Morphological Methods for Evaluation of Myocardial Protection Victor J. Ferrans, M.D., Ph.D.

ABSTRACT A review is presented of histological, histochemical, and electron microscopical methods considered useful in the morphological evaluation of intraoperative myocardial protection. Transmural samples of myocardium should be studied since the response of the ventricular walls to ischemic injury is not homogeneous. Collection of samples should be continued until the injury reaches a stable end-point. Emphasis is placed on the fact that ischemic injury is modified considerably by reflow phenomena. Ultrastructural studies are indispensable and histological methods are of limited value in the morphological evaluation of early myocardial injury.

T

his report summarizes information concerning morphological methods for the evaluation of myocardial protection during cardiac operative procedures. Methods of tissue sampling are discussed and histological, histochemical, and ultrastructural techniques useful for the study of myocardium are described. T h e types of information that such techniques may yield are reviewed.

Methods TISSUE SAMPLING

When myocardial protection is being evaluated in experimental animal models, efforts should be made to obtain serial biopsies. The response of the ventricular walls to ischemic cardiac arrest is not homogeneous [3], and it is therefore necessary to examine transmural samples of myocardium. It is known that the left ventricular subendocardium, particularly the papillary muscles, is preferentially damaged by ischemic arrest. Our experience has been that tissues obtained by either drill biopsy or scalpel cut show less extensive artifacts than tissues obtained by needle biopsy. Konno-Sakakibara types of catheters yield only subendocardial tissue, and for technical reasons these instruments ordinarily are used only for biopsies of the right side of the ventricular septum. Serial samples should be obtained from comparable sites, the site of choice being the free wall of the left ventricle halfway from apex to base, and serial sampling should be continued until a stable end-point is reached. It should be emphasized that it is simply not enough to show that the myocardium “looks better” at one or more given times during the procedure; one must determine carefully to what extent cardiac damage is actually prevented or minimized by the protective procedure. This damage is eventually manifested by cardiac muscle cell necrosis and interstitial fibrosis, two changes From the Section of Pathology, National Heart and Lung Institute, National Institutes of Health, Bethesda. Md. 20014.

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FERRANS that follow different time courses. Cellular necrosis becomes fully developed within a few days after the insult, whereas fibrosis progresses for several weeks afterward. Thus it is highly desirable to allow the animals to recover fully from the operative procedure before final cardiac function studies are made just prior to the time they are killed. Pathological examination of the whole heart can then proceed. HISTOLOGICAL STUDIES

An ice-cold solution of 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, is our fixative of choice for both histological and electron microscopical study. Tissue sarnples are placed directly in this solution without a preliminary rinse in saline solution. Phosphate buffer is preferable to cacodylate buffer in that it produces better visualization of glycogen particles by electron microscopy, i.e., they are more darkly stained with lead citrate than are ribosomes. T h e reasons for this are not known. Glutaraldehyde penetrates rather poorly into tissues, and because of this one of the dimensions of the tissue sampled should not exceed 1 mm. Glutaraldehyde-fixed tissues absorb eosin more readily, and hematoxylin less readily, than d o tissues fixed with formalin. These changes can be corrected by appropriate modifications in tissue staining procedures. Most routine histological staining procedures, including Masson’s connective tissue trichrome, elastic-van Gieson’s for elastic fibers, and Movat’s pentachrome stain, can be easily adapted for use with glutaraldehyde-fixed tissues. The following procedures are recommended for histological study: ( 1) hematoxylin-eosin; (2) Masson’s trichrome for demonstration of collagen; (3) elastic-van Gieson’s for staining of elastic fibers (useful in the study of endocardial thickening); (4)von Kossa’s stain for deposits of calcium; ( 5 ) periodic acid-Schiff, with and without amylase predigestion, for study of glycogen; and (6) phosphotungstic acid-hematoxylin for detection of fibrin deposits. Additional hisiological procedures may be added to this list as required by special situations. All staining procedures for the demonstration of lipid droplets require the use of frozen sections, and separate provisions must be made for this whenever fat stains are required. Alternatively, the presence of lipid droplets can be studied adequately in ordinary preparations for electron microscopy. HISTOCHEE4ICAL STUDIES

Histochemical staining reactions are available that demonstrate a large variety of tissue components. It should be remembered, however, that many of these reactions are technically complicated and require special fixation; almost all of them should be carried out with strict control procedures to confirm the validity of the results. Histochemical reactions for enzymes are usually designed to demonstrate {.he maximal activity that the amount of enzyme present in a tissue section can attain under a specific set of conditions, as close to the ideal as possible, rather th2.n to demonstrate the level of activity that existed in vivo. Quantification and interpretation of such results can be difficult, and expert technical advice should be obtained when planning histochemical experiments. T h e following factors can be evaluated in histological sections prepared by

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the methods described above: ( 1 ) pattern of arrangement of the muscle cells; (2) degree of interstitial fibrosis; (3) degree of interstitial edema; (4)degree of intracellular edema; ( 5 )necrosis, degeneration, or calcification of muscle cells; (6) amount of glycogen present in the muscle cells; and (7) presence and type of inflaimnatory cells. T h e state of blood vessels and capillaries should be evaluated and the degree of hypertrophy estimated by measuring transverse diameters of the muscle cells. Although this information is extremely useful in evaluating late changes, several hours may elapse from the onset of injury before certain changes, such as those of coagulation necrosis, are clearly evident on histological study. Therefore, detailed morphological studies of early myocardial injury must be made by electron microscopy. ELECTRON MICROSCOPY

Electron microscopical study of ischemic myocardium has provided much useful information about early changes [8]. Nevertheless, a number of problems remain, including the identification and exclusion of artifactual changes in the myocardium, difficulties in sampling, and the expensive and time-consuming nature of the data-gathering process. The need for standardization and inclusion of control tissues in all these procedures cannot be overemphasized, particularly when dealing with changes that differ from the normal only in subtle ways. Detailed information is available on: the evaluation of artifacts in myocardial biopsies [7]; quantitative (morphometric) techniques of electron microscopy [lo, 161; and the use of tracers, such as peroxidase, in analyzing changes in permeability of damaged cardiac muscle cells [18]. Fixation of tissues by intracoronary perfusion with glutaraldehyde minimizes artifacts and greatly facilitates meeting the conditions necessary for morphometric studies. It is obvious, however, that such fixation is possible only at the time the animal is killed and that it renders the heart useless for any biochemical study. Light and electron microscopy findings should be correlated as closely as possible, and areas for electron microscopical study should be selected on the basis of light microscopical examination of semifine sections of plastic-embedded tissues. This is particularly important in detecting focal changes that might otherwise be missed.

Ultrastructural Changes According to their cause, myocardial ultrastructural alterations observable in myocardial protection experiments may be analyzed in three situations: (1) ischemia alone, (2) ischemia plus reflow, and (3) perfusion without ischemia. ISCHEMIA

T h e earliest clearly detectable ischemic changes caused by interruption of the coronary arterial supply to part of the heart while the heart continues to beat consist of loss of glycogen granules, intracellular edema, relaxation of myofibrils, swelling of mitochondria, disappearance of the normal intramitochondrial dense granules, appearance of flocculent intramitochondrial precipitates, swelling of VOL. 20, NO. 1, JULY, 1975

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FERRANS the sarcoplasmic reticulum, and clumping of the nuclear chromatin [8]. These changes are evident after 15 minutes of ischemia. As the period of ischemia is extended these changes progress rapidly, particularly in the mitochondria; the myofilaments become indistinct, and coagulation necrosis develops. M yofibrillar relaxatiorl in coagulation necrosis persists because the tissue is not being perfused while irreversible injury develops. In an ischemic area of myocardium the percentage of dying cells increases rapidly as the time of ischemia extends beyond 30 minutes [ 1121.Coagulation necrosis is found typically in central areas of myocardial infarcts and also in myocardium subjected to prolonged periods of ischemic arrest. Nevertheless, cardiac damage resulting from temporary ischemic arrest is more likely to be characterized by necrosis with contraction bands, which is a consequence of the combined effects of ischemia plus reflow. ISCHEMIA PLUS REFLOW

T h e iiltrastructural changes that occur in simple ischemia are greatly modified by restoration of blood flow and oxygenation to the ischemic area [9, 11, 14, 191. These events induce a large flux of calcium ions and plasma proteins across damaged, abnormally permeable plasma membranes into the muscle cells, which then undergo severe, irreversible contraction. This leads to the “myofibrillar degeneration” or “myofibrillar damage” type of cardiac necrosis, which we prefer to call necrosis with contraction bands because it is characterized by extreme shortening and prominent disruption of the sarcomeres as well as indistinct appearance and dislocation of the myofilaments. In addition to the changes just described. cells undergoing necrosis with contraction bands usually show intramitochondrial calcium deposits [ 141. This type of necrosis progresses from the stage of contraction band formation, which occurs very rapidly, to a stage of myocytolysis in which the contractile elements disappear, leaving empty sarcolemmal sheaths. The rate at which this progression occurs is highly variable, and very little is known of the biochemical mechanisms by which the lysis of myofibrils is mediated. Necrosis with contraction bands is regarded as a common type of response (of cardiac muscle cells to a wide variety of injuries, including those induced by: temporary occlusion of a coronary artery, potassium deficiency, various catecholamines, mineralocorticoids plus sodium phosphate, intracranial lesions, hemorrhagic shock, magnesium deficiency, cobalt toxicity, exposure to hypoxia in anoxic chambers, electric shock, hypothermia, normothermic ischemic arrest, and various enzymes and antibiotics [17]. In other words, necrosis with contraction bands develops during the evolution of myocardial damage resulting from temporary inadequacy or interruption of tissue perfusion or primary metabolic alterations, with or without associated changes in tissue perfusion. T h e “stone heart” syndrome represents an extreme form of this type of myocardial damage. Ultra structural changes in cardiac muscle cells develop more slowly with normothermic anoxic cardiac arrest (Figs. 1-4) than with regional ischemia [3]. Cardiac muscle cells, particularly those in the subepicardium, can show remarkably little change after 30 minutes of normothermic anoxic arrest; however,

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FIG. 1. Electron micrograph of part of left ventricular subepicardial muscle cell of a dog that died after termination of a 45-minute period of normothermic anoxic cardiac arrest produced by aortic cross-clamping during cardiopulmonary bypass. The mitochondria (M),sarcoplasmic reticulum (SR), and T tubules (T) appear normal. The myofibrik (MF) are slightly separated from one another. Glycogen particles ( G )are present in moderate numbers. ( X 19,000.)

extensive subendocardial hemorrhages and necroses develop when the period of arrest is allowed to exceed 45 minutes [3]. This type of study should be extended to the hearts of animals with different degrees, stages, and types of cardiac hypertrophy, as it is by no means certain that the response of such hearts to ischemic arrest will be the same as that of normal hearts. Buja and associates [ 3 ] and Hoffmann and Buckberg [13] have provided hypotheses to account for the increased vulnerability of the subendocardial layers of muscle to ischemic injury. Buja and colleagues have also demonstrated that highly unusual morphological changes, including the appearance of intramitochondrial glycogen deposits [ 11 and formation of intracytoplasmic junctions [2], develop as late as two to four weeks after the episode of anoxic cardiac arrest in nondamaged cardiac muscle cells. CARDIAC PRESERVATION

Several morphological studies have been made of hearts preserved by perfusion, with or without hypothermia, either in situ or in the isolated state. These studies [ 5 , 61 have shown that varying degrees of interstitial and intracellular edema develop in preserved hearts even if adequate provisions are taken to maintain proper conditions of perfusion and to avoid hypoxia and glycogen loss. The reasons for the marked variations in response of different hearts to the

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FERRANS perfusion procedure are not clear. The etiology of the edema in preserved hearts is also unknown. Nevertheless, this edema can be minimized or prevented and cardiac structural preservation improved by perfusion with hyperosmolar solutions [ 5 , 6 ] .Infusion of hyperosmolar solutions can also reduce the damage caused by myocardial ischemia [4]. It is known, too, that perfusion at high pressures, particularly at normothermic temperatures, can produce considerable hemorrhage, capillary congestion, and myocyte damage and edema [6]. These observations emphasize the need for strict controls in experiments involving myocardial preservation procedures.

Conclusions It is important to remember the following practical considerations: (1) necrosis with contraction bands is the type of damage that most commonly occurs in association with ischemic cardiac arrest followed by reperfusion; (2) the initial damage caused by ischemia is greatly accelerated by reflow and possibly also by reoxygenation (peroxidation of membrane lipids?);(3) the exact manner in which cardiac arrest is induced may be important in determining the extent of subsequent damage; and (4) perfusion with certain types of solutions during the period of elective cardiac arrest can decrease this damage considerably.

FIG. 2. Eiectron micrograph of parts oftwo cardim muscle cells in the lgt ventricular posterornedinl papillary muscle of the same dog m in Figure 1 . Cellular structure i.s severely disrupted arid large contraction b a n k [ire present (paired arrowheads). Some mitochondricr contain minute electroiidense deposits presumed to be of calcium. ( x 6,300.)

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Morphologzcal Evaluation of Myocardial Protection

FIG. 3. Electron micro

Morphological methods for evaluation of myocardial protection.

Morphological Methods for Evaluation of Myocardial Protection Victor J. Ferrans, M.D., Ph.D. ABSTRACT A review is presented of histological, histoche...
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