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Local haemorrhage induced byBothrops jararaca venom:relationship to neurogenicinflammation

Luis Roberto C. Gonçalves1,CA and Mario Mariano2

1Laboratorio de Fisiopatologia, Instituto Butantan,Av. Vital Brazil, 1500, 05503–900; and 2Disciplina deImunologia, Universidade Federal de Sao Paulo,Escola Paulista de Medicina, Sao Paulo-SP, Brazil

CA Corresponding AuthorTe l: 55 11 813–7222 ex t. 2164Fax : 55 11 813–7222 ex t. 2162Email: [email protected]

WE in vestigated m orphological alterations in ducedby s .c . in jection of 2.5 m g of Both ro p s jararac a venomin rats . In tense disorgan isation of collagen fibres w asobserved 1 m in afte r the venom in jection , particularlyat r egions near vesse ls and nerves . Mast ce lls weredegranulated, and e ryth rocyte s w ere seen leavin gvenules th roughout the endothelial junctions. At th istim e, dam aged endothe lial ce lls were not observed. Inrats envenom ed as above, but im m ediately afte rcardiore spiratory fa ilure induced by deep etheranae sthe sia, alterations in the connective tissue s truc-tures , as previously described, were not observed.Th e m ediation of th is haem orrhage w as investigatedby injecting th e venom in to the foot pad of m ice andcom pared to th e m ediation of oedem a. Local haem or-rhage w as s ign ifican tly reduced in m ice pre-treatedw ith capsaicin or guane th idine or subm itted to asurgical section of sciatic and s aphenous nerve s . Inthe se anim als, oedem a w as not affe cted. Groupstreated w ith m ethyse rgide or m orph in e showed bothhaem orrhage and oedem a s ign ifican tly reduced.In dom ethacin or dex am ethasone pre-treatm ents s ig-n ifican tly reduced the oedem a, but not the haem or-rhage. Moreover, in an im als tr eated w ith prom-ethazin e or m epyram ine , oedem a and haem orrhagew ere not affected. Th ese data sugges t that localhaem orrhage induced by Both ro p s jararaca venomis partially con trolled by seroton in and neurohu-m oral m ediator s . Furtherm ore, r esults in dicate thathaem orrhage and oedem a are m ediated by differentpharm acological system s .

Key w ords : Snake venom, Bo th ro ps ja ra ra ca , Localhaemorrhage , Oedema, Neurogenic inflammation

Introduction

Haemorrhage and oedema, besides blood coagulationdisturbance s, are the main s igns of envenoming byBo thro p s snake venoms.1,2 Despite serum therapybeing an effective tre atment for the control ofsystemic symptoms in this type of envenoming, itdoe s not control the oedema and local haemorrhagicand necrotic lesions w hich deve lop at the site ofvenom inoculation.1 ,3 – 5

Among the tox ins re sponsible for these localeffects, haemorrhagic factors w ere isolated fromvarious viperid venoms.6 Even though some of the sehaemorrhagic fac tors are devoid of proteolytic ac tiv-itie s,7–11 the y are charac terised as metalloprotei-nases .6 Some of the se factors digest prote ins from theex trace llular matrix , and this property has be enre lated to the pathogeny of haemorrhagiclesions .6,1 2 –17

On the other hand, e fforts have also been made tocharac terise the pharmacological mediators involvedin oedema formation,18 – 23 pain2 4 and other para-

meters of the inflammatory response2 5 – 29 induced byvipe rid venoms. Neve rthe less , all this information isnot suffic ient to draw a comprehensive mechanism toex plain the local le sions observed in this type ofenvenoming.

In the pre sent study it is show n that oedema andhaemorrhage induced by B. ja ra ra ca venom havedistinc t pharmacologic al mediation and our re sultssuggest that haemorrhage is partially controlled byse rotonin and neurogenic mediators.

Methods

Venoms

A pool of lyophilised venom obtained from adultspecimens of B. ja ra ra ca snake s, supplied by theLaboratory of Herpe tology, Instituto Butantan, w asused throughout this inve stigation. The venom waskept at –20°C, and venom solutions w ere pre pared(w /v ) w ith sterile saline just before use .

ISSN 0962-9351 print/ISSN 1466-1861 online/00/020101-07 © 2000 Taylor & Francis Ltd 101

Research Paper

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

Animals

Male Wistar rats (220–250 g ) or male Sw iss mice(18–22 g ) w ere used. The animals w ere maintainedw ith w ater and food a d libitum in appropriateenvironment conditions and used under e thic al condi-tions, according to guide lines of the InternationalSocie ty of Tox inology.30

Morphology of venom-induced lesions

Rats, maintained under e ther anaesthe sia, w ere inje c-te d into the subcutaneous tissue of the sc rotal bagw ith 2.5 m g of venom (50 m l) in saline . Control ratsreceived the same volume of saline. One minute afterthe inje ction, animals w ere killed and samples oftissue from inje cte d areas w ere obtained. In orde r toevaluate the role p layed by blood circulation in theevolution of the venom-induced lesions, the sameprocedure as above w as performed in rats w hoseheartbeats had just stopped due to prolonged e theranae sthesia. Tissue samples from the sites of venominjection were collec ted 1 min afte r the injec tion andfix ed in 2.5% buffered glutaraldehyde . Routine doublefix ation, embedding in Araldite and staining w ithuranyl ace tate and lead citrate w ere employed. Theproce ssed samples w ere analysed in a Phillips (EM–201) e lectron microscope.

Evaluation of haemorrhage and oedema

Local haemorrhage and oedema w ere s imultaneouslyevaluated using a modification of the methods de scri-bed by Ow nby e t a l.3 1 and by Yamakaw a e t a l.3 2 Micew ere injected s.c. in the right foot pad w ith 50 m l ofthe venom solution. The contra-lateral foot padreceived the same volume of sterile saline. Threehours later (time determined by a time-course ex peri-ment for haemorrhage and oedema), the animals w eresacrificed in an ether chamber. The paw s w ereremoved at the leve l of the tibio-tars ic junction,w eighted, fragmented and put in tube s containing4 ml of von Kampen–Zijlstra re agent modified byMatsubara e t a l.33 (200 mg K3[Fe (CN)6], 50 mg KCN,120 mg KH2PO4 , 50 mg NaCl and 1 ml non-ionicdete rgent per litre ). The tissue w as homogenised andthe tube s we re centrifuged at 3000 g for 30 min. Thesupe rnatant w as filtered in Millipore membranes(0.8 m pore s ), and the absorbance at 540 nm w asdete rmined in a spec trophotomete r (Micronal B382,Braz il).

The haemoglobin concentration w as dete rmined asfollow s:

[Hb] = A540 nm ´ 64·458

44 ´ d ´ 1000

w here : [Hb]= haemoglobin concentration; A540nm=absorbance obtained; 64·458= molecular w eight of

haemoglobin; 44= 44 mmol–1 /cm= molecular absorp-tion of haemoglobin; d=1= cuvette thickness ; 1000=converting factor (litre to millilitre ); 4= volume of vonKampen–Zijlstra reagent.

In order to e liminate the influence of oedema, theconcentration of haemoglobin obtained in the pawinjected w ith venom w as divided by the w eight of thepaw injec te d w ith saline . The difference betw eenhaemoglobin content of the paw injected w ith venomand of that injected w ith saline w as the e stimatedhaemorrhage, ex pressed as mg Hb /g of tissue. Theminimal haemorrhagic dose (MHD) w as defined asthe minimal concentration of venom able to inducean inc rease of three times the haemoglobin concen-tration in re lation to that of a control tissue. Finally,oedema, ex pre ssed in mg, w as evaluated by thedifference in w eight be tw een one paw injec ted w ithvenom and the othe r inje cte d w ith saline.3 2

Treatment of animals

Groups of 6 to 10 mice w ere injected s.c. into thefoot pad w ith 5 MHDs of the venom (5 m g in 50 m l )after the follow ing tre atments: (1) dex amethasone(corticosteroid, phospholipase A2 inhibitor: Deca-dron®, Promade, Brazil ), 1 and 0.4 mg/kg, i.p., 24and 1 h before the venom; (2) indomethacin (inhib-itor of the prostaglandin-forming cycloox igenase:Sigma), 30 mg/kg, s .c., 30 min before the venom; (3)methysergide (5HT re ceptor antagonist: Sigma),0.8 mg/kg, s .c., 30 min before the venom; (4) prom-ethazine (H1 re ceptor antagonist: Rhodia Pharma,Brazil), 10 mg/kg, i.p ., 30 min be fore the venom; (5)mepyramine (H1 re ceptor antagonist: Sigma), 5 mg/kg, i.p., 30 min before the venom; (6) morphine(opioid: Merck ), 30 mg/kg, s.c., 30 min be fore thevenom; (7) guane thidine (peripheral post-ganglionicadrenergic neurone inhibitor; sympathetic blockade:Ciba Geigy ), three dose s of 30 mg/kg, s.c ., w ith 24 hinte rvals , be fore the venom inje ction; (8) chronicdene rvation: mice w ere anaesthetised w ith a Keta-lar® (Park–Davis ) – Rumpum® (Baye r ) solution(1:2 v /v, diluted 1:4 v /v in saline ), i.p., 0.1 ml/10 gbody w eight, and the sciatic and saphenous nerve sof the right leg w ere surgically se ctioned and thevenom inje cte d into the right foot pad 7 days later;(9) capsaic in (substance P re leaser from sensoryafferent neurones: Merck ), 50 mg/kg, s.c., on thesecond day after birth (in neonate animals, c apsai-cin treatment cause s degeneration of sensory affe r-ent neurone s ) (animals w ere used w hen they ach-ie ved the appropriate we ight); (10) re spectivecontrol groups re ceived saline by the same route.In the case of the chronic denervation, controlanimals w ere sham operated, and in the case of thecapsaicin, control group rece ived the capsaicinsolvent.

L. R. C. Go nça lve s a nd M. Mariano

102 Mediators of Inflammation · Vol 9 · 2000

Statistics

Results w ere analysed by Student’s t-test and con-sidered significantly different w hen p< 0.05.

Results

Morphology of the envenomed connectivetissue

Disorganisation of collagen fibres, particularly nearblood vessels and nerve bundles (Fig. 1A and 1B,respective ly ), mast ce ll degranulation (Fig. 1C) andintense vascular congestion (Fig. 1D) w ere observed1 min afte r venom inje ction into the subcutaneoustissue of the sc rotal bag of rats. Morphologic alalterations in venular endothe lial ce lls we re notclearly detected, although red blood cells w e re se eingsqueezing out through open endothelial cell junc-tions, characte rising a pe r diapede s is type of haemor-rhage (Fig. 2 ). Alte rations such as disorganisation ofcollagen fibres and mast ce ll degranulation w ere notobserved w hen the venom w as injected into the s.c.tissue of clinically dead animals.

Time-course of haemorrhage and oedemainduced by the venom

Haemorrhage induced by 1.25 m g of venom show ed apeak at the 3rd hour after injec tion, pers isting up to6 h. Oe dema w as max imal from 30 min up to 3 h,decre as ing afte r 6 h of venom inje ction. As bothhaemorrhage and oedema w ere max imal 3 h aftervenom inje ction, this time w as se lecte d to evaluatesome parameters of the pharmacologic al mediation ofthese processes.

The haemorrhagic activity w as dose-dependent,and the MHD obtained w as 0.95 m g. A dose of 5 m g ofvenom w as used for the study of the pharmacologic almediation of the le sions.

Mediation of venom-induced haemorrhage andoedema

With the dose of venom used, only local e vents w ereevaluated, as it w as considered that envenomed mic edo not present such systemic effe cts as bloodincoagulability or thrombocytopenia (data notshow n ). Dex amethasone and indomethac in treat-

Bo thro ps ja ra ra ca veno m and lo ca l ha em o rrha ge

Mediators of Inflammation · Vol 9 · 2000 103

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 collagenfibres (bar = 0.5 m m); (C) mast cell degranulation near a nerve bundle (bar= 1 m m); and (D) vascular congestion andhaemorrhage (bar= 1 m m).

ments did not alte r the haemorrhagic pattern inducedby the venom, but significantly inhibited oedema (Fig.3). Pre-treatment w ith morphine or methyse rgideinhibited both haemorrhage and oedema (Fig. 4 ), andguane thidine , surgic al denervation and capsaicintreatments significantly inhibited haemorrhage, butnot oedema induced by the venom (Fig. 5 ). Inpromethazine or mepyramine treated animals,oedema and haemorrhage were not affe cte d.

Discussion

Local haemorrhage induced by snake venoms iscredited to metalloprote inases present in the se ven-oms.6 This effe ct has been associated w ith the ac tivityof the se tox ins on prote ins of the ex tracellularmatrix .6,12

In the pre sent study, the injection of a low dose ofB. ja ra ra ca venom into the connective tissue of thesc rotal bag of rats induced prompt and significantalterations to the morphology of this tissue. Thepremature manifestation of these alterations contrastw ith the long time (hours ) needed for the in v itrodegradation of prote ins of ex tracellular matrix byhaemorrhagic factors isolated from viperid ven-



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

FIG. 3. Effect of treatments with (A) dexamethasone and (B)indomethacin on B. jararaca-induced haemorrhage andoedema. Mice treated with (A) dexamethasone (1 and0.4 mg/kg, i.p., 24 and 1 h before the venom) or (B)indomethacin (30 mg/kg, s.c., 30 min before the venom)received 5 m g of B. jararaca venom into the foot pad.Haemorrhage and oedema in treated animals (dark bars)were compared with results obtained in saline treatedcontrol groups (white bars). Mean ± SE. *p<0.05.

oms.1 3 –1 7 Moreove r, the absence of alterations such asdisorganisation of collagen fibre s and degranulation ofmast c ells, w hen the venom w as inje cte d into theconnective tissue of rats immediately after ceasingheartbeat, suggest that such an e ffec t depends on themediation of some fac tor(s ) libe rated from inje cte dtissue, and possibly from the ac tive bloodcirculation.

The possible participation of mediators from thedamaged tissue in the pathogene sis of local haemor-rhage induced by the venom w as tested in paw ofmice submitted to pharmacological or surgical treat-ments and compared w ith the mediation of theoedema induced by the venom in the same paw. Asshow n in the Results sec tion, the major mediators ofoedema induced by this venom w ere derivatives ofarachidonic ac id, since treatments w ith dex ametha-sone (phospholipase A2 inhibitor ) or indomethacin(cycloox igenase inhibitor ) w ere e ffec tive in inhibitingthis effect. These results are in agre ement w ith datafrom Tre bien and Calix to1 9 and Pe rales e t a l.21 Theparticipation of the se mediators in the proce ss w asalso demonstrated for othe r viperid venom-inducedoedema.18 ,23 While histamine se ems to play a minorrole in oedema induced by B. ja ra ra ca venom inrats,19 in mic e this vasoactive amine doe s notparticipate at all in the process, confirming previousobservations made by Pe rales e t a l.21 By contrast tothe observations of the se authors, w e found thatse rotonin partially mediate s venom-induced oedema,

cons idering that pre-treatment of the animals w ithmethysergide dec reased the intensity of the process.

The oedema induced by B. ja rara ca venom seemsto be also mediated by opioid re ceptors, consideringthat treatment of the animals w ith morphine inhibitsthe proce ss. This inhibitory effect of morphine w asalso demonstrated for oedema caused by othervipe rid venom (Trim ere s u ru s f lavov iridis )20 , as we llas that caused by other flogistic agents such ascarrageenan.3 4 Neverthe le ss, the mechanisms w hichunderlie the anti-oedematogenic effect of morphineare not yet understood.

Our re sults indicate that the mediation of oedemaformation differs from that involved in the inductionof haemorrhage . Based on results obtained in mic epre-treated w ith dex amethasone or indomethacin,arachidonic acid de rivatives seems not to participatein the local haemorrhage induced by B. ja ra ra cavenom, confirming previous observations by Perale se t a l.21 Converse ly, in mice pre-treated w ith guane thi-



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. jararacavenom injection (5 m g) into the foot pad. Haemorrhage andoedema in treated animals (dark bars) were compared withresults obtained in saline treated control groups (white bars).Mean ± SE. *p<0.05.

FIG. 5. Effect of treatments with (A) capsaicin, (B) guanethi-dine and (C) surgical denervation on B. jararaca-inducedhaemorrhage and oedema. Mice were treated with (A)capsaicin (50 mg/kg, s.c., on the 2nd day after the birth), (B)guanethidine (3 doses of 30 mg/kg, with 24 h intervals,lasting 24 h before the venom) or (C) surgical denervation bysciatic and saphenous nerve section, 7 days before thevenom injection. Animals received 5 m g of B. jararaca venominto the foot pad. Haemorrhage and oedema in treatedanimals (dark bars) were compared with results obtained insaline treated control groups (white bars). Mean ± SE.*p<0.05.

dine, capsaicin or surgic al denervation, local haemor-rhage but not oedema w as attenuated. On the othe rhand, methyse rgide or morphine significantly dimin-ished both oedema and haemorrhage, indicating that,at least in mice, serotonin and opioid receptors arepartially implicated in both effects. The fact thatpartial inhibition w as obtained w ith these treatmentssuggests that haemorrhage and oedema are indeedmultimediated phenomena.

The partic ipation of opioid receptors in inflamma-tory events, as we ll as the re lationship betw eenantidromic stimulation of sensitive ne rve fibre s andvascular permeability disturbance , have been demon-strate d.2 0,3 4 – 43 It is know n that neuropeptides, partic-ularly substance P, can induce a direc t vasculardisturbance or may act indirec tly by induc ing libe ra-tion of histamine from mast cells.3 9 Added to thishypothesis is the fact that the blockade of opioidreceptors (e .g. tre atment w ith morphine ) can preventthe liberation of substance P.44 ,45 These results , andthe inhibition of haemorrhage obse rved in animalstre ated w ith capsaic in or w hich are chronicallydene rvated, strongly indicate that haemorrhageinduced by the venom could be class ified as aneurogenic type of haemorrhagic inflammatoryresponse. Re inforcing this interpretation is the fac tthat neurogenic vascular disturbance s can be inhib-ited by sectioning nerve routes or by chemicaldepletion of neuropeptides after capsaicintre atment.3 6

Taken togethe r, the se data might ex plain theex plosive alterations in the connec tive tissue of rats1 min afte r venom injec tion. Malucelli and Mariano4 0

also observed a similar phenomenon of instantaneoushaemorrhagic re sponse on the diaphragm of guineapigs after e le ctric or chemical stimulation of thephre nic ne rve. These authors described a pe r diape-de s is haemorrhage in diaphragmatic vessels. Fur-thermore, this type of haemorrhage w as alsoobserved after T. fla vo viridis venom inje ction.4 6

Conversely, afte r intramuscular injection of variousvipe rid c rude venoms or isolated haemorrhagic fac-tors, the usual feature is a pe r rhex is haemorrhageobserved in capillarie s.4 7– 4 9 Whethe r these differ-ences are due to the dose of venom injected, theroute of inje ction or other peculiar charac teristics ofthe microvasculature in these tissue s remains to beinvestigated. It must be considered that pe r diape de-s is haemorrhage is mainly observed in venules w hilepe r rhex is haemorrhage is reported to occur mainly incapillaries . Nevertheless, only vessels greater than20 m m in diameter, like venules, are innervated.5 0 Thisfact and results here de sc ribed indicate that pe rdiapede s is haemorrhage induced by B. ja ra ra cavenom can be controlled by neurogenic fac tors.Furthe r studie s should be made in orde r to identifythe spec ific neurogenic fac tor(s ) involved in thisproce ss.

In conclusion, our re sults sugge st that local haem-orrhage induced by B. ja ra ra ca venom is partiallymediated by se rotonin and by neurogenic fac tors.Furthe rmore , they show that oedema and localhaemorrhage induced by this venom have distinc tchemical mediation.

ACKNOWLEDGEMENTS. The authors thank Mrs Maria Christina Gaviolli forthe electron microscopy preparations. This w ork w as a partial re quirementfor obtaining MSc de gre e by LRCG, at the Department of Pathology, School ofVeterinary Medic ine, Unive rsity of Sao Paulo, Brazil.

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Accepted 1 May 2000



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