An experimental investigation to correlate changes in plasma fibrinolytic activity with isoprenaline induced myocardial damageKK Saxena, B Gupta, VK Kulshrestha, DN Prasad
Department of Pharmacology, L.L.R.M, Medical College, Meerut-250102, India
Administration of different doses of isoprenaline in albino rats produced varying grades of myocardial necrosis with concomitant changes in euglobulin clot lysis time which is a measure of plasma fibrinolytic activity (PFA). A positive and strong correlation was observed between changes in PFA and severity of myocardial necrosis. Experiments in young rats and propranolol pre-treated animals rule out the possibility of isoprenaline being responsible in bringing about changes in PFA. The measurement of PFA appears to be of prognostic significance in myocardial infarction as discussed.
That the fibrinolytic activity in blood undergoes changes in various diseases is well known. It has been demonstrated to be deranged in various pathological conditions viz. occlusive vascular disease, , ,,, ,,,,,, diabetes mellitus, ,, obesity, ,  pulmonary vascular disease,  blood dyscrasias,  abnormal pregnancy, , trauma  and neoplastic diseases. 
Following myocardial infarction fibrinolytic activity has consistently been demonstrated to decline. ,,,,, On the other hand serum enzymes like glutamic oxalo-acetic transaminase (GOT),lactate dehydrogenase (LDH), a-hydroxy-butyrate dehydrogenase (α- HBDH) and creatine phosphokinase (CPK) have been claimed to rise after myocardial damage ,,,,, Recently severity of myocardial damage has been shown to be reflected in the rise of serum enzymes , and their levels in blood have been used as a prognostic indicator of survival of patients of myocardial infarction.  However, no such study appears to have been carried out regarding fibrinolytic activity in ralation to myocardial damage. In the present investigation, therefore, an attempt was made to explore any possible relationship between changes in fibrinolytic activity and severity of myocardial damage.
Porter strain rats of either sex weighing 260 ± 10 g (older rats) and young rats of either sex of the same strain and weighing 80 ± 5 g were employed in the study. The animals were housed under controlled conditions of temperature and humidity and were fed a commercial rat diet.
Three independent sets of experiment, were performed to induce myocardia necrosis and to estimate the concomitant changes in the plasma fibrinolytic activity. The experiments were as follows:
(A) Effect of graded doses of isoprenalin in older rats
Older rats were divided into six group of ten animals each. Five groups out of these were administered different dose; of isoprenaline hydrochloride subcutaneously (s.c.) for two consecutive days tc induce myocardial necrosis. Five doses of isoprenaline spaced at equal interval were selected between 85.0 and 0.02 mg/kg (the doses capable of producing maximal and minimal cardiac necrosis, respectively) to induce myocardial necrosis of variable grades.  The sixth group was treated with matching volume of saline and served as control.
(B) Effect of isoprenaline in young rats
Young rats were divided into two groups of ten animals each. Animals of one group were injected with 85.0 mg/kg of isoprenaline s.c. for two consecutive days, while the animals of the other group were injected with the matching volume of saline.
(C) Effect of isoprenaline-challenge following propranolol pre-treatment it older rats
Thirty rats of higher weight were divided into three equal groups. Animals in the first group were administered propranolol hydrochloride 5.0 mg/kg, orally for seven consecutive days and the second group with the matching volume of saline. On day sixth and seventh, animals of both the groups were challenged with isoprenaline (85.0 mg/ kg s.c.).  The animals of the third group received propranolol HC1 (5.0 mg/kg, orally) for seven consecutive days to study the per se effect of propranolol on plasma fibrinolytic activity (PFA).
Fortyeight hours after the first injection of isoprenaline/saline, animals were anaesthetized with pentobarbitone sodium (40.0 mg/kg, i.p.) ; blood (5.0 ml) was collected from the abdominal aorta in a citrated all glass syringe for determination of PFA by estimating euglobulin clot lysis time (ELT) .  The animals were then sacrificed and their hearts were autopsied.
Gradation of myocardial necrosis
Frontal sections of the heart cut at 5µ were stained with haematoxylin and eosin. Based on the gross and microscopic appearances the heart lesions were graded on a five point scale from grade 0 to grade 4. 
(A) Effect of graded doses of isoprenaline on myocardial necrosis and plasma fibrinolytic activity (PFA)
The distribution of grades of lesions and values of euglobulin clot lysis time (ELT) are summarized in [Table 1]. With doses of isoprenaline employed in the present study, varying grades of myocardial necrosis (from 0 to 4) and different values of ELT were obtained. The changes in ELT observed exhibited a steady increase with the severity of necrosis. Pearson's coefficient of correlation (r) between values of ELT and grades of myocardial necrosis was found to be ± 0.8783 (p <0.001) which is highly suggestive of a positive correlation [Figure 1]. Regressiin line of these parameters plotted by the method of least squares demonstrates a linear correlation with slope and intercept being 88.04 and 44.8, respectively. Pearson's coefficients of correlation (r) between doses of isoprenaline and ELT, and between doses of isoprenaline and grades of myocardial necrosis were + 0.5647 (p < 0.001) and + 0.5638 (p < 0.001), respectively. In saline treated animals (control) ELT was 81.1 ± 15.9.
(B) Effect of isoprenaline on PFA in young rats
ELT in the young animals treated with saline only was 74,0± 13.8 while in isoprenaline challenged rats the ELT was 82.0 ± 14.6 (p > 0.05). None of the animals in this group developed myocardial necrosis of any grade.
(C) Effect of propranolol pre-treatment on isoprenaline induced myocardial necrosis and PFA
The distribution of grades of lesions and the changes in ELT following isoprenaline challenge in saline as well as propranolol protected animals are shown in [Table 2]. The grades used to assess the lesions do not conform to a normal distribution curve and hence Fisher's exact probability test;  a nonparametric test, was applied to compare the results, obtained. For application of Fisher's exact probability test, each set of results was arbitrarily arranged into two groups; group A containing 0,1 and 2 grades and group B containing 3 and 4 grades. The results obtained with propranolol pretreatment were then compared with the results of isoprenaline alone and the probability of difference was calculated. Propranolol pre-treatment significantly protected animals from the isoprenaline induced myocardial necrosis (p < 0.001) in these animals ELT also showed corresponding change [Table 2]. Propranolol was devoid of any per se effect on ELT as the ELT in propranolol treated animals was 79.0 ± 16.1 which is not significantly different (p > 0.05) from the control.
A decline in fibrinolytic activity in blood following myocardial infarction was first observed by Hume  and this has been confirmed subsequently by numerous workers. ,,,, However, it appears that no effort, clinical or experimental, has been made to analyse the relationship between the severity of myocardial damage and the decrease in plasma fibrinolytic activity (PFA).
In the present study administration of different doses of isoprenaline produced variable grades of myocardial necrosis in albino rats. The PFA in these animals as measured by recording the euglobulin clot lysis time was found to be significantly decreased depending upon the severity of myocardial necrosis as judged by gross as well as microscopic examination of heart at autopsy. Positive and strong correlation was observed between doses of isoprenaline and grades of myocardial necrosis, grades of myocardial necrosis and PFA and also between doses of isoprenaline and PFA. Pearson's coefficient of correlation (r) between these parameters was found to be + 0.5638 (p < 0.001) + 0.8783 (p < 0.001) and + 0.5647 (p < 0.001), respectively.
The foregoing observations obviously suggest that changes in PFA may be either due to isoprenaline or myocardial necrosis, however, further experiments rule out the possibility of the former. In young rats isoprenaline administration failed to produce myocardial necrosis and FFA was found to be within normal limits. Propranolol pre-treatment in older rats partially protected the animals from isoprenaline induced myocardial necrosis and the PFA exhibited corresponding decline. These observations clearly preclude the possibility of any per se effect of isoprenaline on PFA and the correlation observed between these parameters may be due to dependence of severity of myocardial necrosis on the doses of isoprenaline employed. Thus, the role of isoprenaline being ruled out in influencing PFA, the only other factor responsible for producing changes in PFA in the present experimental set up is myocardial damage. A positive and strong correlation between grades of myocardial necrosis and decline in PFA clearly suggest that the severity of myocardial damage is reflected in PFA. Further, measurement of - ELT indicates the plasminogen activator activity, ,, therefore, it appears that severity of myocardial lesion is also related to plasminogen activator activity in plasma.
[Table 1], [Table 2]