coronary artery ligation in anesthetized rats as a method for the production of experimental...

Coronary Artery ligation in Anesthetized Rats as a Method for the Production of Experimental Dysrhythmias and for the Determination of Infarct Size

COLIN CLARK, MICHAEL I. FOREMAN, KATHLEEN A. KANE,

FIONA M. MCDONALD, and JAMES R. PARRATT

Coronary artery ligation in anesthetized rats was assessed as a method for the

production of experimental dysrhythmias and for the determination of infarct

size. Following occlusion of the left main coronary artery, very marked ventricular

dysrhythmias occurred in two distinct phases, an early and a late phase, at O-30

minutes and at l&--I hours after ligation respectively. Infarct size was measured

at 4 hours after ligation using nitroblue tetrazolium to stain for the depletion of

two cytoplasmic enzymes, lactate dehydrogenase and NADPH diaphorase. In this

model, lignocaine (IO mg/kg plus an infusion of 5 mglkglhr) and ORG 6001 (IO

mg/kg plus an infusion of 2.5 mg/kg/hr) had marked antidysrhythmic activity but

neither drug influenced infarct size. The model, because of its simplicity, re-

producibility, and low cost, should be useful for the screening of potential

antidysrhythmic agents and may also be used to determine infarct size following

coronary artery ligation.

Key Words: Coronary artery ligation; Rat; Dysrhythmias; Infarct size; Ligno-

Caine; ORG 6001.

INTRODUCTION

Coronary artery ligation in large animals, such as the dog, has been widely used as a model for the production of experimental cardiac dysrhythmias. The canine

model is, however, of limited use for the screening of compounds with potential antidysrhythmic activity because of its high cost in terms of time, money, and the

amount of compound needed.

The aim of the present experiments was to investigate in detail the model of

coronary artery ligation in the rat, described by Kenedi and Losonci (1973), and to

assess the feasibility of its use as a screening test for drugs with potential antidys-

rhythmic activity. The effectiveness of two known antidysrhythmic drugs, lignocaine

and ORG 6001 (3cY-amino-5a-androstan-2P-ol-l7-one hydrochloride), a new orally

active agent (Marshall & Parratt, 1975; Vargaftig et al., 1975) are reported in this

model. As it has been claimed that antidysrhythmic drugs such as lignocaine may

From the Biochemistry Research and Development Laboratories, Organon Laboratories Ltd., New-

house, Lanarkshire, Scotland and the Department of Physiology and Pharmacology, University of Strath-

Clyde, George Street, Glasgow Cl IXW, Scotland.

Address reprint requests to Dr. J. R. Parratt, Department of Physiology and Pharmacology, University

of Strathclyde, George Street, Glasgow Cl IXW, Scotland.

Received June 4, 1979; revised and accepted July 17, 1979.

357

Journal of Pharmacological Methods 3, 357-368 (1980)

0 Elsevier North Holland, Inc., 52 Vanderbilt Avenue, New York, NY 10017 0X0-5402/80/04035712$02.25

358 Colin Clark et al.

also be able to salvage ischemic myocardium (Schaub et al., 19771, we investigated

the effects of lignocaine and ORG 6001 on the extent of the infarct following

coronary artery ligation. The amount of infarcted tissue was quantitated using a

method in which nitroblue tetrazolium (NBT) was used to stain for the depletion

of two cytoplasmic enzymes, lactate dehydrogenase and NADPH diaphorase. This

is a modification of the histochemical technique described by Fine et al. (1966). A

preliminary account of some of these studies was presented to the 1979 Birmingham

meeting of the British Pharmacological Society (Kane et al., 1979).

METHODS

Experiments were performed using male rats of the Sprague-Dawley strain,

weighting 250-450 g. Anesthesia was induced with pentobarbitone sodium, 6 mg/

100 g intraperitoneally, with small additional amounts administered intravenously

as required.

Systemic arterial blood pressure was recorded from the left common carotid

artery using a capacitance transducer (Elema-Schonander, Type EMT35). A catheter

was placed in a femoral vein for administration of drugs, and the trachea was

cannulated to allow artificial ventilation. The electrocardiogram (ECG) was recorded

using standard limb leads. Arterial blood pressure and the electrocardiogram were

monitored continuously on an oscilloscope and recorded using a Mingograph 81 ink-jet recorder (Elema-Schonander, Stockholm). Rectal temperature was main-

tained at approximately 38°C. The chest was opened by left thoracotomy at the fifth intercostal space and the fifth and fourth ribs were sectioned approximately 2 mm

from the left margin of the sternum. Immediately after opening the chest, the animals were ventilated with room air using a stroke volume of approximately 2 ml/

100 g and a rate of 54 strokes/min. These ventilation parameters maintained arterial

POP, PCOZ, and pH within the normal range. After opening the pericardium the

heart was exteriorized by gentle pressure on the chest walls, and a 6/O braided silk

suture attached to a 10 mm micro-point reverse cutting needle (Mersilk W812,

Ethicon) was placed under the left coronary artery as described by Selye et al.

(1960). The heart was replaced in the chest cavity and any animal in which this procedure itself produced dysrhythmias or a sustained fall in mean arterial blood

pressure (MABP) to less than 70 mm Hg was discarded from the study at this point. After an equilibration period of 15 minutes the ligature was tied.

EVALUATION OF POSTLIGATION DYSRHY-FHMIAS

Thirty-five animals were used for this study. Of these, five died in ventricular

fibrillation and in one, coronary artery ligation resulted in a profound systemic hypotension and A-V block which progressed to asystole. In the 29 survivors, the hemodynamic effects of coronary artery ligation consisted of a transient fall in MABP (from 90 & 3 mm Hg to 73 2 3 mm Hg at one minute after ligation, p < 0.01).

Heart rate did not change significantly from the preligation level of 451 ~fr 10 beats/

Coronary Artery ligation in Anesthetized Rats 359

A laom n

WX-LK3ATtON

FIGURE 1. Characteristic electrocardiogram and blood pressure trace of an anesthetized rat. Panel A control, panels B and C, 10 and 30 min after ligation, respectively.

min. Around five minutes after ligation, the animals began to show marked ventri-

cular ectopic activity. These dysrhythmias (seen in Fig. 1) included isolated extrasy-

stoles and bursts of ventricular tachycardia (VT-defined as any run of seven or

more consecutive ventricular extrasystoles) and of ventricular fibrillation (VF),

which in the rat can spontaneously revert to sinus rhythm. Figure 1 also shows the

characteristic configuration of the lead one ECG 30 minutes after ligation. The most

striking changes that occurred at this time were the development of deep Q-waves,

the marked ST-segment elevation and the increase in QT-interval. By 30 minutes

after ligation, the animals returned to a stable sinus rhythm. The distribution of

ventricular extrasystoles over the first 30 minutes after ligation is shown in Figure

2. Most of the extrasystoles occurred between 4 and 18 minutes following ligation

with a peak ectopic count of 180 per minute at 10 minutes. In some animals a further period of ectopic activity was observed, usually between 14 and 4 hours

after ligation (Fig. 3). The occurrence and severity of these dysrhythmias was variable, and did not relate to the severity of the early dysrhythmias in any particular

animal. The total extent of dysrhythmic activity in each animal was assessed by

counting the number of ventricular extrasystoles, including those occurring as VT,

and also the total duration of VT and VF. The severity of the dysrhythmias in both

the early (O-30 minutes) and the later (18-4 hours) phases for the control group are shown in Table 1.

The Assessment of Antidysrhythmic Activity

After establishment of the model as described above, a further three groups of

animals were studied to investigate the effects of two known antidysrhythmic drugs,


200

100

0 ii

I IT T

15 20 25 30

TIME POST-LIGATION (MIN)

FIGURE 2. The mean ventricular ectopic count t SE at one minute intervals in the &30 minute postligation period.

lignocaine and ORG 6001, on the dysrhythmias and on infarct size. Group 1: in ten

animals, which served as the control group, the ligature was tied at the end of the

15 minute equilibration period. Group 2: After the equilibration period, ten animals

were given lignocaine HCI, 10 mg/kg iv immediately followed by a continuous intravenous infusion of 5 mg/kg/hr. The ligature was tied five minutes after com-

mencement of the infusion. Group 3: After the equilibration period ten animals

were given ORC 6001, IO mg/kg iv immediately followed by a continuous intrave-

nous infusion of 2.5 mg/kg/hr. In these animals the ligature was tied 15 minutes

after commencement of the infusion. All animals in these three groups were followed for four hours after ligation, and

the dysrhythmias assessed as described above; the drugs were infused throughout the entire period.

The Assessment of Infarct Size

At the end of 4 hours, the animals were killed with an anesthetic overdose and the hearts rapidly removed. These were rinsed under running water, dried, and

then frozen in a mixture of acetone and solid CO,. The hearts were sectioned from base to apex, two slices of 20 pm thickness being taken at 1 mm intervals. Thus for


0 60 120 180 240

TIME POST-LIGATION (MIN)

FIGURE 3. The mean ventricular ectopic count + SE at 15 minutes intervals in the B-4 hr period.

each heart, two series of slices were obtained, one of which was stained for lactate dehydrogenase and the other for NADPH diaphorase by incubating for 1 hour at 37°C in 30 ml of O.lM phosphate buffer, pH 7.6, containing 15 mg NBT plus substrate. The substrates used for the enzymes, lactate dehydrogenase and NADPH diaphorase, were 50 mg DL-lactic acid plus 30 mg NAD, and 30 mg NADPH respec-

TABLE 1 Total Number of Ventricular Extrasystoles, Together with the Incidence and Duration of Ventricular Tachycardia (VT) and Ventricular Fibrillation (VF) in both the Early and Late Phases Following Coronary Artery ligation in Control Rats. The Mean Values k SE are Derived from Those Animals in each Group in which the Particular Dysrhythmia Occurred.

VENTRICULAR ECTOPIC VT VF

T COUNT (SEC) (SEC)

O-30 min Total 29 1262 k 153 73 f 12 62 f 14

% incidence 100 100 62

11-4 hr Total 24 1031 2 282 75 A 33 39 2 13

% incidence 71 29 46


tively. After incubation the slices were placed in 10% formaldehyde in saline solu-

tion at 4°C to stop the reaction, washed in running tap water and mounted in

glycerine jelly.

An image analysing computer, the Quantimet 720D, was used to measure in each

slice the total area and the normal area, as defined by the presence of the formazan precipitate that indicated the presence of either lactate dehydrogenase or NADPH

diaphorase. The difference between these two measurements was taken as the area

of infarcted tissue, which was then expressed as a percentage of the total area in

each slice. Mean infarct size for each heart was calculated as a percentage of total

heart volume. The statistical significance of differences between mean ectopic counts and the

percentage incidences of fibrillation were calculated by a one-way analysis of var-

iance together with a Studentized range test and by a chi-square test respectively.

A paired t-test was used to compare the dual measurements of infarct size obtained

within each heart by the loss of both enzymes and a one-way analysis of variance

used for the analysis of the statistical difference between infarct size in the control

and drug-treated groups.

RESULTS

Effects of Lignocaine and ORC 6001 on the Dysrhythmias Following Coronary

Artery ligation

Both lignocaine and ORG 6001 markedly reduced the number of ventricular

extrasystoles, the duration of ventricular tachycardia, and the incidence of ventri-

cular fibrillation occurring in the first 30 minutes following coronary artery ligation

(Table 2). ORG 6001 also reduced the incidence and severity of the later dysrhyth-

mias and completely prevented the occurrence of fibrillation (Table 2). In contrast lignocaine did not significantly reduce the incidence of the late ventricular dys-

rhythmias, although only one of the ten treated rats fibrillated.

Effects of Lignocaine and ORG 6001 on Mean Arterial Blood Pressure and Heart

Rate

Both drugs caused a reduction in MABP and heart rate. MABP returned to ap-

proximately pretreatment levels prior to ligation but heart rate remained depressed,

being reduced by 19% and 10% from the pretreatment levels of 461 rf- 20 and 458 + 14 beats/min for the lignocaine and ORG 6001 groups respectively. The changes

in MABP and heart rate that occurred on ligation were qualitatively similar in all three groups. After a transient fall at one minute after ligation, MABP was 87 ? 7, 87 & 7, 103 ? 8 mm Hg in Groups 1, 2, and 3 respectively at 30 minutes after ligation, and there were no significant changes in MABP from pretreatment values

throughout the remainder of the four hour period. No significant changes in heart rate occurred upon ligation in any group and at four hours following ligation, heart

rate was still reduced in the treated groups, the respective values for Groups 1, 2, and 3 being 464 ? 17, 418 5 16, 374 f 23 beats/min.


TABLE 2 Dysrhythmias Occurring in Anesthetized Rats Subjected to Acute Coronary Artery ligation. There were Ten Animals in each of the Three Groups (Control, Lignocaine-treated, Org BOO+treated), the Mean Values * SE Given for Ventricular Ectopic Count, Duration of Ventricular Tachycardia (VT) and of Ventricular Fibrillation (VF) are derived only from Those Animals in Which the Particular Dysrhythmia Occurred. The Percentage Incidence of each Type of Dysrhythmia is Given in Parentheses.

VENTRICULAR ECTOPIC

COUNT VT (SEC) VF (SEC)

Control (O-30 min) 1483 + 293 (100) 73 k 20 (100) 56 + 15 (70)

Lignocaine 532 + 145’ (100) 22 k 6 (60*) 10.6, 17.4 (20’) ORG 6001 469 + 216’ (90) 33 k 18 (70) 17 (IO’) Control (30-240 min) 1457 + 451 (80) 84 ? 50 (40) 43 t 25 (50)

Lignocaine 1461 + 463 (80) 44 k 17 (50) 11 (10)

ORG 6001 2249, 590 (20*) 1.2, 56.4 (20) 0 (0’)

* p i 0.005. Individual values are quoted when N < 3.

Effects of Lignocaine and ORC 6001 on Infarct Size

The differential staining of the normal and ischemic myocardium as identified by

the loss of NADPH diaphorase and lactate dehydrogenase can be seen in Figure 4.

Table 3 shows the infarct size (as a percentage of total heart volume) as estimated

by the loss of both enzymes in the control and drug-treated groups. In the control

group, infarct size, as estimated from the loss of lactate dehydrogenase, was sig-

nificantly higher (p < 0.05) than that measured by loss of NADPH diaphorase.

However, neither lignocaine nor ORG 6001 had any significant effect on infarct size

as measured by the loss of either enzyme. In Figure 5, the infarct size for each

animal has been plotted against the total number of ventricular extrasystoles that

occurred up to four hours following ligation. As can be seen, there is no correlation

between the severity of the dysrhythmias and infarct size.

DISCUSSION

In these studies we have shown that coronary artery ligation in the anesthetized

rat provides a relatively simple and reproducible model for the production of

TABLE 3 Mean Percentage Infarct Size k SE as Estimated by the Loss of lactate Dehydrogenase (LDH) and NADPH Diaphorase in the three Groups.

GROUP 1 GROUP 2 CROUP 3 CONTROL LIGNOCAINE ORC 6001

% Infarct size NADPH 49.8 c 3.0 43.7” 1.3 46.3 2 1.3

% Infarct size LDH 56.3 + 3.3 53.0 I 2.4 61.7 2 3.9


FIGURE 4. Adjacent transverse slices of a control rat heart after occlusion of the left main coronary artery stained for the presence of NADPH diaphorase (A) and lactate dehydrogenase (B). In both slices part of the left ventricle including the anterior papillary muscle is infarcted, as shown by the lack of stain, whereas the ventricular septum and right ventricle are not.

experimental cardiac dysrhythmias. The dysrhythmias occurred in two distinct

phases. In the O-30 minute postligation period (the early phase) very marked

dysrhythmias were observed in all the control animals and 62% of them fibrillated.

A later phase of dysrhythmias also occurred, between lb and 4 hours after ligation,

in 71% of the animals, but their severity and duration were more variable than

those of the early phase. A similar pattern of the occurrence of ventricular dysrhythmias has been described in the dog subjected to coronary artery occlusion by the Harris technique (Harris, 1950), although the delayed dysrhythmic phase in the dog reaches its peak 16-24 hours after ligation. As our studies were limited to four

hours following ligation, it is not known if the peak of the later dysrhythmias does indeed occur earlier in the rat than in the dog and experiments are planned to further investigate this.


60 i 0 0 0

00 0

CONTROL

40 - a 0 0

20 -

0

I I I I

60 ORG 6001

00 0

40 0

OI 1000 2000 3000 4000 5000

TOTAL VENTRICULAR EXTRASYSTOLES (0 - 4 h. I

FIGURE 5. Infarct size plotted against the total number of ventricular extrasystoles occurring in the &4 hour postligation period, in Groups l-3 rats.


In this model ORG 6001 (IO mg/kg plus an infusion of 2.5 mg/kg/hr) markedly

reduced the number of ventricular extrasystoles and the incidence of fibrillation in

both the early and late phases. These results are in agreement with those obtained

on the early phase dysrhythmias following coronary artery ligation in the dog and

pig (Marshall and Parratt, 1975; Verdouw et al., 1978). Lignocaine, in a dose (IO mgi

kg plus an infusion of 5 mg/kg/hr) that had a marked antidysrhythmic effect in the

early postligation period, had no significant effect on the development of the later dysrhythmias. This might indicate a difference in the sensitivity of the early and

late arrhythmias to this drug although, as lignocaine is a short acting drug (Harrison

and Alderman, 1971), its lack of effect on the later dysrhythmias may have resulted

from an insufficient infusion rate to maintain a therapeutically active concentration of the drug.

It has been postulated that different mechanisms may underlie the genesis of the

early and late dysrhythmias in the dog (Friedman et al., 1973; Lazzara et al., 1973)

and although investigations into both types of dysrhythmias may be relevant to the

clinical situation, we conclude from our initial study of the method described, that

for use as a screening test for drugs with potential antidysrhythmic activity, obser-

vations should be limited to the early dysrhythmic phase. This adds to the model’s

advantages over a larger animal infarct model of low cost in terms of time as well

as money and the amount of compound needed.

Coronary artery ligation in the rat has also been shown to be a useful technique

for the assessment of interventions that reduce infarct size (Maclean et al., 1976).

Our study of infarct size, at four hours following ligation, showed that the model

is very severe with 49.8% and 56.3% of the heart volume being infarcted, as defined

by the loss from the tissue of NADPH diaphorase and lactate dehydrogenase re-

spectively. These results suggest that lactate dehydrogenase is lost more rapidly

from ischemic tissue than NADPH diaphorase and are in agreement with those of

Nachlas and Shnitka (1963), who showed that there is a differential loss of enzymes

from the infarcted canine myocardium, lactate dehydrogenase being one of the first enzymes to be lost. However, neither lignocaine nor ORG 6001, in the doses

used, significantly changed infarct size as measured by the loss of either enzyme. Heart rate and blood pressure are known to influence infarct size (Maroko and

Braunwald, 1975). In these experiments over the four hour period blood pressure

was similar in all three groups whereas heart rate was reduced in both the drug-

treated groups. These differences in heart rate between groups were not consid-

ered to be of importance in the measurement of infarct size, since if anything, a

decrease in heart rate would tend to reduce infarct size. Lignocaine has previously

been shown in the dog to reduce the ultrastructural damage induced by a short time (45 minutes) of cardiopulmonary bypass and it was suggested that the protec-

tion resulted from a stabilization of plasma and/or mitochondrial membranes by lignocaine (Schaub et al., 1977). However, we found no evidence in these experiments to suggest that lignocaine stabilized plasma membranes to prevent the loss of cytoplasmic enzymes. It remains a possibility that higher doses of lignocaine

may have a membrane stabilizing effect. Less direct evidence to support the hy- pothesis that ORG 6001 may also have a beneficial effect on ischemic myocardium,

Coronary Artery ligation in Anesthetized Rats

independent of its antidysrhythmic effect, was obtained from the observations of

Marshall and Parratt (1977), in which ORG 6001 was shown to reduce ST-segment

elevation following transient coronary artery occlusion. In the present experiments,

however, ORG 6001 at a dose that had a very marked antidysrhythmic effect

throughout the four hour period, did not appear to have any effect in preventing

the loss of enzymes. A comparison of the severity of the dysrhythmias and infarct

size between and within each group shows that, in these experiments, there was

no correlation between these two parameters. In summary, the advantages of coronary artery ligation in anesthetized rats as a

model for the screening of potential antidysrhythmic agents include its simplicity,

reproducibilty, and low cost in terms of time, money, and amount of compound

required. The model can also be used to study both early and late dysrhythmias and the extent of myocardial injury following coronary artery ligation.

We gratefully acknowledge the excellent technical assistance of Mrs. Elizabeth Dunlop in preparing the preparations for the histochemical studies.

REFERENCES

Fine G, Morales AR, Scerpella JR (1966) Experimen- tal myocardial infarction: A histochemical study. Arch Pathol02:4-8.

Friedman PL, Stewart JR, Wit AL (1973) Sponta- neous and induced cardiac arrhythmias in sub- endocardial Purkinje fibers surviving extensive myocardial infarction in dogs. Circ Res 33:612- 626.

Harris AS (1950) Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circ 1:1318-1326.

Harrison DC, Alderman El (1971) Relation of blood levels to clinical effectiveness of lidocaine. In Lidocaine in the Treatment of Ventricular Ar- rhythmias, (Proceedings of a Symposium), Ed, DB Scott, DC Julian. Edinburgh: E & S Living- stone Ltd, pp 178-188.

Kane K, McDonald F, Parratt JR (1979) Coronary artery ligation in anaesthetized rats as a model for the assessment of antidysrhythmic activity: the effects of lignocaine, propranolol and Org 6001. Br / Pharmac 66:463-464P.

Kenedi I, Losonci A (1973) Effect of beta adrenergic blocking on the arrhythmias following coronary artery ligation in the rat. Acta Physiol Acad Sci

Hung 44~247-257.

Lazzara R, El-Sherif N, Scherlag BJ (1973) Electro- physiological properties of canine Purkinje cells in one-day-old myocardial infarction. Circ Res

33~722-734.

Maclean D, Fishbein MC, Maroko PR, Braunwald E (1976) Hyaluronidaseinduced reductions in myocardial infarct size. Science 194:199-200.

Maroko PR, Braunwald E (1975) Effects of metabolic and pharmacologic interventions on myocardial infarct size following coronary occlusion. Acta

med Stand Suppl587:125-134.

Marshall RJ, Parratt JR (1975) Antiarrhythmic, haemodynamic and metabolic effects of 3a-amino- 5cr-androstan-2p-ol-17-one hydrochloride in greyhounds following acute coronary artery ligation. Br / Pharmac 55:359-368.

Marshall RJ, Parratt JR (1977) The antidysrhythmic amino steroid ORG 6001, reduces the ST-segment elevation produced by coronary occlusion in the dog. Br / Pharmac 61:315-317.

Nachlas MM, Shnitka TK (1963) Microscopic iden- tification of early myocardial infarcts by altera- tions in dehydrogenase activity. Amer / Path

42: 397-406.

Schaub RG, Stewart G, Strong M, Ruotolo R, Lem- ole G (1977) Reduction of ischemic myocardial

367


damage in the dog by lidocaine infusion. Amer 17-one hydrochloride) a steroidal anti-ar- / Path 87:399-414. rhythmic agent. / fharm Pharmac 27:697-699.

Selye H, Bajusz E, Grassos S, Mendell P (1960) Verdouw PD, Schamhardt HC, Remme WJ, De Simple techniques for the surgical occlusion of long JW (1978) Antiarrhythmic, metabolic and coronary vessels in the rat. Angiology 11:398- haemodynamic effects of Org 6001 (3a-amino- 407. 5a-androstan-2/%ol-17-one hydrochloride) after

Vargaftig BB, Sugrue MF, Buckett WJ, Van Reizen coronary flow reductions in pigs. I Pharm Exp

H (1975) Org 6801 (3a-amino-Sa-androstan-2P_oC Ther 204:634-644.

coronary artery ligation in anesthetized rats as a method for the production of experimental...

Documents