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Acta Cirurgica Brasileira

Print version ISSN 0102-8650On-line version ISSN 1678-2674

Acta Cir. Bras. vol. 13 n. 4 São Paulo Oct./Nov./Dec. 1994

http://dx.doi.org/10.1590/S0102-86501998000400011 

THE MOUSE AS AN EXPERIMENTAL MODEL FOR TITYUS SERRULATUS SCORPION ENVENOMING1

 

Mônica de Mônico Magalhães2
Gisele Viana2
Rosa Maria Esteves Arantes3
Tasso Moraes Santos4
José Renan Cunha-Melo5

 

 

MAGALHÃES, M.M.; VIANA, G.; ARANTES, R.M.E.; SANTOS, T.M.; CUNHA-MELO, R.R. - The mouse as an experimetal model for tityus serrulatus scorpion envenoming. Acta Cir. Bras. 13(4):205-000, 1998.

SUMMARY: The scorpion toxin induces a number of physiological parameters alterations, as disturbance of cardiac rhythm, heart failure, shock, pancreatic hypersecretion, abortion, respiratory arrhytmias and pulmonary edema. As the purification of the venom fractions is a laborious process, one alternative for this would be the utilization of small animals. We utilized in the present study thity-six mice that received progressive doses of scorpion toxin TsTX), i.p. or i.v., and were observed for three hours or sacrificed, and the pulmonary alterations were determined by the lung-body index and by histological analysis of the lungs in order to determine if the mouse can be an esperimental model for scorpion envenomation. The data were analyzed by One Way analysis of variance with p<0,05 indicating significance. These experiments showed no differences in clinical signs of scorpion envenomation between mice and other mammalians, the effects were dose-dependent and the i.v. administration needed less quantity to produce the same changings. In the pulmonary histology we observed septal but not alveolar edema, and we presumed that these differences are due to species-specific variations.
SUBJECT HEADINGS: Scorpion toxin. Scorpion envenomation. Experimental model for intoxication.

 

 

INTRODUCTION

The toxin from scorpion Tityus serrulatus is a neurotoxin that acts on sodium channels of the postganglionic nerve endings of the autonomic nervous sistem (ANS), inducing nerve depolarization5,6,15. It causes a massive simpathetic and cholinergic mediators discharge and subsequent effects are, in their majority, mediated by the release of those neurotransmitters, mainly acetylcholine, catecholamines and substance P3. The action of the mediators on their specific receptors are responsible for the clinical symptoms of scorpion envenomation. Injection of a crude scorpion venom or its purified toxins in several species produce a number of physiological parameters alterations. The cardiovascular manifestations consist of striking effects on the cardiac rhythm (tachicardia,bradicardia, sinusal and auriculo-ventricular block, ventricular and ectopic multifocal beats, bigeminism, sinusal disrhythmia, idioventricular rhythm, and ventricular paroxistic tachicardia), arterial hypertension or hypotension, heart failure and shock2,7,9,10.

Respiratory arrythmias, hyperpnea and "gasping" breath have been described. The "gasping" breath is characterized by a strong contraction of the diaphragm and thoracic inspiratory muscles. The respiratory arrhythmias usally assume the" Cheyne-Stokes" or Biot pattern of breathing2. The mechanism of the hemorrhagic edema that follows the intravenous injection of purified scorpion toxin into unaesthetized rats is still unkown. Nowadys, a multifactorial hypothesis to explain the pulmonary edema has been proposed, including neuroedocrinogenic15, cardiogenic1,7,9, immunological changings12 and direct damage of the lung membranes1,13.

Scorpion toxin also induces pancreatic hypersecretion in several animal species, and acute pancreatitis in humans and rats. Muscular paralysis14 and acute renal failure13 have been described in human beings. The descrition of abortion in the mice females injected by T1 fraction venom and in stung woman suggested an ocitocic action of the scorpion venom11.

The purification of toxins fraction from the crude scorpio’s venom is a laborious and extensive procedure8. If one can utilize smaller amounts of toxin to determine its mechanism of action this would be worthwile, at least to the economical viewpoint. One alternative for this would be the utilization of smaller animals for experimental purposes, that could be intoxicated very low doses of toxin.

The aim of the present sudy is to investigate the clinical symptoms of mice inoculated with the purified fraction T1 from the scorpion tityus serrulatus toxin, and the pulmonary alterations as determined by the lung-body index and by histological analysis of the lungs in order to determine if the mouse can be used as an experimental model for scorpion envenomation.

 

METHOD

Animals: Thirty six male "swiss" mice, weighting from 29,5 to 38,6 g., were distributed in six groups of six animals each. The purified scorpion toxin (tityustoxin, TsTX) was obtained from the venom of the Brazilian scorpion Tityus serrulatus by a combination of extraction and chromatographic techniques, using a sephadex column, according to the procedure described by Gomez & Diniz (1966)10. The quantity of venom or saline solution administered to each animal, and the via, were as follows:

Group 1 - controls injected with 0.05ml of saline solution, i.p.
Group 2 - animals inoculated with 175,5µg/100g of TsTX, i.p.
Gorup 3 - animals inoculated with 187,5µg/100g of TsTX, i.p.
Group 4 - animals inoculated with 200µg/100g of TsTX, i.p.
Group 5 - animals inoculated with 225µg/100g of TsTX, i.p.
Group 6 - animals inoculated with 100µg/100g of TsTX, i.v.

The groups from 1 to 5 were inoculated through the intraperitoneal (i.p.) route. The last group, through the intravenous (i.v.) route, using a tail vein.

After the toxin inoculation mice were observed for thee hours. The behavior, type of breathing, salivations, clinical status and death were observed and reported in a sheet of paper for each mouse. The mice that survived after this period of time were sacrificed by decapitation in a guillotine (beheaded).

Lung/body index: The lungs were dissected free from trachea and weighed. The lung/body index was calculated according to the formula: lung weightx100/body weiht.

Light microscopy: A tranversal cut, passing through the pulmonary hilus, was made, and the lungs were immersed in both 10% neutral formalin and Bouin solution and examined under light microscopy after embedding in paraffin and staining with hematoxilin-eosin.

Statisticalo analysis: The data were analyzed by the One Way analysis of variance, with p<0,5 indicating significance.

 

RESULTS

The clinical observation showed that the scorpion toxin treated rats presented tachypnea, pilorection and hypereactivity as early as five minutes after toxin injection. Runs along the cage, rapid mouvements with the paws and involuntary strong muscular contractions of the dorsal muscles. These symptoms progressively became worse and at twenty minutes an ataxic march, sialorhea, milky uretral secretion, increase irritation and erect posture on the rear paws could be noticed. At twent five minuts, they developed severe dispnea and "bloody tears" corresponding to the Hardi Glands secretion and some of them died. The number of deaths per group was proportional to the toxin dose (table I). The death rate changed according to the dose and the administration route, showing a direct proportion between the dose and severity of poisoning. The dose of 100µg/100g of toxin injected into the tail vein killed the same number of animals as an intraperitoneal dose of 175µg/100g.

 

Table I - Dose-related deaths of mice injected with scorpion toxin

Groups (n=6) Deaths: number (%) Route of venom administration Time elapsed to death (minutes)
1 - Saline - (Control) 0 0 i.p. 0
2 - Toxin - 175.0µg/100g 2 33 i.p. 27,5
3 - Toxin - 187.5µg/100g 4 66 i.p. 36
4 - Toxin - 200.0µg/100g 5 83 i.p. 9,3
5 - Toxin - 225.0µg/100g 6 100 i.p. 30,8
6 - Toxin - 100.0µg/100g 2 33 i.v. 22,5

i.p = intraperitoneal route, i.v. =intravenous route.

 

All lung from mice injected with scorpion venom showed some anatomopathological alterations. These were peripheral and central multifocal venous congestion (peribronchiolar hemorrhage, mainly peri-hilar, focus of the intraalveolar hemorrhage, and septal edema). Its intensity varied from light to pronounced, but it could not be related with dose, survival time or death of the animals. Furthermore, focal intraparenchymatous hemorrhage was more frequent in naturally dead animals than in sacrificed ones. The observed edema was septal, peribronchial and peribronchiolar, but not alveolar (table II). In spite of the above findings there was no statistical differences between the various calculated indexes.

 

Table II - Lung-body index of mice poisoned with TsTX.

Group and toxin dose (µg/100g) Via of inoculation Lung-weight (g) (mean ± S.E.M.) Body weight (g) (mean ± S.E.M.) Lung-body index (x 10-3)
1 - Control i.p. 0,26 ± 0,052 30,7 ± 4,79 8,4 ± 1,7
2 - 175.0 i.p. 0,23 ± 0,024 29,9 ± 3,82 7,6 ± 1,3
3 - 187.5 i.p. 0,23 ± 0,039 32,5 ± 4,47 7,2 ± 1,5
4 - 200.0 i.p. 0,25 ± 0,045 38,0 ± 3,16 6,9 ± 0,8
5 - 225.0 i.p. 0,21 ± 0,015 29,5 ± 2,25 7,0 ± 0,7
6 - 100.0 i.v. 0,30 ± 0,079 38,6 ± 3,78 7,8 ± 2,2

  i.p. = intraperitoneal and i.v. = intravenous.

  

No significant statistical differences where observed between the lung-body indexes among the groups.

 

DICUSSION

The clinical data observed in these experiments showed that there were no differences betwere the scorpion poisoning in mice and others mammalians, concerning the clinical signs of evenomation2,5,9. The effects were dose-dependent and the endovenous administration needed less venom than intraperitoneal route to induce comparable effects. The intensity of the symptoms showed no correlation with the death rate, e.g. in the group injected by i.v. route the symptoms were exuberant, but the death rate was oly 33%. This rate was the same observed in the group injected by 125µg/100g i.p., where the symptoms were milder.

The histological alteration observed in the mice lung did not correlate with those observed in rats1,4,12,13,15. The scorpion venom inoculated in rats shows eosinophilic substance in the alveolar lumen and an intersticial edema more pronounced around the intrapulmonary vessels. A more dicrete intra-alveolar and local perivascular bleeding were also seen. In the mice, it was observed septal but no alveolar edema. It is presume that these differences are due to species-specific variations, the mice being more resistant than rats to envenomation. For example, the injection of a dose of 100µg/100g of toxin i.v. in awaked rats killed 100% of animals in a few minutes, with pulmonary edema in almost all of the animals.

The mouse in our hands did not constitute a good model for studies on the scorpion envenomation, mainly for the toxin-induced pulmonary edema, although on the clinical point of view the signs and symptoms are similar to those observed in other species including men.

 

CONCLUSION

The mice can be used as a model for the sudy of the clinical manifestations of scorpion toxin evenomation, but is not as good as the rat for studying the pathophysiology of scorpion envenoming.

 

REFERENCES

1. AMARAL, C.F.S.; BARBOSA, A.J.A.; LEITE, V.H.R.; TAFURI, L.; REZENDE, N.A. - Scorpion sting-induced pulmonary oedema: evidence of increased alveolocapillary membrance permeability. Toxicon, 32:999-1.003, 1994.         [ Links ]

2. BARROS, E.F. - Aspectos clínicos da intoxicação escorpiônica. Mem. Inst. Biol. Ezequiel Dias 2:101-287, 1983.         [ Links ]

3. CUNHA-MELO, J.R.; FREIRE-MAIA,L.; TAFURI, W.L.; MARIA, T.A. - Mechanism of action of purified scorpion toxin. T. Serrulatus on the isolated rat intestine. Toxicon 11:81-4, 1973.         [ Links ]

4. FREIRE-MAIA, F.; ALMEIDA, H.O.; CUNHA-MELO, J.R.; AZEVEDO, A.D.; BARROSO, J. - Mechanism of the pulmonary edema induced by intravenous injection of scorpion toxin in the rat. Agents Actions 8:113-8, 1978.         [ Links ]

5. FREIRE-MAIA,L.; CUNHA-MELO, J.R.; FUTURO-NETO, H.A.; AZEVEDO, A.D.; WEINBERG, J - Cholinergic and adrenergic effects of tityustoxin. Gen. Pharmacol. 7:115-21.         [ Links ]

6. FREIRE-MAIA,L. & DINIZ, C.R. - Pharmacologic action of a purified scorpion toxin in the rat. Toxicon 8:132, 1970.         [ Links ]

7. FREIRE-MAIA,L.; PINTO, G.I.; FRANCO, I. - Mechanism of the cardiovascular effects produced by purified toxin in the rat. J. Pharmacol. Exp. Ther. 188:207-13, 1974.         [ Links ]

8. GOMEZ, M.V. & DINIZ, C.R. - Separation of toxin components from the Brazilian scorpion (T. Serrulatus) venom. Mem. Inst. Butantan 33:890-9.         [ Links ]

9. GUERON, M.; ILIA, R.; SOFER, S. - The cardiovascular system after scorpion envenomation. J. Toxicol Clin. 30:245-58.         [ Links ]

10. MALHOTRA, K.K.; MIRDEHGAN, C.M.; TANDON, H.D. - Acute renal failure following scorpion sting. Am. J. Trop. Med. Hyg. 27:623-6, 1978.         [ Links ]

11. MENDONÇA, M.; PROFETA DA LUZ, M.; FREIRE-MAIA,L.; CUNHA-MELO, J.R. - Effect of scorpion toxin from Tityus serrulatus on the contraction of isolated rat uterus. Toxicon 33:355-61, 1995.         [ Links ]

12. QUELUZ, T.T.; BRUNDA, M.; BLADUTIL, A.O.; BRENTJENS, J.R.; ANDRÉS, G. - Morphological basis of pulmonary edema in mice with citokine-induced vascular leak sindrome Exp. Lung. Res. 17:1095-108, 1991.         [ Links ]

13. ROSSI, M.A.; FERREIRA, A.L.; PAIVA, S.M. - Fine strutures of pulmonary changes induced by Brazilian scorpion venom. Arch. Path. 97:284-8, 1974.         [ Links ]

14. SMITH, L.R.; POTGIETER, P.D.; CHAPELL, W.A. - Scorpion sting producing severe muscular paralylis: a case report. S. Afr. Med. J. 64:69-70, 1983.         [ Links ]

15. VISHER, M.B.; HADOY, F.J.; STEPHEN, G. - The physiology and pharmacology of lung edema. Pharmacol. Rev. 8:369.         [ Links ]

 

 

MAGALHÃES, M.M.; VIANA, G.; ARANTES, R.M.E.; SANTOS, T.M.; CUNHA-MELO, R.R. - O camundongo como modelo experimental de envenenamento pelo escorpião tityus serrulatus. Acta Cirurgica 13(4):205-000, 1998.

RESUMO: A toxina do escorpião induz a várias alterações fisiológicas, como disturbio do ritmo cardíaco, insuficiência cardíaca, choque, hipersecreção pancreática, aborto, arritmias respiratórias e edema pulmonar. A purificação de frações do veneno é um processo trabalhoso. Como alternativa utilizam-se animais pequenos. No presente estudo utilizou-se 36 camundongos que receberam doses progressivas de toxinas do escorpião (TsTX), intraperitoneal ou intravenosa e foram observados por tres horas ou sacrificados. As alteraçòes pulmonares foram determinadas pela fórmula peso do pulmão x 100/ peso corporal e pela análise hitológica dos pulmões a fim de determinar que o camundongo pode ser um modelo experimental do envenenamento pelo escorpião. Os dados foram analizados pela análise de variância considerando-se p<0,05 indicando significancia. Os experimentos não mostraram diferença nos sinais clínicos do envenenamento comparando-se o camundongo com outros mamíferos. Os efeitos foram dose-dependente e que pela via venosa necessita-se menos quantidade para produzir as mesmas alterações. Nos aspectos histológicos pulmonares observou-se edema septal e não alveolar. Presume-se que as diferenças observadas são devidas a variações específicas das espécies.
DESCRITORES: Toxina de escorpião. Envenenamento por escorpião. Modelo experimental de intoxicação.

Department of Surgery, Faculty of Medicine and GEN-CAD Service, HC, Federal Univercity of Minas Gerais, Belo Horizonte - MG, Brazil.

 

 

 

Address for reprints:
Professor José Renan Cunha-Melo
Departamento de Cirurgia FM - UFMG
Av. Alfredo Balena, 190, Santa Efigênia
CEP30130-100
Belo Horizonte, MG, Brasil
Tel/Fax: (031)273-6530
E-mail: jremelo@medicina.ufmg.br.

Financial Support: CNPq, FAPEMIG

Accepted for publication: october 05, 1998

 

 

1 - Article from the Departament of Surgery, Faculdade de medicina da Universidade Federal de Minas Gerais (UFMG).
2 - MD.
3 - Assistant-Professor of Pathology, ICB, UFMG.
4 - Full Professor, School of Pharmacy, UFMG.
5 - Full Professor of Surgery, School of Medicine, UFMG, Financial Support: Cnq, FAPEMIG.

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