nm w as afferent neurones: Merck), 50 mg/kg, s.c., on the determined in a spectrophotometer (Micronal B382, second day after birth (in neonate animals, capsai- Brazil). cin treatment causes degeneration of sensory affer- The haemoglobin concentration w as determined as ent neurones ) (animals w ere used w hen they ach- follow s: ieved the appropriate weight); (10) respective A540 nm´ 64·458 control groups received saline by the same route. [Hb] = In the case of the chronic denervation, control 44´ d´ 1000 animals w ere sham operated, and in the case of the w here: [Hb]= haemoglobin concentration; A540nm= capsaicin, control group received the capsaicin absorbance obtained; 64·458= molecular w eight of solvent.

102 Mediators of Inflammation · Vol 9 · 2000 Bo thro ps ja ra raca veno m and loca l ha em o rrhage

FIG. 1. Electron micrographs of the connective tissue of scrotal bag of a rat 1 min after injection of 2.5 m g of B. jararaca venom. Arrows indicate: (A) disorganisation of perivascular collagen fibres (bar= 0.25 m m); (B) disorganisation of perineural collagen fibres (bar = 0.5 m m); (C) mast cell degranulation near a nerve bundle (bar= 1 m m); and (D) vascular congestion and haemorrhage (bar= 1 m m).

Statistics Time-course of haemorrhage and oedema induced by the venom Results w ere analysed by Student’s t-test and considered significantly different w hen p< 0.05. Haemorrhage induced by 1.25 m g of venom show ed a peak at the 3rd hour after injection, persisting up to 6 h. Oe dema w as maximal from 30 min up to 3 h, Results decreasing after 6 h of venom injection. As both haemorrhage and oedema w ere max imal 3 h after Morphology of the envenomed connective venom injection, this time w as selected to evaluate tissue some parameters of the pharmacological mediation of these processes. Disorganisation of collagen fibres, particularly near The haemorrhagic activity w as dose-dependent, blood vessels and nerve bundles (Fig. 1A and 1B, and the MHD obtained w as 0.95 m g. A dose of 5 m g of respectively), mast cell degranulation (Fig. 1C) and venom w as used for the study of the pharmacological intense vascular congestion (Fig. 1D) w ere observed mediation of the lesions. 1 min after venom injection into the subcutaneous tissue of the scrotal bag of rats. Morphological alterations in venular endothelial cells were not Mediation of venom-induced haemorrhage and clearly detected, although red blood cells w ere seeing oedema squeezing out through open endothelial cell junctions, characterising a per diapedesis type of haemor- With the dose of venom used, only local events w ere rhage (Fig. 2). Alterations such as disorganisation of evaluated, as it w as considered that envenomed mice collagen fibres and mast cell degranulation w ere not do not present such systemic effects as blood observed w hen the venom w as injected into the s.c. incoagulability or thrombocytopenia (data not tissue of clinically dead animals. show n ). Dexamethasone and indomethacin treat-

Mediators of Inflammation · Vol 9 · 2000 103 L. R. C. Go nça lves a nd M. Mariano

FIG. 2. Electron micrograph of a venule 1 min after injection of 2.5 m g of B. jararaca venom showing an erythrocyte escaping through an endothelial junction (bar=0.5 m m).

ments did not alter the haemorrhagic pattern induced by the venom, but significantly inhibited oedema (Fig. 3). Pre-treatment w ith morphine or methysergide inhibited both haemorrhage and oedema (Fig. 4), and guanethidine, surgical denervation and capsaicin treatments significantly inhibited haemorrhage, but not oedema induced by the venom (Fig. 5). In promethazine or mepyramine treated animals, oedema and haemorrhage were not affected.

Discussion Local haemorrhage induced by snake venoms is credited to metalloproteinases present in these venoms.6 This effect has been associated w ith the activity of these toxins on proteins of the extracellular matrix .6,12 In the present study, the injection of a low dose of B. ja ra ra ca venom into the connective tissue of the FIG. 3. Effect of treatments with (A) dexamethasone and (B) scrotal bag of rats induced prompt and significant indomethacin on B. jararaca-induced haemorrhage and oedema. Mice treated with (A) dexamethasone (1 and alterations to the morphology of this tissue. The 0.4 mg/kg, i.p., 24 and 1 h before the venom) or (B) premature manifestation of these alterations contrast indomethacin (30 mg/kg, s.c., 30 min before the venom) w ith the long time (hours ) needed for the in vitro received 5 m g of B. jararaca venom into the foot pad. Haemorrhage and oedema in treated animals (dark bars) degradation of proteins of extracellular matrix by were compared with results obtained in saline treated haemorrhagic factors isolated from viperid ven- control groups (white bars). Mean ± SE. *p<0.05.

104 Mediators of Inflammation · Vol 9 · 2000 Bo thro ps ja ra raca veno m and loca l ha em o rrhage

FIG. 4. Effect of treatments with (A) methysergide and (B) morphine on B. jararaca-induced haemorrhage and oedema. Mice were treated with (A) methysergide (0.8 mg/kg, s.c.) or (B) morphine (30 mg/kg, s.c.), 30 min before B. jararaca venom injection (5 m g) into the foot pad. Haemorrhage and oedema in treated animals (dark bars) were compared with results obtained in saline treated control groups (white bars). Mean ± SE. *p<0.05.

FIG. 5. Effect of treatments with (A) capsaicin, (B) guanethidine and (C) surgical denervation on B. jararaca-induced 13 –17 haemorrhage and oedema. Mice were treated with (A) oms. Moreover, the absence of alterations such as capsaicin (50 mg/kg, s.c., on the 2nd day after the birth), (B) disorganisation of collagen fibres and degranulation of guanethidine (3 doses of 30 mg/kg, with 24 h intervals, mast cells, w hen the venom w as injected into the lasting 24 h before the venom) or (C) surgical denervation by sciatic and saphenous nerve section, 7 days before the connective tissue of rats immediately after ceasing venom injection. Animals received 5 m g of B. jararaca venom heartbeat, suggest that such an effect depends on the into the foot pad. Haemorrhage and oedema in treated mediation of some factor(s ) liberated from injected animals (dark bars) were compared with results obtained in saline treated control groups (white bars). Mean ± SE. tissue, and possibly from the active blood *p<0.05. circulation. The possible participation of mediators from the damaged tissue in the pathogenesis of local haemorrhage induced by the venom w as tested in paw of considering that pre-treatment of the animals w ith mice submitted to pharmacological or surgical treat- methysergide decreased the intensity of the process. ments and compared w ith the mediation of the The oedema induced by B. ja rara ca venom seems oedema induced by the venom in the same paw. As to be also mediated by opioid receptors, considering show n in the Results section, the major mediators of that treatment of the animals w ith morphine inhibits oedema induced by this venom w ere derivatives of the process. This inhibitory effect of morphine w as arachidonic acid, since treatments w ith dexametha- also demonstrated for oedema caused by other 20 sone (phospholipase A2 inhibitor) or indomethacin viperid venom (Trim eresu ru s flavoviridis ) , as well (cyclooxigenase inhibitor ) w ere effective in inhibiting as that caused by other flogistic agents such as this effect. These results are in agreement w ith data carrageenan.34 Nevertheless, the mechanisms w hich from Trebien and Calixto19 and Pe rales et a l.21 The underlie the anti-oedematogenic effect of morphine participation of these mediators in the process w as are not yet understood. also demonstrated for other viperid venom-induced Our results indicate that the mediation of oedema oedema.18 ,23 While histamine seems to play a minor formation differs from that involved in the induction role in oedema induced by B. ja ra ra ca venom in of haemorrhage. Based on results obtained in mice rats,19 in mice this vasoactive amine does not pre-treated w ith dexamethasone or indomethacin, participate at all in the process, confirming previous arachidonic acid derivatives seems not to participate observations made by Pe rales et a l.21 By contrast to in the local haemorrhage induced by B. ja ra ra ca the observations of these authors, w e found that venom, confirming previous observations by Perales serotonin partially mediates venom-induced oedema, et a l.21 Conversely, in mice pre-treated w ith guanethi-

Mediators of Inflammation · Vol 9 · 2000 105 L. R. C. Go nça lves a nd M. Mariano dine, capsaicin or surgical denervation, local haemor- In conclusion, our results suggest that local haem- rhage but not oedema w as attenuated. On the other orrhage induced by B. jara ra ca venom is partially hand, methysergide or morphine significantly dimin- mediated by serotonin and by neurogenic factors. ished both oedema and haemorrhage, indicating that, Furthermore, they show that oedema and local at least in mice, serotonin and opioid receptors are haemorrhage induced by this venom have distinct partially implicated in both effects. The fact that chemical mediation. partial inhibition w as obtained w ith these treatments ACKNOWLEDGEMENTS. The authors thank Mrs Maria Christina Gaviolli for suggests that haemorrhage and oedema are indeed the electron microscopy preparations. This w ork w as a partial re quirement multimediated phenomena. for obtaining MSc degree by LRCG, at the Department of Pathology, School of The participation of opioid receptors in inflamma- Veterinary Medicine, University of São Paulo, Brazil. tory events, as well as the relationship between antidromic stimulation of sensitive nerve fibres and References vascular permeability disturbance, have been demon- 1. Rosenfeld G. Symptomatology, pathology, and treatment of snake bites in 20,3 4 – 43 strate d. It is know n that neuropeptides, partic- South America. In: Bücherl W, Buckley E, eds Ve no m o us Anim a ls a nd Their Veno m s. New York: Academic Press, 1971: 345–84 ularly substance P, can induce a direct vascular 2. Cardoso JLC, Fan HW, França FOS. Randomized comparative trial of three disturbance or may act indirectly by inducing libera- antivenoms in the treatment of envenomation by lance-headed viper 39 (Bo throps ja raraca ) in São Paulo, Brazil. Quart J Med 1993: tion of histamine from mast cells. Added to this 86:315–25 hypothesis is the fact that the blockade of opioid 3. Gutiérrez JM, Chaves F, Bolanos˜ R, Cerdas L, Rojas E, Arroyo O, Portilla E. Neutralizacion´ de los efectos locales del veneno de Bothrops a sper por receptors (e.g. treatment w ith morphine ) can prevent un antiveneno polivalente. Toxico n 1981: 19:493–500 44 ,45 the liberation of substance P. These results, and 4. Gutiérrez JM, Rojas G, Lomonte B, Gené JA, Cerdas L. Comparative study of the oedema-forming activity of Costa Rican snake venoms and its the inhibition of haemorrhage observed in animals neutralization by a polyvalent antivenom. Co m p Bio che m Physiol 1986: treated w ith capsaicin or w hich are chronically 85C:175–80 5. 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