Cardiovascular Research, 1975, 9, 797-806.
Quantitation of isoprenaline-induced changes in the ventricular myocardium' P . C O L L I N S , c . G . B I L L I N G S , G. R . B A R E R , J . J . D A L Y , and A . J O L L Y From the Department of Experimental Medicine, The Royal Hospital, West Street, Shejield
Rona er a1 (1959) first described the production of myocardial necrosis and hypertrophy in the rat by means of intermittent subcutaneous isoprenaline hydrochloride. The extent of the necrosis was shown to be roughly related to the dose of isoprenaline employed. Their work has been confirmed in the rat by other workers (Wexler and Kittinger, 1963; Stubelt and Breining, 1964; Stanton et a/, 1969; Hattori et al, 1969) and in other animals (Handforth, 1962; Maruffo, 1967; OSkidal et al, 1968). Isoprenaline-induced cardiac necrosis carries a low mortality, is reproducible (Chappel et al, 1959a), and resembles spontaneously occurring myocardial infarcts seen in atherosclerotic animals (Wexler and Kittinger, 1963). It therefore presents a highly satisfactory model for the study of cardiac necrosis and its aggravating or ameliorating factors (Chappel et al, 1959b; Rona et al, 1959b; 1961; Balazs et al, 1962; Rona et al, 1963; Wenzel and Stark, 1966; Stanton and Schwartz, 1967; Lehr, 1969). Previous work has not paid great attention to This work was supported by a grant from the endowment fund ofthe United Sheffield Hospitals (Grant No. 192). Reprint requests to P.C., c/o Dr Daly's Secretary, The Royal Hospital, West Street, Sheffield.
1
objective quantitative measurements of the changes produced in the myocardium. Rough methods such as arbitrary grading have been criticized by Stanton (1966), who attempted to overcome these deficiencies by the use of ridit values (Bross, 1958). Ridit values allow the application of statistical methods and relate to the probability that groups of animals will be more or less severely affected than a standard reference group. Isoprenaline is used therapeutically in situations when the myocardium is already in jeopardy (Nissen and Thomsen, 1965; Brown et al, 1966; Mueller et al, 1968; Resnekov et a/, 1968). With this in view we decided to study doses approaching the pharmacological range. We have also extended the period of administration in order to study the temporal effects of repeated small doses of isoprenaline on the myocardium. The objectives of the present study were threefold. (1) To examine the effect of several dose schedules of isoprenaline on ventricular weight and histology. (2) To obtain objective measurements of the amount and distribution of the histological changes in the myocardium due to isoprenaline by the application of a point counting technique. (3) To clarify the aetiological
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A U T H O R S ' S Y N O P S I S Small doses of isoprenaline sulphate given intermittently produce a characteristic cardiopathy consisting of subendocardial scarring and myocardial hypertrophy. A morphometric technique was successfully applied to the quantitation of these changes. This technique improves the use of the isoprenaline model for the study of cardiac necrosis as statistical analysis can be applied and objective comparisons made. No hypertrophy was seen in the absence of myocardial necrosis which suggests that it is at least in part compensatory.
798 Collins, Billings, Barer, Daly, and Jolly relationship between the myocardial hypertrophy and necrosis.
Grade Z Histological changes confined to granularity of sarcoplasm and impaired striation of myocardial fibres, but without scarring. Grade IZ Focal scarring confined to the inner oblique layer of the left ventricular and septal myocardium. Grade III Focal scarring with confluence of some adjacent areas, Grade I V Extensive confluent scarring confined to the inner oblique layer of the left ventricular and septal myocardium. Grade V Scarring outside the inner oblique layer of the myocardium of the left ventricle and septum.
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Methods Male albino Wistar rats were used. Litter mates were divided into experimental and control groups and were housed in sterilized cages in equal numbers. Water and commercial rat food was allowed ad libitum. Four dose regimes of isoprenaline hydrochloride (Pharmax Ltd) were studied. (1) 0.2 pg/lOO g body weight for 4 weeks; (2) 2.0 pg/lOO g body weight in one group for 2 weeks and a second for 6 weeks; (3) 10 pg/llO g body weight for 4 weeks; (4) 100 yg/lOO g body weight for 3 weeks. An identical protocol was employed for each Myocardial fibre diameter was measured in the middle layer of the ventricles and septum by means experiment. Each animal was weighed daily. The experimen- of a calibrated eye-piece micrometer with a x 10 tal animals then received the appropriate dose of eye-piece and x 40 objective. This layer was chosen isoprenaline sulphate by subcutaneous injections, because the fibres are longitudinally sectioned and whereas the control animals received 0.85% saline, it is therefore possible to select fibres with parallel borders for measurement. Fibres in the other layers under light ether anaesthesia. At the conclusion of each experiment the rats are obliquely sectioned and therefore present were anaesthetized by ether to the point of respira- smaller and more variable areas for measurement. tory arrest. The thorax was then opened, and the Myocardial fibre diameter was taken as the mean of heart removed. The ventricles were carefully five measurements for the right ventricle, left trimmed to remove the atria and great vessels, ventricle, and septum. Measurements were made at blotted, and weighed. ‘Wet’ weights were used the base, middle, and apex of the ventricles. throughout, as further studies were intended. The ventricles were then fixed in formol saline and Quantitative histological analysis was carried out on divided transversely into basal, middle, and apical the 5 p transverse sections of the ventricle stained portions, and, after embedding in paraffin wax, by the van Gieson method in order to facilitate the two 5 y transverse sections were cut with a micro- recognition of collagen. The base, middle, and apex tome from the basal aspect of each one third. of each ventricle was sampled as described below. These sections were subsequently stained, the The method of quantitative analysis is based on one with haematoxylin and eosin and the second by the work of Delasse (1848) in geological studies. the van Gieson method. In hearts which were not Briefly, this states that in a complex mineral area preserved for histological examination, the right proportions of a section are equivalent to volume ventricle was carefully dissected free from the left proportions. Chayes (1 954), substantiated this ventricle and septum, and was weighed separately. concept mathematically, and proved that if a The left ventricle and septum were weighed to- substance was sampled by numerical scoring of its gether. These ventricles were then preserved cold components sampling in relation to a reference and dry for subsequent biochemical studies (to be point grid, point score sums for each component reported separately). gave satisfactory estimates of their relative areas. Chalkley (1943-1944) applied this technique to Histological study was carried out on all regimes histological studies and Dunnill (1962) employed with the exception of the animals which had these principles to the study of normal and diseased received isoprenaline, 2 pg/lOO g body weight daily lung. He used a 25-point reference grid for point for 6 weeks. In this particular experiment all counting of areas sampled and found that 500 to ventricles had been divided into right and left halves 1000 points counted per section gave consistent and were therefore not suitable for histology. results. The proportions of the sums of point An assessment of the degree of scarring was counts for individual tissues were taken to equal made on the histological sections from each third of their volume proportions. each ventricle by grading as follows: We have applied a similar point counting technique to the ventricular myocardium of rats Grade 0 No abnormality on light microscopy.
799 Quantitation of isoprenaline-induced changes in the ventricular myocardium
Z
tissue, corrected z = -x y+z. This is a (100-Y) correction of a proportion and not of absolute area or volume measurement, and was applied to each individual histological tissue. We considered that sampling at three levels in the ventricle would increase the accuracy of relating area to volume proportions and would also give information on regional changes, as it was evident from light microscopy that isoprenaline induced changes were not uniform throughout the ventricles. The fibres of the ventricular myocardium are arranged into three distinct layers, an inner and outer oblique layer and a middle layer, the fibres of which are arranged parallel to the ventricular circumference. Isoprenaline induced scarring is virtually confined to the inner layer in the dosages which we have employed. Taking this striatified and nonhomogeneous arrangement into consideration, it was felt that random sampling might not give representative results, in addition to being difficult to apply to these relatively small sectional areas. In order to achieve representative proportional sampling of all layers, and yet keep selection to a minimum, we decided to sample contiguous fields across the
diameter of the transversely sectioned myocardium. The only selection made was the first field in the subepicardial myocardium of the right ventricle. From this point contiguous fields were sampled moving across the right ventricle, septum, and left ventricle emerging at the epicardial surface of the latter. Point scrores were recorded for the RV, LV, and septum as a whole, and also for the six subepicardial fields of the LV and right ventricular side of the septum, which allowed study of non scarred myocardium. Relative area proportions were deduced from corrected point score sums for each individual component tissue at each of the three ventricular levels sampled. The means of their scores were then determined and relative volume proportions were deduced for the ventricles as a whole, and for the RV and LV and septum individually. An estimate of the mass of each component tissue was made by multiplying the weight of the ventricles and septum by the mean volume proportion of that tissue. In addition, it was known that the ratio of the weight of the left ventricle and septum to the right ventricle was constant and not altered by isoprenaline. Therefore, an estimate of the weight of these could be deduced from the total weight and an estimate of the mass of the individual component tissues of the right ventricle and left ventricle and septum could be calculated. This is a n approximation as it assumes constant density.
Results Effect of isoprenaline on heart weight and body weight This is summarized in Table 1. There was n o significant difference between the mean body weights of the experimental and control animals in any of the four dosages used with the exception of doses of 2.0 pg for 14 days.
Isoprenaline 0.2 pgll00 g body weight daily for 29 days There was n o difference between control and experimental animals in terms of mean total ventricular weight or the mean weights of the right ventricle plus septum. The weight ratios of right ventricle t o left ventricle plus septum were also similar in the two groups. Isoprenaline 2.0 pg/IOO g body weight daily for 14 days The mean ventricular weight of the experimental animals were significantly greater than that of
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treated with 10 pg and 100 pg isoprenaline per 100 g body weight and to their controls. We did not consider that the two groups could be compared to assess dosage effects as they were of different age at death and had been treated with isoprenaline for different periods of time. We employed a graticule containing 42 regularly placed points (Wild 10 x BK) inserted into a x 10 eyepiece with a x 40 objective. Point scores were made by identification of the histological tissue related to each of the 42 reference points for each field sampled. Four tissues were identified: sarcoplasm; myocardial fibre nucleus ; collagen; and non myocardial fibre nucleus (fibroblasts, macrophages, and endothelial cells). A small number of points were related to erythrocytes or debris. In addition, some reference points were related to artefactual clefts between myocardial fibres, particularly in the subendocardial myocardium, of both experimental and control ventricles. These were the result of histological preparation and section. Correction was made by the assumption that the relative point score proportions, for the tissues identified, would be the same in these artefactual clefts as in the remainder of the myocardium. Therefore if y = % of the total point count falling on these clefts and z the percentage of the total point count for a particular
(9)
RV LV
BW
BW
%RV
BW
RV (g) LV (g) RV+LV (g) %RV+LV
BW
0.057k0.01 0.205f0.01
>0.1
> 0.1 >0.1
0.065&0.01 0.235 f0.02 0.27k0.00
0.079 f0.01 0.284 50.03
0.068 f0.01
0.237 fO.01 0.28 fO.028
< 0.005
0.363 k 0.03
0.305 & 0.01
0.29 5 0.022
0.272 fO.02
< 0.005 < 0.005 < 0.005
186.4 f21.2 0.145 f0.02 0.526 k0.05 0.672 f 0.06
183.4f23.1 0.l2Sk0.01 0.44 k0.05 0.564 k 0.06
> 0.1
.c 0.005
0.23 kO.04
0.225 f 0.02
0.05 fO.01
188.78f 19.9 0.091 kO.O1 0.41 f0.06 0.52 f 0.07
> 0.1
(0.01
Control
P
27.5f0.01
< 0.005
< 0.025 < 0.025
0.334f 0.03 0.065 f 0.01 0.265 k 0.03 0.25 f0.04
0.494f 0.06 0.316f0.02
< 0.025
179.28f 15.88 0.112fO.01 0.465 k 0.06 0.602k 0.06
>0.1
0.448fO.02
0.753 kO.1
167.8f25.2
157.11k21.6 >0.1 < 0.025
>O.l
Experiment
Control
P
O.l
P
>0.1
0.1
100 pgg/fOO g BW for 20 days
27.7k0.02
0.22+0.00
0.067k0.01
Experiment
I0 pg/IOO g B W f o r 27 days
0.245k0.05
0.225 k 0.01
0.06-eO.00
< 0.005 0.282 fO.01
0.262 f 0.01
>0.1
0.3k0.01
0.29 f0.00
(0.05
0.1 < 0.05 O.l
>0.1
>0.1 >0.1 >0.1 > 0.1
P
2.0 pg/100 g B W f o r 14 days
Control
10 pg/lOo g B W for 28 days
0.262k0.24
0.23 k0.03
0.216f0.01
0.316k0.08
0.06f0.02
0.29f0.02
0.285k0.01
0.068k0.01
202.7f21.5 0.117f0.03 0.453k0.04 0.580 & 0.04
Experiment
198.7k18.7 0.133f0.02 0.419k0.03 0.567 f 0.05
Control
0.2 lrg/IOo g BW
The efect of isoprenaline dosage on mean body weight ( BW ) and mean right and left ventricular ( R V, L V ) weights
TABLE I
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L'.
2
&
9 s
B
"2
B
B
"
ta =: 5
"2
9 s
0
801 Quantitation of isoprenaline-induced changes in the oentricular myocardium
the controls. However, mean body weight was also greater and when mean ventricular weight was related to mean body weight there was no difference between the two groups. The correlation coefficient of body weight and ventricular weight for rats in this age range is 0.898. Thus ventricular hypertrophy was not seen, and the apparent increase in ventricular weight of the experiment group merely reflected larger experimental animals.
Effect of isoprenaline on ventricular gross and microscopic pathology Macroscopic changes were not marked in the small dosage levels which we employed, and were limited to occasional pallor at the apex of the ventricles in those animals treated with a daily dose of isoprenaline of 100 pg/lOO g body weight. Macroscopic necrosis and haemorrhages as described by Rona et a1 (1959) were not seen. Microscopic abnormalities were not seen in those .experimental animals which showed no increase in mean ventricular weight, ie, those which
TABLE 2
Mean regional scores obtained by the histological grading of isoprenaline-induced changes in the ventricular myocardium IsoprenafinellOO g B W
Base Middle Apex
I0 wg
100 wg
1.1 k0.22 2.2 f 0.84 3.9+ 1.52
I .75 f0.50 4.00k1.15 4.25 2 1.50
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received isoprenaline 0.2 pg/ 100 g body weight daily and the group treated with 2.0 pg isoprenaline daily for 14 days. All control animals had a normal myocardium histologically. The ventricles of those experimental animals treated with 2.0 pg isoprenaline per 100 g body weight daily for 42 days had been divided into right and left halves and were not suitable for histological examination. Characteristic changes were seen in the experimental groups that were treated with a daily dose of isoprenaline of 10 pg and 100 Isoprenaline 2.0 pg/IOO g body weight daily ,for pg/IOO g body weight. The myocardial fibres showed less distinct margins with impaired 42 days This group revealed significant increase in the striation and granular sarcoplasm. No characmean total and individual ventricular weights of teristic changes were seen in the myocardial fibre the experimental animals as compared with their nuclei. The diameters of the myocardial fibres litter mate controls, absolutely and also when appeared increased when compared with their related to mean body weight. The relative weight controls. No abnormality was seen in the intraratio of RV to LV and septum was not disturbed mural vascular walls, nor was there evidence of by isoprenaline administration, indicating that vascular occlusion. Focal areas of the inner the degree of hypertrophy in the two ventricles oblique layer of the left ventricular and septa1 myocardium were replaced by a reticulum of was similar. cellular collagen. This scarring was more proIsoprenaline I0 pg/IOO g body weight for 27 and nounced at the apex of the ventricle where 28 days and 100 pg/IOO g body weight daily j o r adjacent areas tended to become confluent. Occasional foci of scarring were also seen out20 days side the inner oblique layer towards the apex of These experiments revealed uniform increase in the mean ventricular weights of the experimental the ventricle. No scarring was seen in the animals both absolutely and when related to mean myocardium of the right ventricle. body weight. As with the previous experiment, these larger doses of isoprenaline did not disturb Necrosis scores. Arbitrary grading of necrosis the relative weight ratio between the right and scarring, as described previously, is shown ventricle and the left ventricle and septum. The in Table 2. There is significant increase in scores ratio of mean ventricular weight to mean body between base and apex (P0.01-0.05). weight increased with increasing dosage (P < Myocardial jibre diameter (Table 3). There was 0.005). no significant increase in myocardial fibre
802
Collins, Billings, Barer, Daly, and Jolly TABLE 3 The effect of isoprenaline administration on mean myocardial fibre diameter Myocardial fibre diameter ( w ) ~
Isoprenaline (100 wgilOO g B W )
Isoprenaline (10 vg/IOO g B W )
RV LV
Septum Septum+ LV Mean
Control
Experiment
10.79f0.79 1 l.56f 1.33 11.19 f0.94 11.69f1.12 11.39f0.53
10.79k0.7 12.54k 1.19 12.68 k 1.02 12.61 kl.09 12.02f1.05
P
Control
Experiment
P
>0.1
12.9 f I .22 14.48f 1.53 15.12f 1.6
< 0.005
0.1-0.05
11.3 f 1.03 12.93f0.53 12.89k0.34
0.1-0.05 >0.1
12.36f0.79
14.16+ 1.08
0. I > 0.I > 0.1 0.1 > 0. I > 0. I 0. I < 0.05 0. I < 0.01
Control
Experiment
(fSD)
(kSD)
0.21 fO.01 0.006 f 0.003 0.003 k0.003 0.003 f 0.005 0.228k0.025
0.27f0.02 0.005 f0.000 0.038 k0.013 0.006 f 0.003 0.331 f0.011
P 0. I -=0.005 > 0.1 0.05). However,
2.5 L
2.0
3
8
15
L
c ._ g 1.0
4
0.5 0
F I G . 2 Isoprenaline-induced changes in the percentage of the total point count occupied by ventricular collagen. White columns are control and black columns are experimental values.
5
3 Changes in the mean point count percentages occupied by myocardial fibre nuclei in the ventricles of isoprenaline-treated rats (black columns) compared with their controls (white columns). FIG.
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in the middle and basal one thirds as compared with their controls. This reduction in proportion was greater at the apex than at the base of the ventricle (P 0.025). The ratio of myocardial sarcoplasm proportion to myocardial nuclear proportion was increased, as compared with controls, in the septum plus left ventricle and also for the outer non-scarred septum plus left ventricle. The increase in this ratio for the right ventricle did not reach significance. (Fig. 4) In the animals which had received the 10 pg isoprenaline schedule there was a significant increase in this ratio for the right ventricle, but the increase for septum plus left ventricle was not significant.
to the objective quantitation of myocardial necrosis and scarring. The increase in the collagen content of the left ventricle and septum is the most striking feature. Comparison of its proportion in the outer experimental left ventricular and septa1 myocardium (2.7 +_ 2.3 of total count) with that of the whole thickness (11.9% k 5.1 of the total point count) reflects its subendocardial distribution. Also, area proportion measurements at the base, mid-ventricle, and apex indicate greater scarring at the base of the left ventricle and septum. Similarly the measurable increase in nonmyocardial fibre nuclei, which reached significance on the smaller dosage regime only, showed subendocardial distribution, which reflects the cellularity of the collagen scars. Changes in the proportion of sarcoplasm were also confined to the inner portion of the left ventricle and septum. Reduction in the volume proportion of sarcoplasm was demonstrated to be inversely proportional to collagen content, which is consistent with necrosis and loss of sarcoplasm and its replacement by scar tissue. There was also a reduction in the volume proportions of myocardial fibre nuclei, which may be partly explained by loss of myocardial fibre. However, the calculated mass of myocardial fibre nuclei was similar in experimental and control animals. This suggests that there has been hypertrophy or hyperplasia of the residual myocardial nuclei, but not to the same extent as the increase in sarcoplasmic mass, as the ratio of the proportions of sarcoplasm to myocardial nuclei is increased. Myocardial hypertrophy was suggested by three measurements. The ventricular weights were significantly increased in those animals which received 10 pg and 100 pg isoprenaline per 100 g body weight. Calculation of the weight of the individual components of myocardium showed that the greatest component of this weight increase count be attributed to increase in sarcoplasmic mass, with a lesser contribution from collagen. This conflicts with the conclusions of BartoSovi et a1 (1969), who claimed that isoprenaline administration in a dose of 1 mg/kg daily, increasing by daily increments of 1 mg to a total of 15 mg/kg at 15 days, only increased the growth of collagen. This conclusion could not be
805
Quantitation of isoprenaline-induced changes in the ventricular myocardium studies by Stanton and Schwartz (1967), and Stanton et a1 (1969), using larger doses of isoprenaline, have revealed a constant concentration of DNA despite increase in heart size, and suggested that the increase in mass represents hypertrophy rather than hyperplasia. Fanburg (I97 I), reported similar observations for a variety of methods used to induce cardial enlargement. In addition he demonstrated by autoradiography that DNA increased only in interstitial cells. We did not see myocardial hypertrophy in the absence of necrosis. This would suggest that the hypertrophy is at least in part compensatory, as a result of loss of subendocardial myocardium by necrosis. Nevertheless increase in mass of sarcoplasm is greater than that required to achieve control values. Changes in functional demand due to tachycardia, or alteration in the geometry of myocardial contraction as a result of subendocardial scarring, may be important in this respect. In addition it is possible that isoprenaline has a direct anabolic action on the myocardium (Stanton and Schwartz, 1967). Several hypotheses have been advanced to explain the pathogenesis of isoprenaline induced myocardial necrosis and scarring (Rona et al, 1959; Handforth, 1962; Raab, 1963; Rosenmann et al, 1964; Rosenblum et all 1965; Fleckenstein, 1971). Our work (in preparation) supports that of Handforth (1962), and suggests that the lesion represents myocardial necrosis due to isoprenaline-induced impairment of subendocardial perfusion. We wish to thank Professor J. Richmond for the use o f laboratory facilities.
References Alpert, N . R . (1971). Curdioc Hypertrophy. Academic Press, New York and London. Balazs, T., Sahasrabudhe, M. R.. and Grice. H. C. (1962). The influence o f excess body fat on the cardiotoxicity of isoproterenol in rats. Toxicology and Appliedl'hartnacology, 4, 6 13-620. BartoSova, D., Chvapil, M., Korecky, B., Poupa, 0.. RakuSan, K.. Turek, Z., and Vizek, M . (1969). The growth of the muscular and collagenous parts o f the rat heart in various forms of cardiomegaly. Joirrnul of Physiology, 200, 285-295. Bross, I . (1958). How to use ridit analysis. Eiotiierrics, 14, 18-38.
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drawn from their data, which merely inferred an increase in collagen concentration. We have repeated their regime, which produced a highly significant increase in ventricular weight of the experimental animals. Light microscopy of histological sections of these ventricles showed no qualitative differences from the subendocardial scarring, and obvious increase in size of myocardial fibres, seen with the dose of 100 pg/lOO g body weight (A. Jolly, unpublished observations). Second, there was an increase in the ratio of sarcoplasmic proportion to myocardial fibre nuclear proportion and third, an increase in the diameter of the myocardial fibres, with the exception of those of the animals treated with the smaller dose. Ventricular weights showed no evidence of hypertrophy in those animals which had received 0.2 pg isoprenaline or 2.0 pg/100 g body weight for 2 weeks. However, there was significant increase in the ventricular weights of those animals treated with 2.0 pg isoprenaline per 100 g body weight for 6 weeks. This indicates a temporal component in isoprenaline induced myocardial hypertrophy, so that doses which are too small to produce changes over short periods of intermittent exposure, may do so if exposure is continued. This has obvious implications to clinical practice and the use of catecholamines in aerosols. The histological changes in myocardial fibre, and the myocardial scarring were not seen in the experimental animals treated with 0.2 pg isoprenaline daily per 100 g body weight, or in those given 2 pg daily for 2 weeks. No histology was performed in those treated with 2.0 pg/loO g body weight daily for 6 weeks. Well-marked changes were seen in the 10 pg and 100 pg/l00 g body weight daily groups and were similar to those described by Rona et a1 (1 959a). However, unlike Sarnoff and Wexler (1970), and Cox and Wexler (1968), we saw no evidence of vascular occlusions. Examination of myocardial fibre nuclei showed no evidence of mitoses to suggest myocardial fibre hyperplasia, but it is not possible to exclude this by the technique which we have employed. Myocardial fibre hyperplasia is unusual after the early stages of extrauterine growth (Alpert, 1971), but may occur should the ventricular mass exceed a critical value (Linzbach, 1960). However, previous biochemical
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Mueller, H., Giannelli, S., Ayres, S. M.,Conklin, E. F., and Gregory, J. J. (1968). Effect of isoproterenol on ventricular work and myocardial metabolism in the post operative heart. Circulation, 37 and 38, Suppl. 11, 146-153. Nissen, N. I., and Thomsen, A. C. (1965). Oral treatment of A-V block and other bradycardias with sustained action isoprenaline. British Heart Journal, 21, 926-931. Oriidal, B., Rychterovi, V., and Poupa, 0. (1968). Isoproterenol-induced acute experimental cardiac necrosis in the turtle. American Heart Journal, 16, 645-649.
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Resnekov, L., Fordham, R., and Ross, D. (1968). Haemodynamic effects of isopropylnoradrenaline sulphate (isoprenaline) following aortic valve homograft replacement. British Heart Journal, 30, 38-43. Rona, G., Chappel, C. I., Balazs, T., and Gaudry, R. (1959a). An infarct-like myocardial lesion and other toxic manifestations induced by isoproterenol in the rat. Archives of Pathology, 61, 443-455.
Rona, G.. Chappel, C. I., Balazs, T., and Gaudry, R. (1959b). The effect of breed age and sez on myocardial necrosis produced by isoproterenol in the rat. Journal of Gerontology, 14, 169-173.
Rona, G., Chappel, C. I., and Gaudry, R. (1961). Effect of dietary sodium and potassium content on myocardial necrosis elicited by isoproterenol. Laboratory Inuestigation, 10, 892-897.
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Rosenmann, E., Gazenfield, E., Laufer, A., and Davies, A. M. (1964). Isoproterenol-induced myocardial lesions in the immunized and non-immunized rat. Pathologica et microbiologia (Basel), 27, 303-309. Sarnoff, J., and Wexler, B. C. (1970). Isoproterenol-induced myocardial infarction in rats (distribution of corticosterone). Circulation Research, 21, 1101-1109. Stanton, H. C. (1966). Estimation of grossly detectable isoproterenol induced myocardiopathies in rats using the ridit transformation. Toxicology and Applied Pharmacology, 9, 2 18-224.
Stanton, H. C., Brenner, G., and Mayfield, E. D. (1969). Studies on isoproterenol-induced cardiomegaly in rats. American Heart Journal, 11, 72-80.
Stanton, H. C., and Schwartz, A. (1967). Effects of a hydrazine monoamine oxidase inhibitor (phenelzine) on isoproterenol-induced myocardiopathies in the rat. Experimental Therapeutics, 151, 649-658. Stubelt, 0.. and Breining H. (1964). Zur Pathogenese und pharmakologischen Beeinflussung der durch Isoprenalinvergiftung hervorgerufenen Myocdrdnekrosen. Arzneimittelforschung, 14, 1196-1 198.
Wenzel, D. G., and Stark, L. G. (1966). Effect of nicotine on cardiac necrosis induced by isproterenol. American Heart Journal, 11, 368-370. Wexler, B. C., and Kittinger, G. W. (1963). Myocardial necrosis in rats: serum enzymes, adrenal steroid, and histopathological alterations. Circulation Research, 13, 159-1 71.
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American Heart Journal, 81, 447-450.
Fleckenstein, A. (1971). In Calcium and the Heart, edited by P. Harris and L. Opie, pp. 135-188. Academic Press, New York and London. Hattori, E., Yatsuki, K., Miyazaki, T., Sata, T., and Nakamura, M. (1969). Adenine nucleotides of myocardium from rats treated with isoproterenol and o r magnesium or potassium deficiency. Japanese Heart Journal. 10, 2 18-224. Handforth, C. P. (1962). lsoproterenol-induced myocardial infarction in animals. Archives of Pathology, 13, 161-165. Lehr, D. (1969). Tissue electrolyte alteration in disseminated myocardial necrosis. Annals of the New York Academy of
Raab, W. (1963). Neurogenic multifocal destruction of myocardial tissue. Review of Canadian Biology, 22, 217-
Quantitation of isoprenaline-induced changes in the ventricular myocardium' P . C O L L I N S , c . G . B I L L I N G S , G. R . B A R E R , J . J . D A L Y , and A . J O L L Y From the Department of Experimental Medicine, The Royal Hospital, West Street, Shejield
Rona er a1 (1959) first described the production of myocardial necrosis and hypertrophy in the rat by means of intermittent subcutaneous isoprenaline hydrochloride. The extent of the necrosis was shown to be roughly related to the dose of isoprenaline employed. Their work has been confirmed in the rat by other workers (Wexler and Kittinger, 1963; Stubelt and Breining, 1964; Stanton et a/, 1969; Hattori et al, 1969) and in other animals (Handforth, 1962; Maruffo, 1967; OSkidal et al, 1968). Isoprenaline-induced cardiac necrosis carries a low mortality, is reproducible (Chappel et al, 1959a), and resembles spontaneously occurring myocardial infarcts seen in atherosclerotic animals (Wexler and Kittinger, 1963). It therefore presents a highly satisfactory model for the study of cardiac necrosis and its aggravating or ameliorating factors (Chappel et al, 1959b; Rona et al, 1959b; 1961; Balazs et al, 1962; Rona et al, 1963; Wenzel and Stark, 1966; Stanton and Schwartz, 1967; Lehr, 1969). Previous work has not paid great attention to This work was supported by a grant from the endowment fund ofthe United Sheffield Hospitals (Grant No. 192). Reprint requests to P.C., c/o Dr Daly's Secretary, The Royal Hospital, West Street, Sheffield.
1
objective quantitative measurements of the changes produced in the myocardium. Rough methods such as arbitrary grading have been criticized by Stanton (1966), who attempted to overcome these deficiencies by the use of ridit values (Bross, 1958). Ridit values allow the application of statistical methods and relate to the probability that groups of animals will be more or less severely affected than a standard reference group. Isoprenaline is used therapeutically in situations when the myocardium is already in jeopardy (Nissen and Thomsen, 1965; Brown et al, 1966; Mueller et al, 1968; Resnekov et a/, 1968). With this in view we decided to study doses approaching the pharmacological range. We have also extended the period of administration in order to study the temporal effects of repeated small doses of isoprenaline on the myocardium. The objectives of the present study were threefold. (1) To examine the effect of several dose schedules of isoprenaline on ventricular weight and histology. (2) To obtain objective measurements of the amount and distribution of the histological changes in the myocardium due to isoprenaline by the application of a point counting technique. (3) To clarify the aetiological
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A U T H O R S ' S Y N O P S I S Small doses of isoprenaline sulphate given intermittently produce a characteristic cardiopathy consisting of subendocardial scarring and myocardial hypertrophy. A morphometric technique was successfully applied to the quantitation of these changes. This technique improves the use of the isoprenaline model for the study of cardiac necrosis as statistical analysis can be applied and objective comparisons made. No hypertrophy was seen in the absence of myocardial necrosis which suggests that it is at least in part compensatory.
798 Collins, Billings, Barer, Daly, and Jolly relationship between the myocardial hypertrophy and necrosis.
Grade Z Histological changes confined to granularity of sarcoplasm and impaired striation of myocardial fibres, but without scarring. Grade IZ Focal scarring confined to the inner oblique layer of the left ventricular and septal myocardium. Grade III Focal scarring with confluence of some adjacent areas, Grade I V Extensive confluent scarring confined to the inner oblique layer of the left ventricular and septal myocardium. Grade V Scarring outside the inner oblique layer of the myocardium of the left ventricle and septum.
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Methods Male albino Wistar rats were used. Litter mates were divided into experimental and control groups and were housed in sterilized cages in equal numbers. Water and commercial rat food was allowed ad libitum. Four dose regimes of isoprenaline hydrochloride (Pharmax Ltd) were studied. (1) 0.2 pg/lOO g body weight for 4 weeks; (2) 2.0 pg/lOO g body weight in one group for 2 weeks and a second for 6 weeks; (3) 10 pg/llO g body weight for 4 weeks; (4) 100 yg/lOO g body weight for 3 weeks. An identical protocol was employed for each Myocardial fibre diameter was measured in the middle layer of the ventricles and septum by means experiment. Each animal was weighed daily. The experimen- of a calibrated eye-piece micrometer with a x 10 tal animals then received the appropriate dose of eye-piece and x 40 objective. This layer was chosen isoprenaline sulphate by subcutaneous injections, because the fibres are longitudinally sectioned and whereas the control animals received 0.85% saline, it is therefore possible to select fibres with parallel borders for measurement. Fibres in the other layers under light ether anaesthesia. At the conclusion of each experiment the rats are obliquely sectioned and therefore present were anaesthetized by ether to the point of respira- smaller and more variable areas for measurement. tory arrest. The thorax was then opened, and the Myocardial fibre diameter was taken as the mean of heart removed. The ventricles were carefully five measurements for the right ventricle, left trimmed to remove the atria and great vessels, ventricle, and septum. Measurements were made at blotted, and weighed. ‘Wet’ weights were used the base, middle, and apex of the ventricles. throughout, as further studies were intended. The ventricles were then fixed in formol saline and Quantitative histological analysis was carried out on divided transversely into basal, middle, and apical the 5 p transverse sections of the ventricle stained portions, and, after embedding in paraffin wax, by the van Gieson method in order to facilitate the two 5 y transverse sections were cut with a micro- recognition of collagen. The base, middle, and apex tome from the basal aspect of each one third. of each ventricle was sampled as described below. These sections were subsequently stained, the The method of quantitative analysis is based on one with haematoxylin and eosin and the second by the work of Delasse (1848) in geological studies. the van Gieson method. In hearts which were not Briefly, this states that in a complex mineral area preserved for histological examination, the right proportions of a section are equivalent to volume ventricle was carefully dissected free from the left proportions. Chayes (1 954), substantiated this ventricle and septum, and was weighed separately. concept mathematically, and proved that if a The left ventricle and septum were weighed to- substance was sampled by numerical scoring of its gether. These ventricles were then preserved cold components sampling in relation to a reference and dry for subsequent biochemical studies (to be point grid, point score sums for each component reported separately). gave satisfactory estimates of their relative areas. Chalkley (1943-1944) applied this technique to Histological study was carried out on all regimes histological studies and Dunnill (1962) employed with the exception of the animals which had these principles to the study of normal and diseased received isoprenaline, 2 pg/lOO g body weight daily lung. He used a 25-point reference grid for point for 6 weeks. In this particular experiment all counting of areas sampled and found that 500 to ventricles had been divided into right and left halves 1000 points counted per section gave consistent and were therefore not suitable for histology. results. The proportions of the sums of point An assessment of the degree of scarring was counts for individual tissues were taken to equal made on the histological sections from each third of their volume proportions. each ventricle by grading as follows: We have applied a similar point counting technique to the ventricular myocardium of rats Grade 0 No abnormality on light microscopy.
799 Quantitation of isoprenaline-induced changes in the ventricular myocardium
Z
tissue, corrected z = -x y+z. This is a (100-Y) correction of a proportion and not of absolute area or volume measurement, and was applied to each individual histological tissue. We considered that sampling at three levels in the ventricle would increase the accuracy of relating area to volume proportions and would also give information on regional changes, as it was evident from light microscopy that isoprenaline induced changes were not uniform throughout the ventricles. The fibres of the ventricular myocardium are arranged into three distinct layers, an inner and outer oblique layer and a middle layer, the fibres of which are arranged parallel to the ventricular circumference. Isoprenaline induced scarring is virtually confined to the inner layer in the dosages which we have employed. Taking this striatified and nonhomogeneous arrangement into consideration, it was felt that random sampling might not give representative results, in addition to being difficult to apply to these relatively small sectional areas. In order to achieve representative proportional sampling of all layers, and yet keep selection to a minimum, we decided to sample contiguous fields across the
diameter of the transversely sectioned myocardium. The only selection made was the first field in the subepicardial myocardium of the right ventricle. From this point contiguous fields were sampled moving across the right ventricle, septum, and left ventricle emerging at the epicardial surface of the latter. Point scrores were recorded for the RV, LV, and septum as a whole, and also for the six subepicardial fields of the LV and right ventricular side of the septum, which allowed study of non scarred myocardium. Relative area proportions were deduced from corrected point score sums for each individual component tissue at each of the three ventricular levels sampled. The means of their scores were then determined and relative volume proportions were deduced for the ventricles as a whole, and for the RV and LV and septum individually. An estimate of the mass of each component tissue was made by multiplying the weight of the ventricles and septum by the mean volume proportion of that tissue. In addition, it was known that the ratio of the weight of the left ventricle and septum to the right ventricle was constant and not altered by isoprenaline. Therefore, an estimate of the weight of these could be deduced from the total weight and an estimate of the mass of the individual component tissues of the right ventricle and left ventricle and septum could be calculated. This is a n approximation as it assumes constant density.
Results Effect of isoprenaline on heart weight and body weight This is summarized in Table 1. There was n o significant difference between the mean body weights of the experimental and control animals in any of the four dosages used with the exception of doses of 2.0 pg for 14 days.
Isoprenaline 0.2 pgll00 g body weight daily for 29 days There was n o difference between control and experimental animals in terms of mean total ventricular weight or the mean weights of the right ventricle plus septum. The weight ratios of right ventricle t o left ventricle plus septum were also similar in the two groups. Isoprenaline 2.0 pg/IOO g body weight daily for 14 days The mean ventricular weight of the experimental animals were significantly greater than that of
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treated with 10 pg and 100 pg isoprenaline per 100 g body weight and to their controls. We did not consider that the two groups could be compared to assess dosage effects as they were of different age at death and had been treated with isoprenaline for different periods of time. We employed a graticule containing 42 regularly placed points (Wild 10 x BK) inserted into a x 10 eyepiece with a x 40 objective. Point scores were made by identification of the histological tissue related to each of the 42 reference points for each field sampled. Four tissues were identified: sarcoplasm; myocardial fibre nucleus ; collagen; and non myocardial fibre nucleus (fibroblasts, macrophages, and endothelial cells). A small number of points were related to erythrocytes or debris. In addition, some reference points were related to artefactual clefts between myocardial fibres, particularly in the subendocardial myocardium, of both experimental and control ventricles. These were the result of histological preparation and section. Correction was made by the assumption that the relative point score proportions, for the tissues identified, would be the same in these artefactual clefts as in the remainder of the myocardium. Therefore if y = % of the total point count falling on these clefts and z the percentage of the total point count for a particular
(9)
RV LV
BW
BW
%RV
BW
RV (g) LV (g) RV+LV (g) %RV+LV
BW
0.057k0.01 0.205f0.01
>0.1
> 0.1 >0.1
0.065&0.01 0.235 f0.02 0.27k0.00
0.079 f0.01 0.284 50.03
0.068 f0.01
0.237 fO.01 0.28 fO.028
< 0.005
0.363 k 0.03
0.305 & 0.01
0.29 5 0.022
0.272 fO.02
< 0.005 < 0.005 < 0.005
186.4 f21.2 0.145 f0.02 0.526 k0.05 0.672 f 0.06
183.4f23.1 0.l2Sk0.01 0.44 k0.05 0.564 k 0.06
> 0.1
.c 0.005
0.23 kO.04
0.225 f 0.02
0.05 fO.01
188.78f 19.9 0.091 kO.O1 0.41 f0.06 0.52 f 0.07
> 0.1
(0.01
Control
P
27.5f0.01
< 0.005
< 0.025 < 0.025
0.334f 0.03 0.065 f 0.01 0.265 k 0.03 0.25 f0.04
0.494f 0.06 0.316f0.02
< 0.025
179.28f 15.88 0.112fO.01 0.465 k 0.06 0.602k 0.06
>0.1
0.448fO.02
0.753 kO.1
167.8f25.2
157.11k21.6 >0.1 < 0.025
>O.l
Experiment
Control
P
O.l
P
>0.1
0.1
100 pgg/fOO g BW for 20 days
27.7k0.02
0.22+0.00
0.067k0.01
Experiment
I0 pg/IOO g B W f o r 27 days
0.245k0.05
0.225 k 0.01
0.06-eO.00
< 0.005 0.282 fO.01
0.262 f 0.01
>0.1
0.3k0.01
0.29 f0.00
(0.05
0.1 < 0.05 O.l
>0.1
>0.1 >0.1 >0.1 > 0.1
P
2.0 pg/100 g B W f o r 14 days
Control
10 pg/lOo g B W for 28 days
0.262k0.24
0.23 k0.03
0.216f0.01
0.316k0.08
0.06f0.02
0.29f0.02
0.285k0.01
0.068k0.01
202.7f21.5 0.117f0.03 0.453k0.04 0.580 & 0.04
Experiment
198.7k18.7 0.133f0.02 0.419k0.03 0.567 f 0.05
Control
0.2 lrg/IOo g BW
The efect of isoprenaline dosage on mean body weight ( BW ) and mean right and left ventricular ( R V, L V ) weights
TABLE I
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L'.
2
&
9 s
B
"2
B
B
"
ta =: 5
"2
9 s
0
801 Quantitation of isoprenaline-induced changes in the oentricular myocardium
the controls. However, mean body weight was also greater and when mean ventricular weight was related to mean body weight there was no difference between the two groups. The correlation coefficient of body weight and ventricular weight for rats in this age range is 0.898. Thus ventricular hypertrophy was not seen, and the apparent increase in ventricular weight of the experiment group merely reflected larger experimental animals.
Effect of isoprenaline on ventricular gross and microscopic pathology Macroscopic changes were not marked in the small dosage levels which we employed, and were limited to occasional pallor at the apex of the ventricles in those animals treated with a daily dose of isoprenaline of 100 pg/lOO g body weight. Macroscopic necrosis and haemorrhages as described by Rona et a1 (1959) were not seen. Microscopic abnormalities were not seen in those .experimental animals which showed no increase in mean ventricular weight, ie, those which
TABLE 2
Mean regional scores obtained by the histological grading of isoprenaline-induced changes in the ventricular myocardium IsoprenafinellOO g B W
Base Middle Apex
I0 wg
100 wg
1.1 k0.22 2.2 f 0.84 3.9+ 1.52
I .75 f0.50 4.00k1.15 4.25 2 1.50
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received isoprenaline 0.2 pg/ 100 g body weight daily and the group treated with 2.0 pg isoprenaline daily for 14 days. All control animals had a normal myocardium histologically. The ventricles of those experimental animals treated with 2.0 pg isoprenaline per 100 g body weight daily for 42 days had been divided into right and left halves and were not suitable for histological examination. Characteristic changes were seen in the experimental groups that were treated with a daily dose of isoprenaline of 10 pg and 100 Isoprenaline 2.0 pg/IOO g body weight daily ,for pg/IOO g body weight. The myocardial fibres showed less distinct margins with impaired 42 days This group revealed significant increase in the striation and granular sarcoplasm. No characmean total and individual ventricular weights of teristic changes were seen in the myocardial fibre the experimental animals as compared with their nuclei. The diameters of the myocardial fibres litter mate controls, absolutely and also when appeared increased when compared with their related to mean body weight. The relative weight controls. No abnormality was seen in the intraratio of RV to LV and septum was not disturbed mural vascular walls, nor was there evidence of by isoprenaline administration, indicating that vascular occlusion. Focal areas of the inner the degree of hypertrophy in the two ventricles oblique layer of the left ventricular and septa1 myocardium were replaced by a reticulum of was similar. cellular collagen. This scarring was more proIsoprenaline I0 pg/IOO g body weight for 27 and nounced at the apex of the ventricle where 28 days and 100 pg/IOO g body weight daily j o r adjacent areas tended to become confluent. Occasional foci of scarring were also seen out20 days side the inner oblique layer towards the apex of These experiments revealed uniform increase in the mean ventricular weights of the experimental the ventricle. No scarring was seen in the animals both absolutely and when related to mean myocardium of the right ventricle. body weight. As with the previous experiment, these larger doses of isoprenaline did not disturb Necrosis scores. Arbitrary grading of necrosis the relative weight ratio between the right and scarring, as described previously, is shown ventricle and the left ventricle and septum. The in Table 2. There is significant increase in scores ratio of mean ventricular weight to mean body between base and apex (P0.01-0.05). weight increased with increasing dosage (P < Myocardial jibre diameter (Table 3). There was 0.005). no significant increase in myocardial fibre
802
Collins, Billings, Barer, Daly, and Jolly TABLE 3 The effect of isoprenaline administration on mean myocardial fibre diameter Myocardial fibre diameter ( w ) ~
Isoprenaline (100 wgilOO g B W )
Isoprenaline (10 vg/IOO g B W )
RV LV
Septum Septum+ LV Mean
Control
Experiment
10.79f0.79 1 l.56f 1.33 11.19 f0.94 11.69f1.12 11.39f0.53
10.79k0.7 12.54k 1.19 12.68 k 1.02 12.61 kl.09 12.02f1.05
P
Control
Experiment
P
>0.1
12.9 f I .22 14.48f 1.53 15.12f 1.6
< 0.005
0.1-0.05
11.3 f 1.03 12.93f0.53 12.89k0.34
0.1-0.05 >0.1
12.36f0.79
14.16+ 1.08
0. I > 0.I > 0.1 0.1 > 0. I > 0. I 0. I < 0.05 0. I < 0.01
Control
Experiment
(fSD)
(kSD)
0.21 fO.01 0.006 f 0.003 0.003 k0.003 0.003 f 0.005 0.228k0.025
0.27f0.02 0.005 f0.000 0.038 k0.013 0.006 f 0.003 0.331 f0.011
P 0. I -=0.005 > 0.1 0.05). However,
2.5 L
2.0
3
8
15
L
c ._ g 1.0
4
0.5 0
F I G . 2 Isoprenaline-induced changes in the percentage of the total point count occupied by ventricular collagen. White columns are control and black columns are experimental values.
5
3 Changes in the mean point count percentages occupied by myocardial fibre nuclei in the ventricles of isoprenaline-treated rats (black columns) compared with their controls (white columns). FIG.
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in the middle and basal one thirds as compared with their controls. This reduction in proportion was greater at the apex than at the base of the ventricle (P 0.025). The ratio of myocardial sarcoplasm proportion to myocardial nuclear proportion was increased, as compared with controls, in the septum plus left ventricle and also for the outer non-scarred septum plus left ventricle. The increase in this ratio for the right ventricle did not reach significance. (Fig. 4) In the animals which had received the 10 pg isoprenaline schedule there was a significant increase in this ratio for the right ventricle, but the increase for septum plus left ventricle was not significant.
to the objective quantitation of myocardial necrosis and scarring. The increase in the collagen content of the left ventricle and septum is the most striking feature. Comparison of its proportion in the outer experimental left ventricular and septa1 myocardium (2.7 +_ 2.3 of total count) with that of the whole thickness (11.9% k 5.1 of the total point count) reflects its subendocardial distribution. Also, area proportion measurements at the base, mid-ventricle, and apex indicate greater scarring at the base of the left ventricle and septum. Similarly the measurable increase in nonmyocardial fibre nuclei, which reached significance on the smaller dosage regime only, showed subendocardial distribution, which reflects the cellularity of the collagen scars. Changes in the proportion of sarcoplasm were also confined to the inner portion of the left ventricle and septum. Reduction in the volume proportion of sarcoplasm was demonstrated to be inversely proportional to collagen content, which is consistent with necrosis and loss of sarcoplasm and its replacement by scar tissue. There was also a reduction in the volume proportions of myocardial fibre nuclei, which may be partly explained by loss of myocardial fibre. However, the calculated mass of myocardial fibre nuclei was similar in experimental and control animals. This suggests that there has been hypertrophy or hyperplasia of the residual myocardial nuclei, but not to the same extent as the increase in sarcoplasmic mass, as the ratio of the proportions of sarcoplasm to myocardial nuclei is increased. Myocardial hypertrophy was suggested by three measurements. The ventricular weights were significantly increased in those animals which received 10 pg and 100 pg isoprenaline per 100 g body weight. Calculation of the weight of the individual components of myocardium showed that the greatest component of this weight increase count be attributed to increase in sarcoplasmic mass, with a lesser contribution from collagen. This conflicts with the conclusions of BartoSovi et a1 (1969), who claimed that isoprenaline administration in a dose of 1 mg/kg daily, increasing by daily increments of 1 mg to a total of 15 mg/kg at 15 days, only increased the growth of collagen. This conclusion could not be
805
Quantitation of isoprenaline-induced changes in the ventricular myocardium studies by Stanton and Schwartz (1967), and Stanton et a1 (1969), using larger doses of isoprenaline, have revealed a constant concentration of DNA despite increase in heart size, and suggested that the increase in mass represents hypertrophy rather than hyperplasia. Fanburg (I97 I), reported similar observations for a variety of methods used to induce cardial enlargement. In addition he demonstrated by autoradiography that DNA increased only in interstitial cells. We did not see myocardial hypertrophy in the absence of necrosis. This would suggest that the hypertrophy is at least in part compensatory, as a result of loss of subendocardial myocardium by necrosis. Nevertheless increase in mass of sarcoplasm is greater than that required to achieve control values. Changes in functional demand due to tachycardia, or alteration in the geometry of myocardial contraction as a result of subendocardial scarring, may be important in this respect. In addition it is possible that isoprenaline has a direct anabolic action on the myocardium (Stanton and Schwartz, 1967). Several hypotheses have been advanced to explain the pathogenesis of isoprenaline induced myocardial necrosis and scarring (Rona et al, 1959; Handforth, 1962; Raab, 1963; Rosenmann et al, 1964; Rosenblum et all 1965; Fleckenstein, 1971). Our work (in preparation) supports that of Handforth (1962), and suggests that the lesion represents myocardial necrosis due to isoprenaline-induced impairment of subendocardial perfusion. We wish to thank Professor J. Richmond for the use o f laboratory facilities.
References Alpert, N . R . (1971). Curdioc Hypertrophy. Academic Press, New York and London. Balazs, T., Sahasrabudhe, M. R.. and Grice. H. C. (1962). The influence o f excess body fat on the cardiotoxicity of isoproterenol in rats. Toxicology and Appliedl'hartnacology, 4, 6 13-620. BartoSova, D., Chvapil, M., Korecky, B., Poupa, 0.. RakuSan, K.. Turek, Z., and Vizek, M . (1969). The growth of the muscular and collagenous parts o f the rat heart in various forms of cardiomegaly. Joirrnul of Physiology, 200, 285-295. Bross, I . (1958). How to use ridit analysis. Eiotiierrics, 14, 18-38.
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drawn from their data, which merely inferred an increase in collagen concentration. We have repeated their regime, which produced a highly significant increase in ventricular weight of the experimental animals. Light microscopy of histological sections of these ventricles showed no qualitative differences from the subendocardial scarring, and obvious increase in size of myocardial fibres, seen with the dose of 100 pg/lOO g body weight (A. Jolly, unpublished observations). Second, there was an increase in the ratio of sarcoplasmic proportion to myocardial fibre nuclear proportion and third, an increase in the diameter of the myocardial fibres, with the exception of those of the animals treated with the smaller dose. Ventricular weights showed no evidence of hypertrophy in those animals which had received 0.2 pg isoprenaline or 2.0 pg/100 g body weight for 2 weeks. However, there was significant increase in the ventricular weights of those animals treated with 2.0 pg isoprenaline per 100 g body weight for 6 weeks. This indicates a temporal component in isoprenaline induced myocardial hypertrophy, so that doses which are too small to produce changes over short periods of intermittent exposure, may do so if exposure is continued. This has obvious implications to clinical practice and the use of catecholamines in aerosols. The histological changes in myocardial fibre, and the myocardial scarring were not seen in the experimental animals treated with 0.2 pg isoprenaline daily per 100 g body weight, or in those given 2 pg daily for 2 weeks. No histology was performed in those treated with 2.0 pg/loO g body weight daily for 6 weeks. Well-marked changes were seen in the 10 pg and 100 pg/l00 g body weight daily groups and were similar to those described by Rona et a1 (1 959a). However, unlike Sarnoff and Wexler (1970), and Cox and Wexler (1968), we saw no evidence of vascular occlusions. Examination of myocardial fibre nuclei showed no evidence of mitoses to suggest myocardial fibre hyperplasia, but it is not possible to exclude this by the technique which we have employed. Myocardial fibre hyperplasia is unusual after the early stages of extrauterine growth (Alpert, 1971), but may occur should the ventricular mass exceed a critical value (Linzbach, 1960). However, previous biochemical
006 Collins, Billings, Barer, Daly, and Jolly Brown, R. S., Carey, J. S., Woodward, N. W., Mohr, P. A., and Shoemaker, W. C. (1966). Hernodynamic effects of amines in clinical shock. Surgery Gynecology and Obstetrics 122, 303-307. Chalkley, H. W. (1943-1944). Method for the quantitative morphologic analysis of tissues. Journal of the National Cancer Institute, 4, 47-53.
Chappel, C. I., Rona, G., Balazs, T., and Gaudry, R. (1959a). Severe myocardial necrosis produced by isoproterenol in the rat. Archiues internationale de pharmncodynamie et de therapie, 122, 123-128. Chappel, C. I., Rona, G., and Gaudry, R. (1959b). The influence of adrenal cortical steroids on cardiac necrosis produced by isoproterenol. Acta endocrinologica, 32, 4 I9 4 2 4 . Chappel, C. I., Rona, G., and Gaudry, R. (1959~).Relationship between thyroid function and cardiotoxic properties of isoproterenol. Endocrinology, 65, 208-215. Chayes, F. (1954). The theory of thin section analysis. Journal of Geology, 62, 92-101. Cox, G. E., and Wexler, 9. C. (1968). Platelet and fibrin thrombi in isoproterenol induced myocardial necrosis. Federation Proceedings, 21J4 13. Delasse, M. (1848). Procede mtcanique pour determiner la composition des roches. Annales des Mines, 13. 378-388. Dunnill, M. S. (1962). Quantitative methods in the study of pulmonary pathology. Thorax, 17, 320-328. Fanburg, 9. L. (1971). Recent studies on cardiac hypertrophy.
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Linzbach, A. J. (1960). Heart failure from the point of view of quantitative anatomy. American Journal of Cardiology, 5, 370-382.
Maruffo, C. A. (1967). Fine structural study of myocardial changes induced by isoproterenol in rhesus monkeys. American Journal of Pathology, 50, 27-34.
Mueller, H., Giannelli, S., Ayres, S. M.,Conklin, E. F., and Gregory, J. J. (1968). Effect of isoproterenol on ventricular work and myocardial metabolism in the post operative heart. Circulation, 37 and 38, Suppl. 11, 146-153. Nissen, N. I., and Thomsen, A. C. (1965). Oral treatment of A-V block and other bradycardias with sustained action isoprenaline. British Heart Journal, 21, 926-931. Oriidal, B., Rychterovi, V., and Poupa, 0. (1968). Isoproterenol-induced acute experimental cardiac necrosis in the turtle. American Heart Journal, 16, 645-649.
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Resnekov, L., Fordham, R., and Ross, D. (1968). Haemodynamic effects of isopropylnoradrenaline sulphate (isoprenaline) following aortic valve homograft replacement. British Heart Journal, 30, 38-43. Rona, G., Chappel, C. I., Balazs, T., and Gaudry, R. (1959a). An infarct-like myocardial lesion and other toxic manifestations induced by isoproterenol in the rat. Archives of Pathology, 61, 443-455.
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