Effect of leukocyte inhibitors benzydamine and cyclophosphamide, on lung injury caused by Tityus discrepans scorpion venom

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Toxicon 50 (2007) 1116–1125

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Effect of leukocyte inhibitors benzydamine andcyclophosphamide, on lung injury caused by

Tityus discrepans scorpion venom

Gina D’Suzea,�, Patricia Dıaza, Victor Salazarb, Carlos Sevcika, Josmary Brazona

aLaboratory on Cellular Neuropharmacology, IVIC CBB, Apdo 20632, Caracas 1020-A, VenezuelabHistology Service, Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientıficas (IVIC),

Apartado 20632, Caracas 1020A, Venezuela

Received 5 June 2007; received in revised form 29 July 2007; accepted 30 July 2007

Available online 6 August 2007

Abstract

We studied the effects of benzydamine (BZ) and cyclophosphamide (CP) on acute lung injury caused by Tityus

discrepans venom. Mice (male, IVIC strain, � 25 g) were pretreated with either BZ ð20mg=gÞ or CP ð100mg=gÞ i.p. or saline.Envenoming (2mg=g mouse) was induced sc. Lung fraction area occupied by fibrin (FF), nuclei (NF), alveolar space (AS)

and parenchyma (PF) were determined. Venom increased FF, NF and PF, significantly, and decreased AS. BZ

antagonised venom’s effect on FF sharply, antagonised partially effects on PF and AS, but was not able to antagonise

effect on NF. CP abolished venom effects on NF, AS and PF, but was not able to antagonise the effect on FF. CP was

slightly less effective than BZ in reducing FF. Fibrin deposition was associated to leukocyte sequestration. Blocking pro-

inflammatory leukocyte metabolic pathways with BZ diminished FF, suggesting that neutrophil activation, inflammation

and coagulation are correlated in the genesis of scorpionism acute lung injury.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Acute lung injury; Neutrophils; Fibrin; Scorpion; Venom; Benzydamine; Cyclophosphamide

1. Introduction

The Venezuelan scorpion Tityus discrepans pro-duces human fatalities. Its poisoning induces asystemic response consisting of hypertension orhypotension, tachycardia, tachypnea, hypothermia,leucocytosis, sialorrhoea, myocarditis, pancreatitisand respiratory distress syndrome (Sequera et al.,1993; Mota et al., 1994; D’Suze et al., 1999; Novoa

e front matter r 2007 Elsevier Ltd. All rights reserved

xicon.2007.07.015

ng author. Tel.: +58 212 5041225;

41093.

ss: [email protected] (G. D’Suze).

et al., 2003). Lung injury and its final outcome, therespiratory distress syndrome, are the most severecomplications, lethal and usually intractable.

Evidence of left ventricular dysfunction in pa-tients with scorpionism and pulmonary oedema hasled to the idea that lung injury in scorpionenvenomation is cardiogenic (Gueron et al., 1990,1992; Amaral et al., 1993; Bucaretchi et al., 1995;Gueron and Ilia, 1996; Mazzei et al., 2002).However, several authors have presented datafavouring a non-cardiogenic origin of scorpionismlung damage (Rahav and Weiss, 1990; Freire-Maiaand Matos, 1993; Mathur et al., 1993; Amaral et al.,

.

ARTICLE IN PRESSG. D’Suze et al. / Toxicon 50 (2007) 1116–1125 1117

1993). Other factors also seem to have an importantrole in the genesis of this damage, the plateletactivating factor (PAF) promotes lung damage, andheparin seems to prevent it (Freire-Maia andMatos, 1993). T. discrepans venom by itself,does not affect isolated lungs, however, the serumof Td envenomed rabbits is able to damage isolatedlungs (D’Suze et al., 1999; Novoa et al., 2003). It hasbeen suggested that chemotactic substances, pro-duced elsewhere, induce lung neutrophil sequestra-tion and this seems to be closely related to fibrindeposition and alveolar damage (D’Suze et al.,2004).

The American-European Consensus Conferencecommittee described acute respiratory distresssyndrome (ARDS) as a ‘‘syndrome of inflammationand increased permeability’’ proposing the termacute lung injury (ALI) to describe the continuum ofpathological responses to pulmonary parenchymalinjury (Bernard et al., 1994). Although ALI isformally considered different from ARDS, it isusually just a precursor to ARDS and both areconsidered as a syndrome of inflammation. Inflam-mation is considered a physiologically protectiveresponse to insult, which sometimes results in injuryand organ dysfunction followed by endothelialinjury.

There is convincing evidence that leukocyte–endothelium interactions resulting from inflamma-tory reaction play an important role in thepathogenesis of ALI and ARDS (Abraham, 2000,2003; Moraes et al., 2003). Yet, a critical linkbetween coagulation and inflammation in ALI andARDS is neutrophil accumulation associated withintravascular and intraalveolar fibrin deposition(Kaplan and Malik, 1987; Abraham, 2000; Welty-Wolf et al., 2002; Idell, 2003; Gando et al., 2004).Td venom induces generation and release of pro-inflammatory cytokines (IL6 and TNF-a) in hu-mans and experimental animals (D’Suze et al., 2003,2004). This venom produces an important damagein several organs but the most striking effect occursin lungs as a diffuse injury of the alveolar capillarybarrier, interstitial and alveolar oedema associatedwith a marked leukocyte infiltration, epithelialtransmigration and marked fibrin deposits respon-sible for thrombi, wall infarct and necrosis (D’Suzeet al., 2004).

The aim of the present study was to exploreleukocyte role in the genesis of scorpionism ALI.Two immunosuppressants benzydamine (BZ) andcyclophosphamide (CP) were used before experi-

mental scorpion envenoming. BZ affects onlyleukocyte pathways implied in the production ofpro-inflammatory cytokines and chaemotacticagents (Riboldi et al., 2003; Kaminska, 2005). CPinhibits the production of new leukocytes andsuppresses neutrophil oxidative metabolism but itis unable to inhibit their degranulation (Cairo et al.,1986; Hirsh et al., 2004).

2. Material and methods

2.1. Animal procedures

Male white mice (Balb/C mice, � 25 g, IVICstrain) were obtained from the central IVIC animalfacility and kept with sterile food and water ad

libitum in our Centre’s animal room. Three groupsof animals (10 animals per group) were studied. Thecontrol group received sterile saline sc for 5 daysand was named the saline pretreated mice (SPM). Asecond group named benzydamine pretreated mice(BPM) received 20mg=g sc benzydamine hydro-chloride [TantumTM, Elmor, 3-(1-benzylindazol-3-yl)oxy-N,N-dimethyl-propan-1-aminehydrochlor-ide] dissolved in sterile saline (Guglielmotti et al.,1997) for 3 days, twice per day. A third groupnamed cyclophosphamide pretreated mice (CPM)received 100mg=g ip cyclophosphamide (Endoxanr,Asta-Medica Frankfurt-Germany, 4-hydroxycyclo-phosphamide) dissolved in sterile saline (Carter andLivingston, 1975) for 5 days once per day. Thepharmaceutical formulation of cyclophosphamidecontained 75mg manitol per 100mg cyclopho-sphamide. After pretreatment with saline or im-munosuppressant, five animals of each of the threegroups received 2mg=g sc T. discrepans wholevenom and were sacrificed after 5 h by cervicaldislocation. Lung fixation was done initially bytracheal perfusion with 10% w/v formaldehyde,buffered with 1

15M phosphate buffer pH 7.60;

fixation was completed by immersion in the samefixative for 24 h at room temperature. The materialwas later dehydrated with ethanol, clarified withxylene and embedded in Paraplast (Luna, 1968).

Lung tissue preparation for morphometric andhistochemical study was done as indicated byReinhardt et al. (2005) with some modification.After intracardiac perfusion with 10% w/v formal-dehyde, with 1

15M phosphate buffer pH 7.60, lungs

were subjected to tracheal instillation of fixative at apressure 20 cmH2O, the trachea was then ligated,and lungs excised. Fixation was completed at 4 1C

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for 12 h in the same fixative, and lungs weretransferred to 15% sucrose in PBS at 4 1C over-night. The tissue was finally rinsed in 50% ethanoland stored in 70% ethanol. Five micrometer tissuessections were dehydrated with ethanol, clarifiedwith xylene and embedded in Paraplast, dewaxed inxylene, and re-hydrated with distilled water. Sec-tions were stained with haematoxylin and eosin toshow tissue architecture and inflammatory cellinfiltration. Fibrin was stained using the Carstairs’method modified by Li et al. (1999). With thismethod fibrin is bright red, collagen is bright blueand red blood cells are yellow.

2.2. Morphometric methods

For morphometry, a random sample was ob-tained by analysing at least five well stained sectionsof each specimen. Sections were viewed using brightfield illumination on a Nikon E600 light microscope(Nippon Kogahu KK, Tokyo, Japan) with a 40plan-apochromatic objective with 0.75 numericalaperture, and digitally photographed with a digitalcamera (Nikon model 5000), the 2560� 1920 pixelimages were saved as a 24-bit colour TIFF files. Theillumination was optimised by Koehler illumina-tion, and the light intensity was standardised bycalibrating the camera threshold using the alveolarspace (AS) in lung sections.

The ImageJ (version 1.34n) public domain Javaimage processing package (National Institute ofHealth, Bethesda, Maryland, USA) was used tocalculate the areas and count particles (Gao et al.,2002). The treatment of images using ImageJ,followed the procedure of Johansson et al. (2001)with several modifications. The images were con-verted from 24 to 8 bits, brightness and contrast ofthe images were increased to separate the haema-toxylin stained nuclei from background, and thecolour range threshold set to exclusively encompassthe nuclei; the images were finally converted to onebyte black and white images. Values above thetarget threshold resulted in white pixels, whereasvalues that fell on or below the target thresholdresulted in groups of black pixels representing thenuclei. The ‘‘Analyze Particles’’ option of ImageJwas set to accept between 1 and 10,000 pixels inorder to include clusters of nuclei that were notseparable from each other (such as is the case ofneutrophil clusters) but to exclude larger non-nuclear structures. Under these conditions ImageJprovided the number, total area, average size

(square microns) of nuclei or nuclear clusters andthe fractional area (percent) occupied by nuclei(NF) or nuclear clusters in the pictures. In lungimages threshold was adjusted for delimitingparenchyma excluding AS, after that, thresholdwas set for nuclei detection as described above,obtaining nuclear parameters in parenchyma only.

The parenchyma and AS area fraction wasestimated by Reinhardt et al. (2005) methodmodified as follows. Sections were photographedusing a 10� objective, pixel size was converted tomicrometers and image analysis was performed.Threshold set using pre-defined RGB criteria forairspace, allowed to differentiated AS from par-enchyma and to estimate the fractional areaoccupied by AS, the parenchymal fractional (PF)area was calculated by subtracting the AS from thetotal area of the section. Profiles corresponding toarteries, veins and bronchioles were manually tracedand their area was subtracted from the total area ofthe field (Magliozzi et al., 2003). All values obtainedfrom individual fields were utilised for calculatingthe mean values, which were submitted to statisticalanalysis.

2.3. Statistical procedures

Data were processed using non-parametric statis-tical procedures. Data are always presented asmedians and their 95% confidence intervals (CI)calculated with the procedure of Hodges andLehmann. Statistical significance of differencesbetween two samples was decided with Mann–Whit-ney (Wilcoxon) test (M–W W test), multiplecomparisons were done using the non-parametricKruskall–Wallis (K–W test) analysis of variance.The ratio between data in two samples and its 95%CI was calculated with the Moses method. SeeHollander and Wolfe (1973) for all details of thesenon-parametric methods. Significance of differencesbetween proportions was assessed with the w2 testwith Yates’ correction for continuity (Fleiss, 1973).Differences between treatments were consideredsignificant if the probability that the null hypothesiswas true was p5% ðPp0:05, two tails).

3. Results

The characteristic features of Td scorpionismwere clearly observable. Fasciculation occurred2min after venom injection, tachycardia and sialor-rhoea after 10min envenomation which persisted

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during the whole experiment (5 h). Severe systemicenvenoming characterised by sweating, piloerection,hypothermia, polyuria, tachypnea and respiratorydistress was observed after 3 h envenoming.

Fig. 1A shows normal mice lung structure. TheSPM group inoculated with Td venom after 5 hdeveloped a strong reduction of AS due tointravascular, intraalveolar and intersticial fibrindeposition, intraalveolar oedema and alveolus walldegradation (Fig. 1B, C). Marked lung leukocyteinfiltration was observed after 2 h envenomation(results not shown) but it is only after 5 h when thisinduced wall collapses, thrombi and necrosis be-came apparent (Fig. 1D). Fibrin deposition was

Fig. 1. Lung light Micrographs from saline pretreated mice (SPM) 5 h

Tityus discrepans whole venom). (A) SPM lung 5 h after the animals r

changes observed 5 h after envenoming: strong fibrin deposition at the a

strong neutrophil sequestration inducing wall collapse (D). Arrows poin

and to leukocyte accumulation (D). Staining: haematoxylin and eosin. B

related to places where neutrophil sequestrationoccurred. Five hours were required, at the studyvenom dose ð2mg=g), to observe a generalisedalveolar fibrin deposition, generalised neutrophilaccumulation and thickening and oedema of alveo-lar septa.

Fig. 2 shows lung light micrographs from BPMafter 5 h subcutaneous saline (A) or venom injection(B, C, D). BZ did not change significantly bronch-iolar or alveolar wall architecture (Fig. 2A). InBPM, scorpion venom induced only a mild leuko-cyte infiltration in alveolar walls (Fig. 2B, C),producing a slight AS reduction. In spite of alveolarwall leukocyte infiltration and leukocyte groups

after subcutaneous saline or scorpion venom injection (2mg=g sc

eceived sterile saline instead of venom. (B–D) Lung histological

lveolar space (B), alveolus wall degradation and oedema (C) and

t at fibrin deposition in the alveolar space (B), alveolar oedema (C)

ar: 12:5mm. Inserts: images at low amplification. Insert bar: 25mm.

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Fig. 2. Lung light Micrographs from benzydamine (20mg=g sc for 3 days, twice per day) pretreated mice (BPM) 5h after subcutaneous

saline or scorpion venom injection (2mg=g sc T. discrepans whole venom). (A) BPM lung 5 h after the animals received sterile saline instead

of venom. (B–D) Lung histological changes observed 5 h after envenoming: a mild leukocyte accumulation at the alveolus wall (arrow),

scarce fibrin deposition (asterisk) (B, C) and leukocyte groups forming wall microthrombi (arrow) (D). No fibrin deposition was observed

in this section Staining: haematoxylin and eosin. Bar: 12:5mm. Inserts: images at low amplification. Insert bar: 25mm.

G. D’Suze et al. / Toxicon 50 (2007) 1116–11251120

forming microthrombi scarce fibrin deposition wasobserved (Fig. 2D).

Fig. 3 shows lung light micrographs from CPMafter subcutaneous saline (A) or venom injection (B,C, D). In CPM, scorpion venom induced abundantfibrin deposition. In spite of scarce leukocyteinfiltration we found abundant fibrin depositionin alveolar wall, blood vessel and capillaries(Fig. 3B–D) inducing reduction of alveolar area.However, in general, AS and wall architecture weremaintained when envenomed and non-envenomedCPM groups were compared.

Table 1 contains data on lung morphometry ofSPM, BPM and CPM groups after subcutaneous

saline or venom injections. Fibrin, nuclei andparenchyma fractional areas increased and ASfractional area decreased highly significantly inenvenomed mice ðP510�6Þ. Fibrin deposits werecompletely absent from non-poisoned mice lungs.As shown in the table, fibrin deposits accounted for14.5% of the lung area in envenomed SPM; BZ andCP both reduced the effect of venom on FFdrastically (Po10�6, M–W W test), and whencompared between the two leukocyte inhibitors,the effect of venom on FF was less in BZ than in CP(P ¼ 0:02, M–W W test). CP completely preventedvenom effects on AS, NF and PF (P40:1, K–Wtest). BZ per se increased lung cellularity from 7.3

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Fig. 3. Lung light Micrographs from cyclophosphamide (100mg=g ip for 5 days, once per day) pretreated mice (CPM) 5 h after

subcutaneous saline or scorpion venom injection (2mg=g sc T. discrepans whole venom). (A) CPM lung 5 h after the animals received sterile

saline instead of venom. (B–D) Lung histological changes observed 5 h after envenoming: alveolar wall fibrin deposition (arrow),

pronounced alveolar space decrease (asterisk) (B), fibrin deposition in blood vessels and capillaries (arrows). Staining: haematoxylin and

eosin. Bar: 12:5mm. Inserts: images at low amplification. Insert bar: 10:0mm.

G. D’Suze et al. / Toxicon 50 (2007) 1116–1125 1121

(6.8, 7.8)% to 15.4 (13.9, 13.0)%, NF was larger innon-envenomed BPM [15.4 (13.9, 16.0)%] than innon-envenomed SPM [9.0 (7.7, 10.2)%] (Po0:001,K–W test). BZ did not modify significantly theeffect of scorpion venom on either NF, AS or PF(P40:1, K–W test).

4. Discussion

Severe scorpion envenoming produces a systemicinflammatory response syndrome that may causeshock, pancreatitis, cardiac dysfunction and pul-monary ALI (Sequera et al., 1993; Bucaretchi et al.,1995; Porras et al., 1994; Rossomando et al., 1997).

In previous work, we showed that in rams, T.

discrepans venom produced an inflammation reac-tion, characterised by neutrophil infiltration andsequestration in different tissues, vasculitis, arteritisand a marked fibrin deposition. Fibrin depositswere found only at places where a markedneutrophil accumulation occurred.

The rapid accumulation of neutrophils in lungs inresponse to any pro-inflammatory stimulus, scor-pion venom in this case, is one of the firstrecognisable events in the pathogenesis of manypulmonary diseases (Worthen and Nick, 1998; Nicket al., 2000). In this work, neutrophil migration andaccumulation were observed into airspaces in the

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Table 1

Lung parameters in mice envenomed with scorpion venom and pretreated with either saline, benzydamine or cyclophosphamide

Tissue components Condition 5 h before

sacrificing the mice

Saline pretreated

mice (SPM)

Benzydaminea

pretreated mice (BPM)

Cyclophosphamideb

pretreated mice (CPM)

Fibrin (FF) Not envenomed 0 0 0

(�0.5, 0.2) (�0.5, 0.4) (�1.7, 0.7)

Envenomed 14.5 2.6 4.6

(13.8, 16.4) (1.1, 4.2) (3.3, 5.6)

Nuclei (NF) Not envenomed 7.3 15.4 9.0

(6.8, 7.8) (13.9, 16.0) (7.7, 10.2)

Envenomed 22.1 23.9 9.4

(20.4, 24.3) (23.1, 24.9) (8.9, 10.1)

Alveolar space (AS) Not envenomed 77.8 74.9 77.7

(72.5, 82.8) (72.6, 77.0) (75.7, 79.3)

Envenomed 62.2 66.0 78.1

(55.1, 68.0) (61.5, 70.6) (76.5, 79.4)

Parenchyma (PF) Not envenomed 22.3 25.1 22.3

(17.2, 27.5) (23.1, 27.5) (20.8, 24.3)

Envenomed 37.9 33.8 23.6

(32.1, 44.9) (29.2, 38.6) (21.5, 25.7)

aTwenty mg=g sc benzydamine hydrochloride for 3 days, twice per day.bOne hundred mg=g ip cyclophosphamide for 5 days, once per day. Not envenomed animals received saline sc 5 h before sacrificing them,

envenomed animals received 2mg=g T. discrepans whole venom 5h before sacrificing them. Data are fractions (as %) of the total lung area

occupied by each tissue component measured in five sections. Data are presented as medians and their 95% confidence interval (between

parentheses). Other details are in the text of the communication.

G. D’Suze et al. / Toxicon 50 (2007) 1116–11251122

setting of ALI after 5 h envenoming. Furthermore,this venom induce the synthesis of pro-inflamma-tory cytokines as it was demonstrated in experi-mental animals and scorpionism victims (D’Suze etal., 2003). Cytokines may serve to amplify andperpetuate the recruitment of leukocytes in the AS(Xing et al., 1993). The aim of the present study wasto explore the leukocyte role in the genesis of ALI inscorpionism. Therefore, two immunosuppressantsBZ and CP were used before experimental scorpionenvenoming.

Lung changes induced by Td venom in SPM (notinmmunosuppressed) involve a significant AS re-duction due to intravascular, intraalveolar andinterstitial fibrin deposition and a marked neutro-phil sequestration, all of which resulted in NF andPF increases. These features are characteristics ofALI. Lung histological micrography from enve-nomed mice of the SPM group showed activatedneutrophils migrating through the epithelium intothe AS, suggesting the activation of inflammation in

situ. Activated neutrophils are able to expressclotting tissue factor and perhaps are responsiblefor the intravascular and extravascular fibrindeposition observed here. Similar events are fre-quently found in the early phase of ALI and ARDS(Idell, 2003; Moraes et al., 2003). Fibrin is a potent

inhibitor of surfactant function, which wouldcontribute to alveolar collapse (Laterre et al., 2003).

In the inmmunosuppressed groups the lungchanges induced by Td venom were significantlydifferent from the SPM group. Lung micrographsfrom envenomed mice in the BPM group showed ascarce presence of fibrin. In spite of neutrophilaccumulation in lungs, when neutrophil’s metabolicpathways associated with up-regulation of pro-inflammatory cytokines and chemoattractants wereblocked by BZ, fibrin deposition was reducedminimising lung injury. BZ a non-steroidal drug,only affects the leukocyte pathways implied in theproduction of pro-inflammatory cytokines andchemotactic agents (Riboldi et al., 2003; Kaminska,2005). Its anti-inflammatory activity has beenrecently related to selective inhibition of pro-inflammatory cytokine production and to chemo-tactic responses by blocking the extracellular signal-regulated kinase (ERK) and p38 mitogen-activatedprotein kinase (MAPK) pathways in polymorpho-nuclears (PMNs) and monocytes (Sironi et al., 1996,1997; Riboldi et al., 2003).

Lung micrographs from envenomed mice of theBPM group showed leukocyte groups formingmicrothrombi, and still, little fibrin deposition wasobserved. These results demonstrate that inhibiting

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leukocyte degranulation changes the course of lunginjury caused by scorpionism. Our results agree withprevious work which indicates that neutrophilactivation was determinative in lung fibrin deposi-tion by scorpionism. Leukocyte recruitment is a keyevent in inflammatory processes, and migration inresponse to chemotactic agonists is a crucialdeterminant of leukocyte trafficking. Our resultsalso agree with those of other researchers whichfound that MAPK inhibitors reduces inflammationin many experimental models including endotoxinshock, collagen induced arthritis (Badger et al.,1996), pulmonary inflammation (Nick et al., 2000)and gastritis (Takahashi et al., 2001). The resultspresented here, demonstrate that BZ, which causes asignificant inhibition of leukocyte’s activation,minimises fibrin deposition associated with scorpionenvenoming.

To explore leukocyte role in the genesis of ALI inscorpionism, another immunosuppressant was used,cyclophosphamide, an immunosuppressive alkylat-ing agent used for the treatment of a broadspectrum of malignancies. Cyclophosphamide in-hibits the production of new leukocytes andsuppresses neutrophil oxidative metabolism but itis unable to inhibit their degranulation (Cairo et al.,1986; Hirsh et al., 2004). Similar to other immuno-suppressive drugs, CP produces neutropaenia in adose-related manner (Bosch and Ferrer, 2002;Montillo et al., 2003). We choose a CP pretreatmentwhich allowed a balance between satisfactory micesurvival with intense neutropaenia (Carter andLivingston, 1975). We have shown that in enve-nomed mice with a CP-induced neutropaenia areduced accumulation of neutrophils in lungsoccurred with fibrin deposition slightly larger thanthe observed in BPM. CP inhibits the production ofnew leukocytes and suppresses neutrophil oxidativemetabolism but is unable to hinder the degranula-tion of existing neutrophiles. CP treatment neitherprevents nor attenuates PMNs functions (Cairoet al., 1986; Hirsh et al., 2004). Residual PMNsin CP neutropaenic subjects preserved their abilityto respond to the Td venom by activation andrelease of granules, and therefore, a final fibrindeposition was observed after 5 h envenoma-tion. According to our results, inducing neutropae-nia (with few neutrophiles available for degranula-tion) is somewhat less effective in preventing lungfibrin deposition after Td envenoming, than inhibit-ing degranulation with BZ with a normal neutrophilcount.

In summary our results show that Td envenom-ing, is able to induce ALI, characterised by amarked neutrophil sequestration associated with aflorid fibrin deposition, which was hindered in BPMand in CPM. Since fibrin deposition in lungs is ofbad prognosis in lung injury, BZs effect on FF isremarkable. Blocking pro-inflamatory leukocytemetabolic pathways with BZ diminished FF,suggesting that neutrophil’s activation, inflamma-tion and coagulation are correlated in the genesis ofscorpionism ALI. Still, our results should not beconstrued as our claim to propose BZ as atherapeutic agent in either scorpionism, ALI orARDS; more evidence and careful clinical valida-tion is required to asses the therapeutic value of BZin any of these pathologies.

Acknowledgements

The authors are indebted to the people of SanAntonio de Los Altos and their Fire Department forscorpion supply. We greatly appreciated the techni-cal assistance of Moises Sandoval. Financed in partby Laboratorios Silanes–Instituto Bioclon (Mexico)and FONACIT Grant S1-2001000908.

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