Upsala J Med Sci 87: 235-242, 1982 

A Paracorporeal Rat Heart Model for Ischemic and 
Reperfusion Studies 

Jan Hultrnan, Jan Ola Forsberg, Hans Erik Hansson and Gunnar Ronquist 
Department of Anaesthesiology, Department of General Surgery, Department of Thoracic and 

Cardiovascular Surgery and Department of Cfinical Chemistry, 
University Hospital, Uppsala, Sweden 

ABSTRACT 

Paracorporeal rat hearts were supplied with blood derived from the abdominal 

aorta of a supporting rat. The circulatory stability and working capacity of 

the supporting animal was analyzed in the experimental situation in terms of 

PO2, SO2, PC02, HC03, pH and electrolytes, all of which were within the normal 

range before and during a 60 min period of paracorporeal perfusion. For evalua- 

tion of ischemic damage in this model studies were made on three groups of ex- 

cised hearts. They were subjected to 10, 15 or 20 min of complete global ische- 

mia at 37OC (ambient temperature) and reperfused for 30 min, including ECG, 
observations of contractility and an analysis of creatine kinase efflux in the 

coronary effluent. The results showed good reproducibility and the data were in 

accordance with reports from similar studies on Langendorff preparations. The 

model, which is easily set up, inexpensive and based upon pulsatile blood per- 

fusion, should be more physiologic than the conventional Langendorff prepara- 

tion. 

INTRODUCTION 

Improved myocardial protection is probably the most important contribution 

to recent advances in open-heart surgery. The clinical improvements are greatly 

attributable to extensive experimental laboratory investigations (1, 2, 13, 16) 

The isolated rat heart model has proved to be very useful in studies of myo- 

cardial ischemia, and experimental experiences have led to clinical innovations 

(10). An often used rat heart model is the Langendorff preparation (15) and its 

modifications (la), in which an isolated heart is perfused with a crystalloid 

solution in a non pulsatile way, usually a Krebs-Henseleit solution. In the 

clinical situation, however, the conditions are different. After the ischemic 

period the aortic cross-clamp is released and a pulsatile coronary blood flow 

is reinstituted. 

The paracorporeal perfusion technique is known from earlier experiments (6, 

17, 20). Gamble et al. (6) modified the paracorporeal heart model in the rat 

235 



but did not use a direct pulsatile retrograde perfusion technique in the aorta 

of the excised heart. Aiming at a situation closer to the clinic we have modi- 

fied the paracorporeal rat heart model. Its reliability was evaluated and data 

on enzyme efflux correlated to ischemic heart damage are presented. 

MATERIAL AND METHODS 

Rats - 
Non-starved, 300 g male Sprague-Dawley rats were used. 

The exoerimental model 

The paracorporeal rat heart model consists of three main parts (Fig. 1): 
1. The supporting rat. 2. The excised heart. 3 .  The retransfusion system. The 

ascending aorta of the excised heart was connected to the abdominal aorta of 

the supporting rat by a tube ("pump tube"). The coronary effluent from the 

excised heart was collected in a temperated water-jacketed funnel and retrans- 

fused to the supporting rat. After the ischemic period pulsatile blood flow was 

established from the supporting rat to the excised heart. The temperature of 

the supporting rat and the excised rat heart was kept at 3 7 O C  C l0C. 

Cannubtion 
obdominol- 
aorto 

Peristaltic 
roller 
pump 

Fig. 1. The experimental model 
The experimental model is composed of three main parts: 
1. The supporting rat 
2. The excised heart 
3 .  The retransfusion system 

236 



Surgical and technical procedures 

administration of InactinR (Byk-Gulden, Germany) , 120 mg-kg 
stomy the right carotid artery was cannulated for continuous blood pressure re- 
cording via an EMT 34 transducer (Elema-SchBnander, Sweden) and a M 34 recorder 
(Elema-SchGnander, Sweden). The right jugular vein was cannulated for transfu- 
sion. Heparin in a dose of 200 IU was given intravenously. The supporting rat 

was breathing air spontaneously. 

Rats intended for supporting function were anaesthetized by intraperitoneal 
-1 . After tracheo- 

The abdominal aorta was cannulated and the cannula was fixed to the aorta by 

silk ligatures. Close to the aorta, the cannula was cut and rejoined by a more 
flexible piece of silicone rubber tubing to permit easy clamping. Connections 

permitted blood sampling and infusion. The body temperature was recorded con- 
tinuously in the abdominal cavity. 

Heart donors were anaesthetized with ether. After 200 IU of heparin intra- 

venously, the blood was rapidly collected from the abdominal aorta. Following 

thoracotomy and ligation of the aortic arch branches the heart-arch preparation 
was excised. The time required for this procedure was 3-5 min. 

The ascending aorta was attached to the cannula from the supporting rat. 

Electrodes for bipolar ECG recording were placed at the apex, the right atrium 
and the aortic arch. During the ischemic period the heart was kept in a water- 

jacketed funnel filled with plain saline at 37OC-38OC. 
The retransfusion system consisted o f  a peristaltic roller pump (Multiperpex 

2115, LKB Products, Broma, Sweden), a temperature controlled water bath and an 
air bubble trap. All connections were of silicon rubber tubing. The system was 

primed with 20 ml of heparinized blood collected from the heart donor and an 
additional rat. 10 ml of saline was added to the system during the first 7 d n  
of reperfusion. Subsequently no extra volume was added. The retransfusion of 

blood was adjusted by regulating the height of the bubble trap. The mean arte- 

rial blood pressure (MABP) of the supporting rat was maintained at 95+10 mu Hg. 

Experimental protocols 

To test the endurance to ischemia, excised hearts were subjected to complete 
global ischemia at 37OC (ambient temperature) for 10, 15 or 20 min. The number 

of hearts examined in these three groups was 6, 8 and 7, respectively. Samples 
of arterial blood from the supporting rat were obtained before reperfusion was 
started and 10 and 30 min after the start. Sampling of the coronary effluent 
from the excised hearts was performed via the funnel after 10, 20 and 30 min of 

reperfusion. No buffer solutions were used. The following recordings were made: 

1. Supporting rat: MABP and heart rate. 
2. Arterial line (pump tube): NABP 

PO2’ S02’ PC02’ PH 

16-822858 237 



HC03-, base excess (BE) 

Electrolytes Na', K+, Ca 
++ , and glucose 

3. Excised heart: ECG (continuous recording) 

4. Coronary effluent: Flow rate 
Creatine kinase (CK) efflux 

The CK activity was determined according to the recommendations given by the 

Scandinavian Committee on Enzymes. For background measurements samples were 

taken from the supporting animal and from the blood prime before the start of 

reperfusion. 

For control six excised hearts were reperfused directly after cannulation 

for determination of CK efflux and to evaluate the influence of enzyme efflux 

from the supporting rat, another six rats were cannulated as supporting animals, 

but with no excised heart in the paracorporeal line. The flow through the cannu- 

la was adjusted to 8 ml-min . Samples taken from the blood prime and from the 
paracorporeal line after 10, 20 and 30 min were analysed for CK activity. 

-1 

RESULTS 

Supporting rat 

Most rats showed a stable condition during the experiment. Their body tempe- 

rature, blood gases, electrolytes and blood pressure had to be within normal 

range for acceptance as supporting animals. 

Sodium, potassium and calcium concentrations in samples from the arterial 

line determined in six cases before reperfusion and after 10, 20 and 30 min 

(ischemic periods 10, 15 and 20 min) were all within the normal range. Arterial 

blood gases analysed before and 10 and 30 min after the start of reperfusion 
showed only minor variations, indicating high stability in the system (Table I). 
For control, additional blood gas analyses performed on blood samples after 5, 

15 and 20 min of reperfusion with the same results. Blood glucose values at 

repeated determinations were within the normal range. 

Table I. Blood gas data for the supporting rats before and after 10 and 30 min 
of reperfusion of excised hearts subjected to 10, 15 and 20 min of 
complete global ischemia at 37OC. A s  there was no significant diffe- 
rence between the three groups, the means and SD were calculated from 
all supporting rats. Significant differences (p<0.05) during support 
compared with pre-supporting values are indicated with an asterisk. 
n = number of animals. 

PH PC02 (kPa) PO2 (kPa) BE so HC03 
(78 ( m o l .  1-5 

Before 
reperfusion 7.40-10.03 5.3k0.6 12.4t1.0 -0.1k1.7 95t1 24t1 

n 19 19 18 18 18 18 
10 min of 
reperfusion 7.37k0.03 5.0k0.5 13.0k1.5 -3.1t2.0* 96t1 2 . 2  2* 

n 20 20 20 19 19 19 
30 min of 
reperfusion 7.38k0.03 5.1k0.5 1 2 . 2 t 1 . 2  -2.3k1.6 95k1.3 231.3 

n 22 22 2 2  2 1  21 21 
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The hemodilutive effect of added saline resulted in a Hb concentration of 

(SM2.1) at 10 and 30 min of reperfusion, -1 13.6 g.1-l (SItt2.1) and 14.4 gel 
respectively, as compared with 17.5 g-1 (SM2.0) before transfusion. -1 

Excised hearts 

All hearts subjected to 10 min of ischemia regained sinus rhythm and a rate 
of at least 140 beatslmin after 10 min of reperfusion. After 15 min of ischemia 

only 113 of the hearts returned to normal rhythm, while the other hearts deve- 
loped serious arrhythmias. After 20 min of ischemia no heart returned to normal 
rhythm (Table 11). 

Table 11. Heart activity after 10 min of reperfusion of hearts subjected to 
10, 15 and 20 rnin of complete global ischemia at 37OC, verified by 
ECG and observed contractility. n = number of examined hearts. 

Duration of pre-reperfusion ischemia 
10 min 15 rnin 20 min 
n = 10 n = 12 n = 9  

Sinus rhythm 

Non-reversible 

140 beatslmin 10 4 - 

8 9 
Heart activity 

arrhythmias - 

The coronary flow rate in the 15-min ischemic group is given in Table 111. 

It was already fairly high during the first minute and increased to a maximum 
of 9 ml/min after 4-5 rnin of reperfusion. After 20 rnin a constant flow rate of 
about 5 ml/min was established. 

-1 Table 111. Coronary flow (ml-min ) during reperfusion of 7 excised hearts 
subjected to 15 min of complete global ischemia at 37OC. Sampling 
periods one min. Mea&SD. 

Duration of 
reperfusion 
(min) 0- 1 2' 4' 10' 20' 30' 

Flow 
ml-min -' 6.e1.7 8.7t2.4 9.222.9 7.1k1.9 5.011.8 4.6? 1.5 

The CK background activity was found to be stable, with a mean of 8.1 
(SD * 2.0 ukat.1 ) in supporting animals (27 animals) and 4.8 (SDt 1.8 
ukat-1-l) in the blood prime (27 experiments). In the control group with no 
heart in the paracorporeal line the background activity was similar. 

-1 

The CK activity in the coronary effluent after 10, 20 and 30 min of reper- 

fusion showed similar patterns depending upon the period of ischemia (Table IV). 

In control samples after 40 and 50 min of reperfusion there was a greater in- 
crease in the accumulation of CK in the 20-min ischemic group compared to the 
other groups. A significant difference (p<0.05) between the CK activity in 

239 



samples from h e a r t s  s u b j e c t e d  t o  10 rnin of i s c h e m i a ,  compared t o  t h e  15- and 20- 

rnin groups, was observed. The d i f f e r e n c e  between t h e  two l a s t  g r o u p s ,  however, 

was i n s i g n i f i c a n t .  I n  t h e  c o n t r o l s  t h e  e f f l u x  of CK was low (Table IV). 

-1 
Table I V .  The e f f l u x  of c r e a t i n e  k i n a s e  ( u k a t - 1  ) i n  t h e  c o r o n a r y  e f f l u e n t  

a f t e r  10, 2 0  and 30 rnin of r e p e r f u s i o n  o f  h e a r t s  s u b j e c t e d  t o  10, 
15 o r  20 min of complete g l o b a l  ischemia a t  37OC. To compare, v a l u e s  
a r e  g i v e n  from a group of e x c i s e d  h e a r t s  r e p e r f u s e d  d i r e c t l y  a f t e r  
c a n n u l a t i o n  and a n o t h e r  group w i t h  no e x c i s e d  h e a r t  i n  t h e  para- 
c o r p o r e a l  l i n e ,  i.e. CR e f f l u x  from t h e  s u p p o r t i n g  r a t .  
Mean 2 SD. n = number of e x p e r i m e n t s .  

R e p e r f u s i o n  of e x c i s e d  h e a r t s  
10 min 20 min 30 rnin 

10 min 1 2 . 3 4 . 1  15. W 4 . 3  1 8 . B 4 . 2  
n 6 6 6 

15 rnin 
n 

19.&5.7 30.W9.6 34.72 8 . 7  
8 8 8 

D u r a t i o n  20 min 25.1210.7 3 7 . B 1 3 . 9  4 6 . 2 1 7 . 8  
of n 7 7 7 
ischemia C o n t r o l  group 

n 6 6 6 

C o n t r o l  group 

n 6 6 6 

( r e p e r f u s e d  d i r e c t l y )  7 . % 0 . 7  11.31.1 1 3 . e 1 . 6  

(no e x c i s e d  h e a r t )  6.120.9 7 . 4 2 1 . 1  10. If 1.1 

DISCUSSION 

I n v e s t i g a t i o n s  u s i n g  d i f f e r e n t  models have i n c r e a s e d  t h e  !<nowledge concerning 

h e a r t  metabolism. I n  many ways t h e  Langendorff model and i t s  m o d i f i c a t i o n s  ( 9 ,  

18) a r e  s u p e r i o r  t o  i n  s i t u  models. Any p e r f u s i o n  medium may be a d m i n i s t e r e d  

and any h e a r t  t e m p e r a t u r e ,  p e r f u s i o n  p r e s s u r e  and d e g r e e  of oxygenation can be 

m a i n t a i n e d .  The p e r f u s a t e  i s  e a s i l y  c o l l e c t e d ,  measured and examined. 

. The Langendorff p r e p a r a t i o n ,  however, c o n s t i t u t e s  a d e n e r v a t e d  h e a r t  which 
i s  devoid of normally o c c u r r i n g  humoral f a c t o r s ,  and most o f t e n  a non-corpuscu- 

l a r  p e r f u s i o n  medium i s  used. T h i s  may i n f l u e n c e  n o t  o n l y  t h e  p h y s i o l o g i c a l  

d i s t r i b u t i o n  of oxygen and s u b s t r a t e  t o  t h e  d i f f e r e n t  p a r t s  of t h e  myocardium 

b u t  a l s o  t h e  removal of m e t a b o l i t e s  (6, 2 1 ) .  

These d i s a d v a n t a g e s  can p a r t l y  be overcome by t h e  u s e  o f  a mechanical pump 

and w i t h  donor blood a s  p e r f u s i o n  medium. However, t h e  b e s t  "pump" q u a l i t i e s  i n  

terms of p u l s e  rate and p r e s s u r e  a r e  o b t a i n e d  by t h e  most s u p e r i o r  pump a v a i l -  

a b l e  - a n o t h e r  h e a r t .  Thus, t h e  p a r a c o r p o r e a l  h e a r t  model (17, 20) i s  a middle 
c o u r s e  optimized i n  terms of pump f u n c t i o n  and pump flow. The p a r a c o r p o r e a l  

h e a r t  can be i n f l u e n c e d  by hormones and s u b s t r a t e s  from t h e  s u p p o r t i n g  r a t ,  

which i s  i m p o r t a n t  f o r  c l i n i c a l  a p p l i c a t i o n s .  V a r i a t i o n s  i n  acid-base and 

e l e c t r o l y t e s  can a l s o  be c o r r e c t e d  by t h e  pump rat. 

240 



To stimulate the clinical reperfusion situation we used a retransfusion sys- 

tem. The model was stable for at least 60 min of paracorporeal circulation but 

30 min was enough to cover our interest in early reperfusion after ischemia. 
A prerequisite for good function of this system was that the supporting ani- 

mal was able to stand the surgical trauma and could increase its cardiac output 

to compensate for the high demands due to the high flow through the excised 

heart. These requirements were fulfilled. Thus their acid-base status, blood-gas 

balance and electrolytes were stable both before and during perfusion of the 

excised hearts. Quickly retransfused volumes neither caused deficient reoxygena- 

tion nor signs of "pump" insufficiency in terms of pulse pressure failure in the 

aortic arch of the experimental heart. Concerning the reliability of the model, 
our findings corroborate the results of other investigators using Langendorff 

preparations. Thus, the critical period of complete global ischemia at 37OC was 

between 15 and 20 min (12). The efflux of CK after different periods of ischemia 
was also in accordance with other reports (12, 22). The model showed a high 
degree of reproducibility. Heart function in terms of electrical activity and 

observed contractility correlated well to the ischemic time. The efflux of CK 

was also proportional to the ischemic time. This model can easily be modified 

to allow the use of a left ventricular latex balloon for isovolumetric contrac- 

tion studies (4, 14, 1 9 ) .  

The mechanisms of ischemic heart damage associated with heart surgery have 

been extensively discussed. Increased attention has been paid to the early 

period of recirculation (7, 11). Significant damage to heart muscle seems to 
occur within a few minutes of recirculation. The role of calcium and eventually 

further energy content depletion during the reperfusion period has been 

stressed. Furthermore, an adverse effect of molecular oxygen, 'Ithe oxygen para- 

dox", has been proposed ( 3 ,  12). This may be based upon a possible toxic effect 
of oxygen-derived free radicals, such as the superoxide (0 -) and hydroxyl 

(OH') radicals (5). 
2 

We believe that the presented model, which is simple and inexpensive, is 

suitable for studies of myocardial ischemia and the reperfusion phenomena. 

Although the present experimental arrangement is still far from the clinical 

situation, it is closer than the Langendorff preparation. 

ACKNOWLEDGEMENTS 

The study was supported by grants from The Tore Nilson Fund for Medical 

Research, The Swedish Society against Heart and Lung Diseases, and Fortia A B .  

24 1 



1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

10. 

11. 

12. 

13. 

14. 

15. 

16. 

17. 

18. 

19. 

20. 

21. 

22. 

REFERENCES 
Bretschneider, J.H., Hubner, G., Knoll, D., Lohr, B., Nordbeck, H. & 
Spieckermann, P.G.: Myocardial resistance and tolerance to ischemia. 
Physiological and biochemical basis. J Cardiovasc Surg 16:241-260, 1975. 
Buckberg, G.D., Brazier, J.R., Nelson, R.L., Goldstein, S.M., McConnell, 
D.H. & Cooper, N.: Studies on the effects of hypothermia on regional myo- 
cardial blood flow and metabolism during cardiopulmonary bypass. I. The 
adequately perfused beating, bifrillating and arrested heart. J Thorac 
Cardiovasc Surg 73:87-94, 1977. 
Demopoulous, H.B.: Free radicals in medicine and biology. Acta Phys Scand 
Suppl 492 Symposia, 1979. 
Fallen, E.L., Elliott, W.C. & Gorlin, R.: Apparatus for study of ventricu- 
lar function and metabolism in the isolated perfused rat heart. J Appl 
Physiol 22:836-839, 1967. 
Fridovich, I.: Quantitative aspects of the production of superoxide anon 
radical by milk xanthine oxidase. J Biol Chem 245:4053-4057, 1970. 
Gamle, W.J., Conn, P.A., Edalju Kumar, A., Plenge, R. & Monroe, R.G.: 
Myocardial oxygen consumption of blood-perfused, isolated, supported, rat 
heart. Am J Physiol 219:604-612, 1970. 
Ganote, C.E., Worstell, J. & Kaltenbach, J.P.: Oxygen-induced enzyme re- 
lease after irreversible myocardial injury. Effects of cyanide in perfused 
rat hearts. h e r  J Pathol 84:327-350, 1976. 
Gordon, K.M. & Visscher, M.B.: The mechanism of failure in the completely 
isolated mammalian heart. Am J Physiol 125:461-473, 1939. 
Hearse, D.J., Humphrey, S.M. & Chain, E.B.: Abrupt reoxygenation of the 
anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme 
release. J Molec Cell Cardiol 5:395-487, 1973. 
Hearse, D.J., Humphrey, S.M. & Garlick, P.B.: Species variation in myo- 
cardial anoxic enzyme release, glucose protection and reoxygenation damage. 
J Molec Cell Cardiol 8:329-339, 1976. 
Hearse, D.J.: Reperfusion of the ischemic myocardium. J Molec Cell Cardiol 
9 : 606-616 , 1977. 
Hearse, D.J. & de Leiris, J. (Eds.) Enzymes in Cardiology. Diagnosis and 
research. D.J. Hearse and J. de Leiris, New York, John Wiley & Sons, 
Chichester, Brisbane, Toronto, 417-444, 1979. 
Kirsch, U., Rodewald, G. & Kalmar, P.: Induced ischemic arrest. J Cardio- 
vasc Surg 63:121-130, 1972. 
Kligfield, P., Horner, H. & Brachfeld, N.: A model of graded ischemia in 
the isolated perfused rat heart. J Appl Physiol 6:1004-1008, 1976. 
Langendorff, 0.: Untersuchungen am iiberlebenden SBugetieren. Pfliigers 
Archiv 61:291-332, 1895. 
Melrose, D.G., Dreyer, E., Bentall, H.H. & Baker, J.B.E.: Elective cardiac 
arrest. Lancet ii:217 1955. 
Mulch, J., Schaper, J., Gottwik, M. & Hehrlein, F.W.: Recovery o f  the 
heart after normothermic ischemia. Thorac Cardiovasc Surg 27:145-152, 1979. 
Neely, J.R., Liebermeister, H., Battersby, E.J. & Morgan, H.E.: Effect of 
pressure development on oxygen consumption by isolated rat heart. Amer J 
Physiol 212:804-814, 1967. 
Opie, L.H.: Effect of extracellular pH on function and metabolism of 
isolated perfused rat heart. Amer J Physiol 209:1075-1080, 1965. 
Sarnoff, S . J . ,  Case, R.B., Welch, G.H.Jr., Braunwald, E. & Stainsby, W.N.: 
Performance characteristics and oxygen debt in nonfailing, metabolically 
supported isolated heart preparation. Amer J Physiol 192:141-147, 1958. 
Taegtmeyer, H., Hems, R. & Krebs, H.A.: Utilization of energy-providing 
substrates in the isolated working rat heart. Biochem J 186:701-711, 1980. 
WaldenstrGm, A.P., Hjalmarsson, A.C., Jodal, M. & WaldenstrGm, J . :  Signifi- 
cance of enzyme release from ischemic isolated rat heart. Acta Med Scand 
201:525-532, 1977. 

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