THE KURUME MEDICAL JOURNAL

Vol.24, No.9', p.207-211, 1977

MYOCARDIAL INFARCTION

HISTOPATHOLOGIC STUDY OF EARLY EXPERIMENTAL

MYOCARDIAL INFARCTION IN DOGS (1)

AKIRA TANIMURA, YASUHIRO NAKAMURA

AND TERUYUKI NAKASHIMA

Second Department of Pathology, Kurume UniversitySchool of Medicine, Kurume, 830, Japan

HIDEO YAMAMOTO

Second Department of Surgery, Kurume UniversitySchool of Medicine, Kurume, 830, Japan

TAIJI YOKOTA

Third Department of Internal Medicine, Kurume UniversitySchool of Medicine, Kurume, 830, Japan

(Received forr publication August 29, 1977)

Various staining methods of experimentally produced myocardial infarc-

tion were examined and compared about their specificity, reliability and

practical use. Results obtained are as follows : ischemic areas showed positive stain-

ing as early as 1 hour after coronary ligation by HBFP staining and later,

PTAH and Azan staining methods revealed pathologic changes of myocar-

dium. Ischemic myocardium was homogenous purpule in colour and Azan

staining is purpulish-red. To detect the effect of early myocardial infarc-

tion, HBFP, PTAH and Azan staining methods are useful.

INTRODUCTION

Though many methods have been reported to detect an early myocardial infarction, no one was reliable enough for practical use.

Recently Lie et al were reported a new method to detect the early myo-cardial infarction by use of basic fuchsin (HBFP, Lie et al., 1971), but about this method there were many con-tr.oversities. In this paper, we tried to

evaluate the reliability and specificity

of Lie's method and otherr staining methods of inf arcted areas by use of experimental materials and autopsy

cases.

MATERIALS AND METHODS

Adult mongrel dogs (35 dogs) weigh-ing about 10-13 Kg were used in this study. The dogs were intravenousely anesthetized with thimyl sodium (Iso-

207

208 TANIMURA, ET AL.

sol). Respiration was maintained with a respirator connected to trachea. Un-der such circumstance, experimental

procedures were performed and then pericardial sac was opened. Double li-gations were placed on the proximal anterior descending coronary artery

just distal to the origin of the circum-flex artery. Immediately after ligation

pericardial sac was closed and skin was sutured. The animals were killed at varying intervals of 1, 3, 6, 12, 24, 48 hours, 5 days and 10 days after ligation. At the time of sacrifice, the chest of the dogs was opened and intravenousely injected thiamyl sodium and then the heart was quickly removed. Each slice was fixed in 10% neutral f ormalin and Carnoi solution. The specimens were cut and stained with H. E, Azan, PAS, PTAH, HBFP and PAS after digestion.

RESULTS

After ligation of anterior descend-ing branch of canine coronary artery,

the animals were sacrificed at intervals

varying from 1 hour to 10 days. The heart were excised quickly, and sections

were taken from the myocardium in-cluding through the anterior wall, lateral wall, posterior wall and septum

of left ventricles. The comparative study was perform-

ed about ischemic areas under varying staining methods (H. E., Azan, PAS, PTAH, HBFP and PAS after diastase digestion).

Gross observation

The hearts that were sacrificed at 1 hour after coronary ligation had no vesible lesions. Ischemic areas became pale in appearance at 3 hours cases (Fig. 1). Ischemic areas being exami-nated after 6 hours were dark red in

colourr in comparison with their adja-cent myocardial tissue. Obvious necrosis appeared in anteriorr wall (especially, anteriorr papillary muscle) within 12 hours. Necrotic areas were dark red-dish yellow in colour.

At 5 days, multiple areas could be found which revealed shrinkage of in-f arcted areas with rubbery centers.

Microscopic examination

The results of histologic observations were described as follows ;

1 hour after coronary ligation : From the usual staining methods such as H. E., Azan and PTAH stains, no remarkable features could be seen in the slides. In HBFP stained sections, reddish colored ischemic areas appeared in individual myocardial fibers but the number of f uchsin stained fibers were not so nu-merous and intensity of the staining were not so strong.

3 hours after coronary ligation : H. E, stained section showed minimal change. In ischemic areas, myocardial cells were eosinophilic or granular and was intermingled with normal ones. Intercellular spaces were widened and edematous but inflammatory cells were not seen. In PTAH stain method, is-chemic myocardium showed obvious changes ; myocardial disarrangement, irregularity, destruction and contrac-tion bands of myofibrils were seen at random (Fig. 2). In HBFP stain, in-tensive, positive staining areas were appeared corresponding with ischemic areas of anterior wall (Fig. 3). Though individual fuchsin stained cells were frequently seen among cardiac fibers that appeared normal, HBFP positive areas which were consisted from the accumulation of pathologic muscle f fi-bers were dermacated from surrounding myocardium. Surrounding myocardial tissue had also some positive areas but

MYOCARDIAL INFARCTION 209

they were usually small. HBFP-positive materials were homogenously present

in ischemic myocardium and positive

staining was most intense in perinuc-

lear area. In degenerated myocardial

cell, myocardial fibrils and striated,

condensed myofibrils were strongly po-sitive.

Fig. I Three hours after ligation of

anterior descending branch.

Fig. 2 Contraction band of myofibrils (PTAH, 3 hours after ligation).

Fig. 3 Intensive, positive staining area in HBFP (black area) (6 hours after liga-tion).

Fig. 4 Prominent contraction bands throughout infarcted area (PTAH) (6 hours after ligation).

Fig. 5 Strongly positive stained area in HBFP (6 hours after ligation).

Fig. s More strongly positive area in HBFP (12 hours after ligation).

210 TANIMURA, ET AL.

At 6 hours after coronary ligation, ischemic areas were more prominent and was sharply demarcated from nor-mal myocardium. There were consider-able separations of individual myofibrils and contraction bands could be seen distributedly throughout infarcted are-as. Contraction band could be seen especially prominent at peripheral areas of infarction in PTAH stain (Fig. 4). HBFP stain was positive more strong-ly than that of 3 hours (Fig. 5).

12 hours after coronary ligation ; In H. E. staining, ischemic areas were homoginously eosinophilic and few neu-trophil leucocytes appeared at the peri-

phery. Distinctive necroses were not prominent. HBFP stain remained st-rongly positive as much as that of 6 hours (Fig. 6).

24 hours and 48 hours after coronary ligation ; Edema and neutrophil leuco-cytic infiltration in interstitial tissue of ischemic areas most prominent. PTAH positivity of ischemic areas dis-appeared irregulary at 24 hours.

After 48 hours, when the myocardi-um was necrotic and neutrophilic in-flammatory infiltration was reaching a peak, positive fuchsin staining dis-appeared in HBFP and necrotic muscles were stained dark brown than that of normal myocardium. Degenerated myo-cardial cells showed extensive destruc-tion of myofibrils, and central areas of ischemic lesions were dark blue and amorphous in PTAH stain. Necrotic areas became more extensive and these areas were not stained by PTAH.

In the materials of 5th day and 10th day, necrotic lesions were sharply de-marcated from normal myocardium with

prominent hyperemia and in its adiacent areas, small number of leucocytes, lym-

phocytes and phagocytes appeared. Necrotic myocardial areas were loosely and irregularly segmented by fibrotic septum. PTAH staining was negative

in necrotic tissue and HBFP staining

also disappeared completely. The pre-sence of PAS positive, diastase resistant

materials were noted in the infarcted muscle at 24 hours and increased in

amount as the time of ischemia was

prolonged. The vicissitudes of above mentioned stainings and materials of

experimental myocardial infarction were

summarized in Fig. 7, 8.

Fig. 7 Staining reactions in early

experimental myocardial infarction.

Fig. 8 Comparison with HBFP and

PTAH stainings in infarcted area.

DISCUSSION

The specificity and reliability of this method are not under discussion. Many

enzymatic and histochemical methods

have been introduced (Brody et al., 1967 ; Cox et al., 1968 ; Fine et al., 1966 ;

Hack and Helmy, 1967 ; Hudson, 1965 ;

MYOCARDIAL INFARCTION 211

Nachlas and Shnitka, 1963; Sybers et al., 1972 ; Zugibe et al., 1965) but neither were practical nor reliable enough for routine purpose. Though electron-mi-croscopical observation seems to be most reliable, it needs fresh materials and can search ischemic lesions in only very small areas.

Experimental myocardial infarction in animals has contributed to under-stand the early ischemia (Fine et al., 1966; Hack and Helmy, 1967). For the

practical application upon human auto-PSg materials, experimentally produced ischemic hearts were studied and dis-cussed about several staining methods. As shown on Table 1, HBFP staining revealed positive results within 1 hour and then PTAH, Azan and H. E. stain-ing methods became positive progres-sively. To determine the early phase of ischemic lesion, PTAH and HBFP stainings are best among above me-thods. After 2 days, however, it seems less effective to determine inf arcted areas than other methods especially PTAH staining. PTAH staining is also thought to be useful to study early lesions since 6 -12 hours after attack.

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