Standardization of an experimental model of parabiotic isolated heart in rabbits

Padronização de modelo experimental de coração isolado parabiótico em coelhos

STANDARDIZATION OF AN EXPERIMENTAL MODEL OF PARABIOTIC ISOLATED HEART IN RABBITS¹

Antônio Sérgio Martins²

Marcos Augusto de Moraes Silva²

Beatriz Bojokian Matsubara³

Flávio Aragon⁴

Carlos Roberto Padovani⁴

Martins AS, Moraes-Silva MA, Matsubara BB, Aragon F, Padovani CR. Standardization of an experimental model of parabiotic isolated heart in rabbits. Acta Cir Bras [serial online] 2000 Jul-Sept;15(3). Available from: URL: http://www.scielo.br/acb.

ABSTRACT: The objective was to standardize an experimental model of parabiotic isolated heart in rabbits, testing their stability and durability for use in cardiovascular research. Sixty-six Norfolk-2000 rabbits were used and divided in 2 groups: donors of the isolated heart and support animals, in a total of 33 preparations. Circulatory support for the isolated heart was established with the aid of peristaltic pumps and the flow was kept constant (16ml/min.). An intraventricular balloon was inserted in the left ventriculum, and was adjusted to produce diastolic pressure of ± 10mmHg. Heart rate was established at 120 beats per minute with the use of a pacemaker. Hemodynamic, laboratory and histopathological parameters were evaluated. Of the 33 preparations, 13 were excluded according to preestablished criteria. Of the 20 remaining preparations, 10 completed the maximum protocol time (180 minutes). There was progressive hemodynamic deterioration with decrease of mean blood pressure (89.30±6.09mmHg->47.50±6.35mmHg) in the support animal. There was hemodynamic stability of the isolated heart for the 10 preparations that completed the 180 minutes of the protocol. Laboratory parameters showed progressive decrease of sodium, potassium and hemoglobin, which is compatible with hemodilution. Histopathology showed greater distance among the fibers, compatible with edema. The present model showed 100% stability and activity of the preparations within 60 minutes, and 50% of the active preparations were progressively lost within 180 minutes. The present model is viable for cardiovascular research.

SUBJECT HEADINGS: Heart. Casts, surgical. Ventricular function.

INTRODUCTION

Reliable experimental models are important for successful experiments. In the cardiovascular field, the isolated heart maintained by nutrient solutions first introduced by LANGENDORFF¹ has gained importance due to its simplicity. HEYMANS and KOCHMANN, in 1904², published a variation of LANGENDORFF's model using a support animal. The lack of effective anticoagulation has limited the use of the method. The use of nutrient solutions is limited by the size of the heart and by the intense edema produced^3,4. The use of blood as perfusate has physiological advantages such as less edema, however support animals have to be used ⁵. The isolated heart model may be used in almost all laboratory animals, and has been reported in dogs^6,7, mice⁸, guinea pigs⁹, pigs¹⁰ and rabbits^11,12. The rabbits showed advantages when compared to other smaller animals, such as a larger heart, which makes surgical instrumentation easier, composition of myosins and calcium kinetics similar to humans, as opposed to mice¹³.

OBJECTIVE

Standardize the experimental model of parabiotic isolated heart in rabbits, testing their stability and durability in cardiovascular research.

METHODS

Sixty-six Norfolk-2000 rabbits of both genders, weighing 2270 to 2700 grams were used. The animals were prepared according to the "Guide for Care Use of Laboratory Animals."¹⁴ written by the United States National Science Academy.

Two animals were randomly used in each experiment. One as support animal and the other as a donor of the isolated heart.

Experimental times

After 15 minutes of perfusion, time zero was established (T0) and then after every 30 minutes(in a total of 180 minutes)establishing times T1, T2, T3, T4, T5 and T6.

Experimental model

The animals were anesthestized with pentobarbital sodium (30mg/Kg i.v.) and heparinized (3mg/Kg).

The isolated heart was perfused, at the root of the aorta, with blood from the support rabbit, obtained from the cannulation of the right carotid artery and pumped by a peristaltic infusion pump. Venous return was carried out by gravity, through the pulmonary artery of the isolated heart, which was previously cannulated in the right jugular vein of the support animal. A latex balloon was inserted in the left ventriculum to measure its function. Two pacemaker electrodes for stimulation (120epm) and a 3 mm polyvinyl cannula to read right atrial pressure were placed in the right atrium. The isolated heart was immersed in a basin with saline solution and circulation of heated water to maintain the temperature at 38^oC.

The left carotid artery of the support animal was catheterized to obtain blood samples and for mean blood pressure monitoring (MBP). The jugular vein was catheterized for drug infusion (Ringer 5ml/kg and 8.4% sodium bicarbonate at 1.0mEq/Kg/hour) and central venous pressure monitoring (CVP).The support animal was placed on a thermal mattress to maintain rectal temperature at 38^o C. The support animal was artificially ventilated with 35 cycles per minute. The isolated heart and support animal were connected to a polygraph (EEP-12 by Electronics for Medicine).

At the end of the experiment, fragments of the left ventriculum of the isolated heart and support animal were obtained for histopathological analysis under light microscopy and H-E staining.

Statistical analysis

Repeated multivariate analysis were performed for the ten preparations completing the protocol time¹⁵.

RESULTS

Of the 33 initial preparations, 13 were excluded according to criteria shown in Table 1.

Of the 20 remaining preparations, 10 completed the total protocol time (180 minutes). From T3 (90 minutes) there was progressive loss of the preparations due to death of the support animal (Table 2.).

Hemodynamic parameters of the support animal.

Mean Blood Pressure (MBP) and central venous pressure (CVP) .

Mean and respective standard deviations at the studied experimental times, as well as the statistical analysis results, are shown in Table 3.

Hemodynamic parameters of the isolated heart.

Developed pressure (DP), diastolic pressure (DP), peak first time derivative of intraventricular pressure (dp/dt+), coronary resistance (CR), right atrial pressure (RAP) and perfusion pressure(PP)

Mean and respective standard deviation at the studied experimental time as well as the statistical analysis results are shown in Table 4.

Laboratory Parameters

Sodium, potassium, calcium, glycose, hemoglobin.

Mean and respective standard deviations at the studied experimental times, as well as the statistical analysis results are shown in Table 5.

pHA, PaO ₂, PaCO₂, HCO₃, Sata.

Mean and respective standard deviations at the studied experimental times, as well as the statistical analysis results are shown in Table 6.

Somatic parameters.

Rectal temperature of the support animal, temperature of the isolated heart, heart rate of the support animal.

Mean and respective standard deviations at the studied experimental times, as well as the statistical analysis results are shown in Table 7.

Histopathology

All of the isolated hearts showed the fibers were further apart and 8 of them had signs of mild interstitial hemorrhage(fig.1).

DISCUSSION

The rabbit has advantages when compared to smaller animals, such as easy racial, age and gender standardization, and availability in vivarium. Due to their size, the surgical manipulation of the heart is easier. Myosin composition and calcium kinetics are similar to human myocardium¹³. Therefore, the rabbit is an adequate choice for cardiovascular research. The isolated heart method may be carried out using nutrient solutions as perfusate. Adequate preparation and stability are required for the results obtained in the experiment to be valid. However, it does not require the use of another animal, which has to be done in the parabiotic model. The blood avoids severe edema, which is usual in nutrient solutions and the support animal works as a perfect homeostatic control¹⁶.

The flow system or constant pressure system may be used, each of them with advantages, disadvantages and in the present study, constant flow system was used according to DORING e DEHNERT's formula¹⁷.

The intraventricular balloon method was used to asses ventricular function¹⁸ with a preestablished diastolic pressure of ± 10mmHg.

Fixed ventricular rate is required for a comparison of the experimental parameters throughout the different experimental times. Based on literature, we chose a rate of 120 beats per minute^{19, 20}.

The major exclusion cause was related to respiratory infection of the support animals, a severe condition leading to early death of the animal. In the present study, 90% of the preparations were active 120 minutes after the beginning of the experiment, an adequate result for most of the existing experimental protocols.²¹ Hemodynamic parameters of the support animal showed progressive decrease of mean blood pressure. Since volemia was kept constant (CVP p>0.05) and knowing that the death of the support animal was the major cause of failure of the preparations, it may be concluded that other factors could explain this condition, such as progressive vasoplegia resulting from extended extracorporeal circulation periods, i.e., the exposure of blood elements to non biological surfaces produces the inflammatory syndrome whose final expression is severe hypotension and death²².

Hemodynamic parameters of the isolated heart were compatible with literature reports²³ and showed stability throughout the protocol.

Laboratory parameters showed progressive hemodilution, probably due to blood loss resulting from manipulation and sample collection. Calcium stability, which was continuously replaced reinforces this idea.

Histopathology showed greater distance among the fibers, indicating edema(fig.1). Signs of interstitial hemorrhage may be explained by perfusion pressure slightly higher than average for the flow used in the study, and heparinization.

CONCLUSIONS

The present model of parabiotic isolated heart showed adequate stability and reliability for use in cardiovascular research.

Acknowledgements

We wish to thank Ms.Rosemary Cristina da Silva for technical support.

Martins AS, Moraes-Silva MA, Matsubara BB, Aragon F, Padovani CR. Padronização de modelo experimental de coração isolado parabiótico em coelhos. Acta Cir Bras [serial online] 2000 Jul-Sept;15(3). Available from: URL: http://www.scielo.br/acb.

RESUMO: O objetivo foi a padronização de modelo experimental de coração isolado parabiótico em coelhos, testando sua estabilidade e durabilidade, para fins de pesquisa cardiovascular. Foram utilizados 66 coelhos raça Norfolk-2000 divididos em grupo doador do coração isolado e animais suporte, totalizando 33 preparações. Mediante auxilio de bombas peristálticas estabeleceu-se suporte circulatório para o coração isolado mantendo-se fluxo constante(16ml/min.). Um balão intraventricular foi inserido no ventrículo esquerdo, sendo ajustado para gerar pressão diastólica de ± 10mmHg. A freqüência foi fixada em 120 batimentos por minuto mediante o uso de marcapasso. Foram avaliadas variáveis hemodinâmicas, laboratoriais e anatomopatológicas. Das 33 preparações, 13 foram excluídas mediante critérios pré-estabelecidos. Das 20 restantes, 10 cumpriram o tempo máximo do protocolo(180 minutos). Com relação ao animal suporte houve deterioração hemodinâmica progressiva c/ queda da pressão arterial média(89,30±6,09mmHg->47,50±6,35mmHg). Com relação ao corações isolado, das 10 preparações que cumpriram os 180 minutos de protocolo, houve estabilidade hemodinâmica. As variáveis laboratoriais mostraram queda progressiva do sódio, potássio e hemoglobina, sendo compatível com hemodiluição. O exame anatomopatológico mostrou espaçamento maior entre fibras, compatível com edema. O presente modelo mostrou estabilidade e atividade de 100% das preparações em 60 minutos, havendo perdas progressivas chegando a 50% das preparações em atividade em 180 minutos. O presente modelo, dentro das limitações estabelecidas é viável para pesquisas cardiovasculares.

DESCRITORES: Coração. Modelos cirúrgicos. Função ventricular.

Address for correspondence:

Caixa Postal 539 Campus Unesp Botucatu

Botucatu - São Paulo

18618-000

e-mail:[e-mail]

Data do recebimento: 31/05/2000

Data da revisão: 16/06/2000

Data da aprovação: 14/07/2000

2. Department of Surgery FMB-UNESP;

3. Department of Cardiology FMB-UNESP;

4. Department of Statistics IB-UNESP.

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1

. Experimental LaboratoryDepartment of Surgery FMB-UNESP;

Publication Dates

• Publication in this collection
  04 Sept 2000
• Date of issue
  Sept 2000

History

• Accepted
  14 July 2000
• Reviewed
  16 June 2000
• Received
  31 May 2000