Colloidal Lanthanum as a Marker forImpaired Plasma ... - Europe PMC

Colloidal Lanthanum as a Marker for Impaired Plasma Membrane Permeability in lschemic Dog Myocardium Sylvia Hoffstein, PhD, Doris E. Gennaro, Arthur C. Fox, MD, Jacob Hirsch, MD, Fritz Streuli, MD and Gerald Weissmann, MD

Colloidal lanthanum salts have an average particle size of 40 A; consequently, this electron-opaque marker remains extracellular and does not cross the intact plasma membrane. The affinity of lanthanum for calcium-binding sites on mitochondrial membranes makes it possible to demonstrate loss of plasma membrane integrity at the cellular level in ischemic myocardium. Biopsies were obtained from infarcted, marginal and normal areas 31 hours after ischemia was produced in 9 anesthetized closed-chest dogs by electrically induced thrombosis of the left anterior descending coronary artery. The tissue was immediately fixed in 4% glutaraldehyde and 0.1 M cacodylate buffer containing 1.3% La(NO8)8, pH 7.4, for 2 hours. In normal control tissue prepared this way the lanthanum tracer, as expected, was confined to the extracellular spaces, including basement membranes, gap junctions and portions of the intercalated discs. Specimens taken near the center of frank infarctions all contained intracellular as well as extracellular lanthanum. Intracellular lanthanum could be seen evenly distributed around lipid droplets and in focal deposits around mitochondria. Only when mitochondria were disrupted did lanthanum gain access to internal sites on mitochondrial membranes. Areas marginal to the infarct contained cells in varying stages of degeneration including many that appeared normal by morphologic criteria alone. Intracellular lanthanum was present in many but not all of the marginal cells in which degenerative changes could be seen. Similarly a few of the cells that appeared morphologically normal contained intracellular lanthanum. The entry of lanthanum into some of these marginal cells and its exclusion from adjacent cells demonstrated that ischemic injury affects the permeability properties of the plasma membrane independently of other intracellular morphologic changes and that lanthanum can be a sensitive indicator of such alteration in membrane permeability. (Am J Pathol 79:207-218, 1975)

THE CELLULAR MECHANISMS by which myocardial ischemia causes irreversible injury and cell death have not yet been clearly defined, although morphologic studies of ischemic myocardial cells have shown that each organelle or subcellular structure undergoes a characteristic sequence of degenerative changes. However, degenerative From the Divisions of Rheumatology and Cardiology, Department of Medicine, New York University School of Medicine, New York, NY. Supported by Grants AM-11949, HL-15140 and NHLI-72-2953 from the National Institutes of Health and by grants from The John Polachek Foundation for Medical Research and 'Te Whitehail Foundation. Accepted for publication January 15, 1975. Address reprint requests to Dr. Sylvia Hoffstein, Division of Rheumatology, Department of Medicine, New York University School of Medicine, 550 First Ave, New York, NY 10016.

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changes do not necessarily occur simultaneously in all organelles of any one cell or synchronously in adjacent cells.'-7 The experimental induction of infarction by occlusion of a branch of a coronary artery results in irreversible injury to a few cells by 21 minutes and to 50% of the cells exclusively supplied by this vessel within 40 minutes.3'4 By 1 to 2 hours, most of the cells in this region are irreversibly injured.5'7 There is better collateral circulation to the marginal or partially ischemic zone of an infarct and myocardial cells of this region exhibit extreme morphologic diversity.'- Intact cells can be seen adjacent to injured cells. Some cells show changes associated with ischemic injury, eg, loss of glycogen and intracellular edema, although their mitochondria appear morphologically intact or only slightly and perhaps reversibly injured.7 However, cells in this marginal population continue to die for at least 12 and up to 24 hours after coronary occlusion." 2'7-9 An early event in the pathogenesis of ischemic injury may be injury to the plasma membrane, causing increased permeability to ions and macromolecules before overt structural changes appear. Indeed the failure of cells to exclude dyes such as eosin Y or trypan blue 10,11 has been equated with cell death, but these light microscopic technics are not useful for studying early changes in ischemic myocardium. Colloidal lanthanum, which has an average particle size of 40 A, penetrates spaces as small as 20 A 12 and has been used as a tracer of extracellular space at the ultrastructural level.'2-'7 In normal muscle, lanthanum remains confined to the extracellular space,'3 but in skeletal muscle fibers injured by heat it penetrates the cell and stains organelles in a characteristic pattern."' Thus, intracellular lanthanum can be used as a marker for plasma membrane injury and its presence in, or absence from cells can be correlated with other ultrastructural parameters of cell damage. This study was undertaken to relate structural alterations in the cytoplasm of ischemic myocardial cells to changes in the permeability of the sarcolemma,. estimated by using colloidal lanthanum nitrate as an ultrastructural probe. Materials and Methods Thrombosis of the left anterior descending coronary artery was produced in 9 closed-chest, lightly anesthetized dogs by passage of DC current through an electrode-tipped catheter.'8 A Jones Catheter (No. 7) was introduced, under fluoroscopic control, into the proximal portion of the artery and its position verified by injection of a small amount of radioopaque dye. A weak current, averaging 0.8 mA was passed through the intercoronary electrode for 15 to 20 minutes, until definite electrocardiographic changes characteristic of acute myocardial infarcation were produced. Thrombi were regularly produced at the site of the electrode; they completely occluded the arterial lumen and were firmly attached to the vessel wall.

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Such thrombi were histologically identical to naturally occurring arterial thrombi (hematoxylin- and eosin-stained sections). Infarcted, "marginal" and remote (normal) samples were obtained from the beating heart with Vim-Silverman biopsy needles (No. 14) at 3Y2 hours after infarction and rapidly fixed for electron microscopy. Selection of biopsy sites of infarcted and normal areas was straightforward, but "marginal" areas could be approximated only by their adjacent relationship to grossly infarcted areas or to the region supplied by the occluded artery. In 2 control dogs, the electrode-tipped catheter was inserted into the coronary artery, but no current was applied. Tissue was sampled from these animals 24 or 96 hours later. Electron Microscopy

A 4% aqueous solution of La(NO3)3 in a narrow-mouthed flask was adjusted to pH 7.8 by adding 0.1 N NaOH slowly through a finely drawn pipette to avoid precipitation of La (CO3),,. The final fixative consisted of one part of freshly prepared 4% La (NO3)3 and two parts of 6% glutaraldehyde (Fisher) in 0.1 M sodium cacodylate buffer, pH 7.4. Biopsy specimens were immediately placed in this fixative and allowed to stand for 2 hours at room temperature, with occasional gentle shaking. While in the fixative, the endocardial and epicardial ends of the specimens were trimmed off to a 2 mm depth, and the remaining tissue was cut into small pieces. The tissue was washed three times within 1 hour in one volume of 10% sucrose in 0.1 M sodium cacodylate buffer, pH 7.4, to two volumes of 4% La (NO3) . The samples were postfixed for 1 hour in 1% osmium tetroxide in 0.1 M cacodylate buffer without added La(NO3)3, then rinsed in distilled water, and dehydrated in acetone as rapidly as possible. The tissue samples were embedded in Epon, polymerized at 70 C for 48 hours and sectioned with a diamond knife on a Sorvall Porter Blum MT 2B. Blocks were trimmed before sectioning so that only cells in the center of the tissue sample were studied. Both unstained and lead-citrate-stained sections were viewed in a Zeiss EM 9S electron microscope.

Results

Normal myocardial cells contained considerable glycogen, most of which was in the cytoplasm between myofibrils and around mitochondria, although some was evident between moderately contracted myofilaments. Mitochondria were numerous, with long cristae and dense matrices. This morphology was not altered by the addition of La( NO3 )3 to the fixative. Lanthanum was excluded from the intracellular spaces in normal myocardial cells, but it readily stained basement membranes and penetrated gap junctions (Figure 1). It was rarely seen inside T tubules in our preparations, probably because it was washed away during processing. As Fahimi and Cotran reported,16 rough handling of the tissue during fixation introduced lanthanum into normal cells at the periphery of the tissue blocks, but under these circumstances the deposition of lanthanum was not selective and all cytoplasmic structures, including myofibrils, were indiscriminately stained. Cells so damaged by handling were easily detected when either stained or unstained sections were scanned at low magnification. Virtually all of these mechanically

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injured cells were eliminated when the plastic-embedded tissue was trimmed for sectioning. Infarcts were invariably present, and irreversibly injured ischemic myocardial cells from the center of the infarcted area lost stainable glycogen and developed intracellular edema. The myofibrils were separated by electron-lucent areas containing a few vesiculated elements of the sarcoplasmic reticulum (SR). The myofibrils in many cells appeared relaxed, with marked I bands, portions of which often appeared eroded. Mitochondria were swollen, with abnormally clear matrices and, in 12 of 14 specimens examined, contained patches of an amorphous lipid-like material (Figure 2). In 10 of the 12 specimens with amorphous mitochondrial patches, cells with relaxed myofibrils were seen. Both configurations were not always seen in the same cells, but the mitochondria in cells with relaxation bands were clearly abnormal, appearing vesiculated and devoid of matrix. In the two other specimens from a single animal there was severe derangement of myofibrillar organization, and sarcomeres were not visible. Mitochondria from these cells were also devoid of matrix and occasionally disrupted (Figure 3). All nuclei from cells with any of these myofibrillar or mitochondrial abnormalities were also abnormal, with condensed chromatin and clear centers (Figure 2). Cells that showed any of these myofibrillar or mitochondrial abnormalities, characteristic of severe ischemic injury, invariably failed to exclude lanthanum. Intracellular lanthanum was most prominent in infarcted cells as nodular deposits bound to outer mitochondrial membranes (Figure 2). The preferential binding of La... to outer mitochondrial membranes was generalized, so that if one mitochondrion in a cell section had lanthanum deposits, all had. The amount bound appeared to be uniform within each cell and from cell to cell in the same specimen (Figure 3). Indeed, very little variation in the intensity of lanthanum staining was seen among different specimens of infarcted tissue. Lanthanum was present within mitochondria only when mitochondrial membranes were ruptured. Other structures in injured cells that occasionally bound lanthanum were lipid droplets and T tubule membranes. Marginal samples invariably contained some injured cells. Of 17 specimens examined, five were indistinguishable from infarcted tissue, and all cells examined from these specimens failed to exclude lanthanum. The remaining 12 specimens showed the morphologic heterogeneity characteristic of marginal tissue. Within individual sections of such marginal tissue, the cell injury ranged from moderate to severe, or normal cells were seen adjacent to moderately injured cells, but normal and severely injured cells were not present together in the same section.

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Severely or irreversibly injured cells in the marginal zone showed one or another of the morphologic changes seen in cells from the center of the infarct after 32 hours of total ischemia. The criteria of irreversible injury included marked myofibrillar relaxation, gross mitochondrial swelling and loss of matrix, and amorphous dense patches within mitochondria. When any of these morphologic criteria of irreversible injury were observed in a cell, that cell invariably failed to exclude lanthanum. Morphologic changes seen in moderately injured cells from the marginal zone include contraction bands, loss of glycogen ranging from partial to complete, a variable degree of separation of myofilament bundles by edema, and mitochondrial changes, including some patchy loss of matrix density or blebbing of the mitochondrial membranes. Many of these cells failed to exclude lanthanum. Nuclei from such cells appeared normal or had only slight margination of the chromatin. In moderately injured cells, intracellular lanthanum had the same distribution as in infarcted cells (Figures 4-6). It was most prominent around mitochondrial membranes, and when one mitochondrion in a cell profile contained lanthanum deposits, all did. Contraction bands were seen in "marginal" specimens from 6 animals. Of 21 cells with contraction bands in these specimens that were photographed at random, all had retained glycogen in the contracted region and showed no intracellular edema (Figure 4). All but three of these cells failed to exclude lanthanum, and these three were adjacent to cells with contraction bands and intracellular lanthanum. Mitochondria in cells with contraction bands appeared normal or with only slight loss of matrix density. Intracellular edema was associated with loss of stainable glycogen from between myofibrils although not necessarily from within the myofibrils. Many such cells were capable of excluding lanthanum (Figures 7 and 8). Some cells with no other ultrastructural indications of injury failed to exclude lanthanum, and adjacent cells were sometimes seen in which one excluded lanthanum and one did not. Myofibrils in cells with intracellular lanthanum were not more electron dense than those in adjacent cells, indicating that lanthanum penetration was not an artifact of preparation. Discussion

These studies have shown that lanthanum nitrate can be used as an ultrastructural probe for ischemically induced permeability changes in the sarcolemma. Permeability to lanthanum invariably accompanied the degenerative changes associated with irreversible injury to ischemic myocardial cells. These changes include: a) swollen, electron-lucent

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mitochondria, b) amorphous dense patches in mitochondria, c) marked relaxation of myofibrils, d) extreme margination of nuclear chromatin. However, cells with less severe ultrastructural evidence of injury also generally failed to exclude lanthanum, as did some cells which appeared otherwise normal. The only type of damage seen in marginal cells still capable of excluding lanthanum was intracelluar edema with loss of glycogen. Several histologic studies of ischemic myocardium 9,19-21 have shown that irreversibly injured cells are characterized by loss of glycogen and the acquisition of diastase-resistant periodic acid-Schiff (PAS) staining properties. Kent,20,21 has shown that this PAS-positive staining is due to plasma glycoproteins which penetrate and bind to injured cells. Albumin and IgG diffuse into dog myocardial cells within an hour after ischemia is induced. Fibrinogen is also found in the center of human myocardial infarcts, but only albumin and IgG are found at the periphery of infarcts at 3 to 4 hours. Our ultrastructural studies have shown that these two morphologic consequences of ischemia, loss of glycogen and altered sarcolemma permeability do not necessarily coincide in marginally injured cells although they do coincide in obviously necrotic cells. Our data suggest that cell injury in the marginally ischemic area and perhaps in the center of the infarct may occur by at least two mechanisms. In one, an early event is the loss of sarcolemmal integrity leading to uncontrolled entry of ions and macromolecules and concomitant loss of soluble cytoplasmic constituents. The second pathway appears to involve early intracellular release of substances (possibly from lysosomes) which exert a glycogenolytic effect. In the presence of an intact sarcolemma these processes may lead to an increase in osmotic pressure and consequent intracellular edema. Whether the operation of either or both of these two consequences of ischemic injury imply irreversible damage is not clear from these studies alone, although Korb and Totovic 2 report that at the periphery of the ischemic zone both contracted and adjacent relaxed cells depleted of glycogen later became necrotic. Indeed, lack of synchrony in these two mechanisms of cell injury may account for some of the morphologic heterogeneity characteristic of ischemic lesions. Further, lanthanum and other tracer studies of short-term occlusions in which the injury is known to be reversible should help clarify the problem. Elucidation of the mechanism of cell death in ischemic myocardium is important since previous work has shown that the tissue immediately peripheral to an infarct may continue to change with time. Histochemical

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studies at the light microscopic level have shown that ischemically injured myocardium surrounding necrotic tissue continues to expand for 18 hours and then decreases in size as part becomes necrotic and part recovers.8 Similarly, experiments with various therapeutic interventions indicate that reduction of infarct size or area of necrosis can be achieved when the intervention occurs as long as 6 hours after infarction.2223 Better understanding of the mechanisms of early ischemic damage might therefore lead to a more rational basis for therapies designed to protect the jeopardized or partially damaged myocardium. References 1. Heggtveit HA: Morphological alterations in the ischaemic heart. Cardiology 56:284-290, 1971/72 2. Korb G, Totovic V: Electron microscopical studies on experimental ischemic lesions of the heart, Ann NY Acad Sci 156:48-60, 1969 3. Herdson PB, Sommers HM, Jennings RB: A comparative study of the fine structure of normal and ischemic dog myocardium with special reference to early changes following temporary occlusion of a coronary artery. Am J Pathol 46:367-386, 1965 4. Jennings RB, Sommers HM, Herdson PB, Kaltenbach JP: Ischemic injury of the myocardium. Ann NY Acad Sci 156:61-78, 1969 5. Kloner RH, Ganote CE, Whalen DA, Jennings RB: Effect of a transient period of ischemia on myocardial cells. II. Fine structure during the first few minutes of reflow. Am J Pathol 74:399-422, 1974 6. Shnitka TK, Nachlas MM: Histochemical alterations in ischemic heart muscle and early myocardial infarctions. Am J Pathol 42:507-527, 1963 7. Jennings RB, Baum JH, Herdson PB: Fine structural changes in myocardial ischemic injury. Arch Pathol 79:135-143, 1965 8. Cox JL, McLaughlin VW, Flowers NC, Moran LG: The ischemic zone surrounding acute myocardial infarction: Its morphology as detected by dehydrogenase staining, Am Heart J 76:650-659, 1968 9. Dusek J, Rona G, Kahn DS: Healing process in the marginal zone of an experimental myocardial infarct: Findings in the surviving cardiac muscle cells. Am J Pathol 62:321-338, 1971 10. Pappenheimer AM: Experimental studies upon lymphocytes. I. Reactions of lymphocytes under various experimental conditions. J Exp Med 25:633650, 1917 11. King DW, Paulson SR, Puckett NL, Krebs AT: Cell death. IV. The effects of injury on the entrance of vital dye in Ehrlich tumor cells. Am J Pathol 35:1067-1080, 1959 12. Revel JP, Karnovsky MJ: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol 33:C7-C12, 1967 13. Doggenweiler CF, Frenk S: Staining properties of lanthanum on cell membranes. Proc Natl Acad Sci 53:425-430, 1965 14. Zacks SI, Saito A: Direct connections between the T-system and the subneural apparatus in mouse neuromuscular junctions demonstrated by lanthanum. J Histochem Cytochem 18:302-304, 1970

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15. Martinez-Palomo A, Benitez D, Alanis J: Selective deposition of lanthanum in mammalian cardiac cell membranes. J Cell Biol 58:1-10, 1973 16. Fahimi HD, Cotran RS: Permeability studies in heat induced injury of skeletal muscle using lanthanum as fine structural tracer. Am J Pathol 62: 143-157, 1971 17. McNutt NS, Weinstein RS: The ultrastructure of the nexus: A correlated thin-section and freeze-cleave study. J Cell Biol 47:666-88, 1970 18. Salazar AE: Experimental myocardial infarction: Induction of coronary thrombosis in the intact closed-chest dog. Circ Res 9:1351-1358, 1961 19. Sommers HM, Jennings RB: Experimental acute myocardial infarction: Histologic and histochemical studies of early myocardial infarcts induced by temporary and permanent occlusion of a coronary artery. Lab Invest 13:14911503, 1964 20. Kent SP: Intracellular plasma protein: A manifestation of cell injury in myocardial ischemia. Nature 210:1279-1281, 1966 21. Kent SP: Diffusion of plasma proteins into cells: A manifestation of cell injury in human myocardial ischemia. Am J Pathol 50:623-637, 1967 22. Maroko PR, Libby P, Ginks WR, Bloor CM, Shell WE, Sobel BE, Ross J Jr: Coronary artery reperfusion. I. Early effects on local myocardial function and the extent of myocardial necrosis. J Clin Invest 51:2710-2716, 1972 23. Libby P, Maroko PR, Bloor CM, Sobel BE, Braunwald E: Reduction of experimental myocardial infarct size by corticosteroid administration. J Clin Invest 52:599-807, 1973

Acknowledgments The authors would like to acknowledge the photographic assistance of Winston Blackett.

Legends for Figures Fig 1-Portions of two adjacent normal myocardial cells fixed with lanthanum as described. Lanthanum (L) is visible only in the intercellular space, not around mitochondria or in T tubules (T). Glycogen (G) can be seen between myofibrils, at the cell margins and to some extent between myofilaments (Uranyl acetate and lead citrate, x 20,400).

Fig 2-Several of the characteristic morphologic changes associated with irreversible ischemic injury can be seen in this portion of a myocardial cell from the center of a 3l/2-hour infarct: a) The cell is completely devoid of glycogen. b) The nuclear chromatin is marginated, leaving clear spaces within the nucleus (N). c) The myofibrils are relaxed, showing wide I (I) bands which are eroded or torn in some places. d) Mitochondria are swollen, without matrix and have vesiculated cristae. Two of the mitchondria have amorphous matrix densities (arrows), and all have black lanthanum deposits at their periphery (Uranyl acetate and lead citrate, x 20,400).

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Fig 3-A low magnification view of portions of several irreversibly injured cells. Two of the cells appear to have undergone an intense contraction, but the myofibrillar organization of the cell at the lower left appears relatively normal. All the m iKochondria, however, have lost matrix substance and have lanthanum deposits clearly visi 13le around their outer margin, even though little or none can be seen between the cells (Uranyl acetate and lead citrate, X 4500). Fig 4-A -pair of adjacent cells from the marginal ischemic zone. Both cells appear to be normal except for a slight, patchy loss of mitochrondrial matrix density. The cell on the right differs from the cell on the left in only two respects: its myofibrils are strongly contracted (C) and intracellular lanthanum bound to mitochondrial outer mem branes is visible (arrows) (Uranyl acetate and lead citrate, X 20,400).

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Fig 5-A portion of another nearly normal cell from the marginal zone showing intracellular lanthanum (arrows) and contracted sarcomeres, together with a normal nucleus (N) and normal mitochondria (Uranyl acetate and lead citrate x 2040. .4 Fig 6-A partially injured cell from the marginal zone Mitochondria have undergone a patchy loss of matrix density. Glycogen (G) is reduced but not absent, sarcomeres are only slightly contracted and lanthanum is present intracellularly (Uranyl acetate and lead citrate, x 20,400).

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