Experimental Studies on the Endothelium Ultrastructure of Heart Capillaries under Moderate (28–30°) and Deep (22–24°) Hypothermia without Perfusion

Microvascular Research 58, 250 –267 (1999) Article ID mvre.1999.2181, available online at http://www.idealibrary.com on

Experimental Studies on the Endothelium Ultrastructure of Heart Capillaries under Moderate (28 –30°) and Deep (22–24°) Hypothermia without Perfusion Galina M. Kazanskaya, Alexander M. Volkov, Alexander M. Karas’kov, Vladimir N. Lomivorotov, and Anatoly V. Shun’kin Laboratory of Electron Microscopy, Department of Anesthesiology and Laboratory of Hypothermia, Research Institute of Circulation Pathology, 630055 Novosibirsk, Russia Received July 27, 1998

Ultrastructural changes in endothelial cells (EC) of myocardial capillaries were studied in 24 dogs which underwent hypothermia without perfusion. Biopsy specimens for electron microscopy were taken from the left ventricle of each dog in the control group, during anesthesia (prior to active cooling), and at the end of moderate (28 –30°) and deep (22–24°) artificial body cooling. The following morphological types of the EC were identified both in the control group and in all test groups: those with moderately dense cytoplasm, light, dark, and irreversibly damaged cells. Dark cells showed increased numbers of plasmalemmal vesicles and appeared to be more transportspecialized as opposed to other types. In all stages of the experiment the amount of dark cells continuously increased (to 23.80, 34.62, and 47.17%, respectively). On cooling to 28 –30°, subcellular manifestation of reduced synthetic activity of organelles (nucleus, Golgi complex, and rough endoplasmic reticulum) was observed in all types of the EC. These changes persisted, or even increased, at the end of deep hypothermia. The transport activity of the EC changed differently in three experimental groups in all cell types. Micropinocytotic activity increased under spontaneous mild hypothermia (34 –35°) during anesthesia and tended to decrease with subse-

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quent artificial lowering of the temperature to 22–24°. These ultrastructural changes seem to make up an integral part of the process of capillary endothelium adaptation to body surface cooling, and they might contribute to the development of tolerance to subsequent ischemic exposure during cardiac arrest. © 1999 Academic Press Key Words: endothelium; capillaries; heart; ultrastructure; hypothermia.

INTRODUCTION Extensive use of perfusionless hypothermia as a method of protection from hypoxia for patients undergoing cardiac surgery (Litasova and Lomivorotov, 1988; Litasova et al., 1994) necessitates a study of changes in the microvascular bed of the myocardium under these conditions. There is a line of evidence according to which topical hypothermia preserves both the coronary endothelium and the myocardial energy sources during ischemic cardiac arrest (Karck et al., 1995). There is another line of evidence indicating that the use of topical hypothermia neither improves postoperative hemodynamics nor reduces 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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perioperative myocardial infarction or the need for inotropes (Allen et al., 1992). While carrying out morphological research in myocardium under hypothermic cardiac arrests followed by heart reperfusion, emphasis was mainly placed on studying the changes taking place in cardiac myocytes. It was demonstrated that the ratio of adaptive and destructive cell changes depends on the nature of myocytes rearrangement at the phase preceding the occlusion of heart main vessels, i.e., under hypothermia when cells turn into the state of hypometabolism (Maksimov and Korostyshevskaya, 1990). Nevertheless, the effect of topical cooling, specifically on vascular endothelial morphology, has not been extensively studied. Additionally, it is not clear what ultrastructural changes in capillary endothelium can be involved in adaptation of the myocardium to cold. In order to resolve such problems, it should be realized that the capillary endothelium, contrary to our former belief, does not represent a homogeneous cell population. There is a wealth of data establishing the morphological, biological, and functional heterogeneity of vascular endothelial cells (EC) within the pulmonary, cerebral, and placental microcirculation (DeFouw, 1988; Karimu and Burton, 1995; Shaver et al., 1990). Morphological heterogeneity of the EC of myocardium capillaries was called attention to a fairly long time ago. In 1968 Epling (1968) showed that in myocardial capillaries of cattle suffering from high mountain disease there were four forms of the EC differing in electron density and ultrastructure. The author considered the discovered heterogeneity of myocardial capillary endothelium a manifestation of the chronic circulatory hypoxia experienced by cattle with terminal high mountain disease. However, other researchers reported on the presence of the EC with a different electron density (dark, light, intermediate, and irreversibly damaged ones) in the capillaries of intact organs and tissues (Chernykh et al., 1975). In a recent study we have found that the above four morphological types of the EC are present in the capillaries of a normal myocardium in mongrel dogs (Volkov et al., 1996). Moreover, all four types can be found after such pathophysiological changes as myocardial ischemia and reperfusion (Kazanskaya et al., 1998). In this case, however, their percentage varies widely.

The present study has been undertaken in order to more adequately characterize ultrastructural changes taking place in myocardial capillaries under moderate (28 –30°) and deep (22–24°) cooling of body temperature. Use has been made of an experimental model of hypothermia without perfusion. As indicated earlier, we have broken down capillary endothelium of the left ventricle of dogs’ hearts into four morphological types according to electron density and ultrastructure. Taking into consideration the fact that the main function of the EC in capillaries is primarily maintained by such structural elements as plasmalemma, plasmalemmal vesicles, and mitochondria (Chan et al., 1991; Wagner and Chen, 1991; Willemsen et al., 1990) we have focused on the analysis of some structural parameters relating to vascular endothelium permeability. The ultrastructure of each morphological type was compared at all steps of hypothermia and with that of the control group. The ultrastructural comparisons were paralleled by analysis of the time course of changes in cell population under moderate and deep hypothermia. The approach used is, to our knowledge, novel because it combines subcellular and cellular-populational analysis of capillary endothelium.

MATERIALS AND METHODS A total of 24 mature mongrel dogs of either sex, weighing 9 –15 kg, were used for the study. Six dogs served as unoperated control; the remainder represented an experimental group. In the control group (group 1) the animals were killed by an overdose of sodium pentobarbital at normal body temperature (36 –37°). In accordance with a previously described technique (Semionova et al., 1985) hearts were rapidly dissected and placed on finely crushed ice in order to stop cardiac contractions. In doing so, care was taken to see to it that the left ventricle region was not in contact with ice. In approximately 30 – 60 s after heart dissection tissue specimens of the left ventricle were taken. During this period of time the temperature of a subepicardial layer of myocardium, the tissue specimens of which were harvested for control, was kept at a level not below 36°.

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The dogs in the anesthesia group (group 2, n 5 6) underwent only operative preparation procedure; they were not subject to perfusionless hypothermia. The purpose of this group was to test the responses to anesthesia, intubation, mechanical ventilation, sternotomy, and mild hypothermia. During this test, which lasted for about 30 – 45 min, the esophageal temperature dropped spontaneously down to 35–34°. The 12 remaining dogs in groups 3 and 4 were surface cooled in an ice bath and were studied at moderate (28 –30°) hypothermia (group 3, n 5 6) or deep (22–24°) hypothermia (group 4, n 5 6).

Kazanskaya et al.

erative care consisted of assisted ventilation with oxygen and analgesia with intermittent doses of trimeperidine hydrochloride. Arrythmias were controlled with lidocaine (1 mg/kg) as necessary. After the volume-controlled respirator was switched off and the dogs started breathing by themselves, they were transferred to postoperative cages for a period of 3 days. Close control over the recovery of all vitally important functions of the animals was exercised during this period. Toward the end of the third day all dogs in groups 3 and 4 would have adequate hemodynamics, take water on their own, urinate, respond to a voice, and move around the cage.

Experimental Design All animals were premedicated with 2% trimeperidine hydrochloride (1 mg/kg) and 0.1% atropine sulfate (0.05 mg/kg) subcutaneously. Anesthesia was induced with pentobarbital sodium (10 mg/kg intravenously) and maintained with 2% trimeperidine hydrochloride (Promedol). The dogs were endotracheally intubated and mechanically ventilated with a volume-controlled respirator on room air supplemented with oxygen. The right femoral artery was catheterized for continuous pressure monitoring. The electrocardiogram was monitored continuously and a bladder catheter was inserted to measure urine output. Esophageal temperature was continuously measured throughout the experiment. After stabilization of arterial pressure and heart rate a thoracotomy was performed. Following full instrumentation of the surgical preparation, the dogs in the second group were subjected to biopsy. The dogs in groups 3 and 4 were cooled in tubs with cold water (1–4°). In addition, their bodies were covered with finely crushed ice. Cooling rates were 1° per 6–7 min and 1° per 7–9 min in the moderate hypothermia (28–30°) and deep hypothermia (22–24°) groups, respectively. At such prescribed levels of esophageal temperatures tissue samples were taken from a free wall of the left ventricle. At the end of the experiment the ice pieces and cold water were removed from the bath. In the case of ventricular fibrillation 10 mg lidocaine was infused intravenously, and the heart was defibrillated. One dog in group 3 and two dogs in group 4 had to have their hearts defibrillated. When a stable rhythm was obtained, the dogs were rewarmed. Postop-

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Processing of Tissue Blocks for Electron Microscopy All biopsy specimens were taken from the wall of the left ventricle (0.25–0.50 cm). Multiple cubes, 1 mm in dimension or smaller, were cut with a razor blade under glutaraldehyde and were transferred to vials containing 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (0.1 mol/L, pH 7.35). After at least 4–8 h of fixation the specimens were rinsed in several changes of cold phosphate buffer and postfixed in a 1% phosphatebuffered osmium tetroxide solution for 2.5 h. Tissue blocks were dehydrated in graded alcohol and acetone. After dehydration the tissue blocks were embedded in Epon 812/araldite (Epon812, 22.4%; dodecenyl succinic anhydride, 56%; araldite M, 19.6%; (dimethylaminomethyl)phenol, 2%) and cured at 60° for 48 h. Tissue blocks were then cut by Reichert (Austria) ultramicrotome. Thick sections mounted on glass slides and stained with 1% toluidine blue were examined with a light microscope to determine the orientation of the tissue. The tissue blocks were oriented so that capillaries were sectioned transversely rather than longitudinally. Thin sections were mounted on copper grids and stained with uranyl acetate and lead citrate. The sections were examined under a JEM 100CX electron microscope (JEOL, Japan).

Morphometric Methods Twenty-four capillaries in cross sections were randomly photographed from each tissue block at a primary magnification of 7.5003 (one block per each

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dog, six blocks per each experimental group). Vessels were identified in accordance with the criteria established by Rhodin (1968). Capillaries with closed lumen were discarded from morphometric analysis. Vessels formed by four or more endothelial cells were also eliminated from morphometry. We first calculated the relative proportions of each morphological EC type in total cellular population of each block. A mean value of this parameter for each group was then estimated. The percentage of the EC with moderately dense cytoplasm 5 (the number of the EC with moderately dense cytoplasm in the profiles of all capillaries in the section)/(the total EC number in the profiles of all the capillaries in the section) 3 100. The calculation procedure for light, dark, and irreversibly damaged cells was identical to that for EC with moderately dense cytoplasm. Morphometric analyses were made from all electron micrographs (144 micrographs per experimental group) which were printed at final magnification of 38,0003. While using a test system consisting of 312 points and 24 lines 2.5 cm long, we determined the diameters of the myocardial capillaries, the length of the luminal and abluminal surfaces of the capillaries, and the average thickness of the EC of each type. The test system records were taken three times by turning the test grid by 60° each time, thereby increasing accuracy. The arithmetic average for three measure values was then calculated and the averages were used in the formulas. The diameters and the length of the luminal and abluminal surfaces of the capillaries, as well as the average thickness of the EC, were estimated by using the formulas of Weibel (1973) modified by Karaganov et al. (1976) specifically for morphometric analysis of microvascular system components. The diameters of the capillaries were calculated by the equation D c 5 2 3 ~~P i 3 A s 1 P j 3 A s!/ p ! 1/2 ,

(1)

where P i( j) is the number of points of the test system contained within the profile of the EC and capillary lumen, respectively. A s 5 ~D/K m/K ng! 2 ,

(2)

where K m is the electron microscope magnification in thousands (in our case K m 5 7.5); K ng is the magnification of negative (in our case K ng 5 5); and D is the distance between test points (in our case D 5 1 cm). The length of the luminal and abluminal surfaces of the capillaries was calculated as L l(al) 5 A L 3 I l(al),

(3)

where I l(al) is the number of intersections formed by the boundary trace of luminal or abluminal counter of the capillaries with a test line system. A L 5 p /2 3 d /K m/K ng,

(4)

where d is the distance between test lines (in our case d 5 0.5 cm). The average thickness (t) of the EC of each type was calculated by the equations

t 5 2 3 A t 3 P i/~I al 1 I l!

(5)

A t 5 2/ p 3 D 2 / d /K m/K ng.

(6)

Standard point-counting techniques were used to determine V V(pv) of plasmalemmal vesicles (volume density is defined as the fraction of cell volume occupied by plasmalemmal vesicles). Volume densities of free plasmalemmal vesicles were measured by counting sampling points falling on EC and plasmalemmal vesicles. The number of free plasmalemmal vesicles per unit volume of endothelial cell was calculated by the equation (Weibel, 1980) N v(pv) 5 1/ b 3 N A(pv) 3/2 /V V(pv) 1/2 ,

(7)

where N A(pv) is the count of vesicle profiles in the sampled area, V V(pv) is the volume density of vesicles, and b is the shape coefficient of vesicular profiles. The numerical density of free plasmalemmal vesicles was calculated by assuming that all vesicular profiles were circular. Additionally, we determined the total volume of cytoplasm per average capillary EC for each morphological type. We analyzed 72 electron microphotographs (24 each from three tissue blocks) per heart as previously described (Mastin et al., 1988). All microphotographs were photographed at 20003 and printed at 85003. We used a test system consisting of 112 lines of 2 cm length (224 points). We first calcu-

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TABLE 1 Population Composition of Capillary Endothelium from the Left Ventricle in Different Groups of Topical Cooling without Perfusion

Groups Group 1 (control 36–37°) Group 2 (anesthesia 34–35°) Group 3 (moderate cooling 28–30°) Group 4 (deep cooling 22–24°)

Number of dogs

EC types ME

LE

DE

IDE

6

65.17 6 3.01

15.17 6 3.23

17.50 6 2.95

2.17 6 1.25

6

62.60 6 3.23

12.20 6 2.81

23.80 6 3.28

1.40 6 0.75

6

52.00 6 1.70* ,**

11.12 6 1.43

34.62 6 2.91* ,**

2.25 6 0.36

6

41.50 6 2.68* ,** ,***

47.17 6 3.04* ,** ,***

2.50 6 0.88

8.83 6 1.60

Note. ME, EC of the main type; LE, light EC; DE, dark EC; IDE, irreversibly damaged EC. Data for control and each experimental groups are expressed as percentages of the total number of cells in capillary endothelium. Data are given as mean 6 standard error of the mean. * Value significantly different from control value; P , 0.05 (Student’s t test). ** Value significantly different from Group 2 value; P , 0.05. *** Value significantly different from Group 3 value; P , 0.05.

lated the volume density of cytoplasm per EC. Then, we calculated the mean volume of an individual capillary EC, which was equal to the volume density of capillary EC in heart tissue (in cm 2/cm 3 of heart tissue) divided by the numerical density of capillary EC in heart tissue (in cells/cm 3). The number of capillary EC per unit volume of heart tissue was calculated as previously described (Weibel, 1973). We next calculated the mean volume of cytoplasm in an average capillary endothelial cell, which was equal to the volume density of cytoplasm per EC multiplied by the mean individual capillary EC volume. The results were expressed in micrometers cubed per cell. The approach to semiquantitative analysis of the EC was essentially similar to that used by Lindal et al. (1988). It was based on an assessment of the extent of ultrastructural changes in each type of EC in accordance with the criteria of Table 4. In our modification of Lindal’s scheme we additionally analyzed two organelles: the Golgi complex and rough endoplasmic reticulum.

Statistical Analysis All results were analyzed by the Student t test and accepted to be statistically significant at the 95% confidence level. Data were presented as mean and standard error.

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RESULTS Ultrastructure of Capillary Endothelium in Control Group (Group 1, T 5 36 –37°) The endothelium surrounding capillaries in control hearts appeared in four forms: endothelium with moderately dense cytoplasm, endothelium with light cytoplasm, endothelium with dark cytoplasm, and irreversibly damaged endothelium. Cells with moderately dense cytoplasm were the most numerous ones, constituting 65.17% of the total EC number (Table 1). This was the main cell type in capillary endothelium. The average thickness of the EC was estimated for the peripheral region of the cell and reached a value of 0.41 6 0.01 (SE) mm. The total volume of cell cytoplasm per average capillary EC of the main type was 151.72 6 5.43 (SE) mm 3 (Table 2). Cytoplasmic organelles were concentrated around the nucleus in the thickest region of the cells. Nuclear chromatin was nonuniformly dispersed and contained distinct clumps of dense chromatin distributed along the nuclear membrane (Fig. 1a). The Golgi complex (GC) was composed of four to five flat cisternae, several vacuoles, and a group of small vesicles. The cisternae were filled with amorphic substance, with forming vesicles seen on their surface. Rough endoplasmic reticulum (RER) was represented by elon-

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TABLE 2 Absolute Volume of Cytoplasm per Average Capillary EC and the Average Thickness of the EC in Different Groups of Topical Cooling without Perfusion Volume of cytoplasm per capillary EC (mm 3) Groups Group 1 (control 36–37°) Group 2 (anesthesia 34–35°) Group 3 (moderate cooling 28–30°) Group 4 (deep cooling 22–24°)

ME

LE

Average thickness of the EC (mm)

DE

ME

LE

DE

151.72 6 5.43

161.26 6 7.09

121.89 6 5.84

0.41 6 0.01

0.50 6 0.01

0.35 6 0.01

139.17 6 7.00

179.85 6 8.82

108.86 6 4.69

0.39 6 0.02

0.47 6 0.02

0.29 6 0.01*

122.43 6 6.09*

138.72 6 4.49* ,**

105.95 6 7.71

0.36 6 0.01*

0.51 6 0.02

0.30 6 0.01*

150.52 6 5.48***

159.78 6 4.35***

119.54 6 4.68

0.38 6 0.02

0.53 6 0.06

0.34 6 0.01

Note. ME, EC of the main type; LE, light EC; DE, dark EC; IDE, irreversibly damaged EC. Data for control and each experimental groups are expressed as percentages of the total number of cells in capillary endothelium. Data are given as mean 6 standard error of the mean. * Value significantly different from control value; P , 0.05 (Student’s t test). ** Value significantly different from Group 2 value; P , 0.05. *** Value significantly different from Group 3 value; P , 0.05.

gated channels, with the surface densely covered by ribosomes (Fig. 1b). Medium-sized mitochondria with a moderate electron-density matrix and few cristae were concentrated around the RER. EC of the main type demonstrated numerous invaginations (usually referred to as attached plasmalemmal vesicles) on both luminal and abluminal aspects of their plasma membranes. Coated pits were occasionally noted. Obviously, they are present everywhere on the plasma membrane. Free plasmalemmal vesicles ranging in size were usually the most prominent organelles in the peripheral regions of the EC. The calculated values of the number of vesicles in the total volume of cytoplasm of the EC and numerical densities for free plasmalemmal vesicles are summarized in Table 3. The percentage of light cells was 15.17% of the total EC number. Light EC were thicker (approximately 0.50 6 0.02 mm) than the EC of the main type. However, the values of total volume of cytoplasm per capillary EC did not differ significantly in light EC and in those of the main type (Table 2). In addition, all cytoplasmic organelles in light EC had a normal appearance (Fig. 1c). Most of the light EC were very similar in morphology of the nucleus, GC, and RER to the EC of the main type described above. In some cells nuclear chromatin was slightly cleared and single cisternae of GC were dilated. In occasional cells the cytoplasm happened to contain mitochondria with short

cristae and partial clearing of the matrix. The distribution of attached plasmalemmal vesicles was approximately the same in the light EC and cells of the main type, while a number of vesicles in the total volume of cytoplasm of the EC and numerical density of free vesicles were significantly (P , 0.05) smaller in the light EC (Table 3). Fusion of vesicles resulting in the formation of vacuoles and rosette-like structures was frequently observed in the light cells. The thickness of dark cells (0.35 6 0.01 mm) and total volume of cytoplasm of these cells (121.89 6 5.84 mm 3) occurred to be significantly lower than those of light cells and the cells of the main type (Table 2). Even though the dark cells appeared to contain cytoplasm of increased electronic density, they had no signs of shrinkage. The majority of the dark EC exhibited morphology which was in general similar to that of the main type. Several sections displayed small remnants of dark EC with morphological alterations such as nuclei with condensed chromatin, moderately swollen mitochondria with partly clear matrix, and GC composed of narrow cisterns with hardly discernible lumina. Characteristic of the dark cells was a high activity of plasmalemma manifesting itself as an abundance of attached and free vesicles (Figs. 1a and 1c). As the estimates of the numerical density of vesicles in a unit of cell volume and the estimates of a number of vesicles in the total volume of cytoplasm of

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FIG. 1. Ultrastructure appearance of heart capillary endothelium in the control group. (a) Endothelium is composed of cells of two types—a cell of the main type (ME) and a dark cell (DE). A large nucleus (N) protruding into capillary lumen (L) is seen in the cell of the main type. Original magnification, 10,2503; (b) A fragment of the cell of the main type. Seen in the perinuclear space of the cell are Golgi complex (GC), mitochondria (M), and elongated channels of rough endoplasmic reticulum (RER). Original magnification, 22,5003; (c) The capillary lumen (L) is surrounded by two cells, one dark (DE), the other light (LE). The cytoplasm is much more electron dense in the dark compared to light cell. Package density of vesicles (V) is considerably higher in the dark cell rather than in the light one. Original magnification, 16,5003.

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TABLE 3 Changes in Numerical Density of Vesicles and Changes in the Total Number per Cell Cytoplasm of Vesicles in the EC of Three Types in Different Groups of Topical Cooling without Perfusion Plasmalemmal vesicles Number of vesicles per total volume of cytoplasm (number of vesicles)

Density/mm 3 cytoplasm (N A(pv)) (vesicles/mm 3) ME

LE

615 6 14

279 6 19

943 6 13

93307 6 3340

44992 6 1979

114942 6 5506

875 6 10*

531 6 17*

1224 6 15*

121776 6 6127*

95498 6 4682*

133242 6 5739*

707 6 27* ,**

403 6 22* ,**

447 6 8* ,** ,***

224 6 11** ,*** 623 6 7* ,** ,***

Groups Group 1 (control 36–37°) Group 2 (anesthesia 34–35°) Group 3 (moderate cooling 28–30°) Group 4 (deep cooling 22–24°)

Note. ME, EC of the main type; and all experimental groups. * Value significantly different ** Value significantly different *** Value significantly different

DE

ME

886 6 29**

86556 6 4306**

LE

55903 6 1810* ,**

67283 6 2451* ,** ,*** 35791 6 975* ,** ,***

DE

93372 6 6618* ,** 74474 6 2917* ,** ,***

LE, light EC; DE, dark EC. Data are given as mean 6 standard error of the mean; n 5 6 for control group from control value; P , 0.05 (Student’s t test). from Group 2 value; P , 0.05. from Group 3 value; P , 0.05.

the EC for each type showed, dark EC contained far more free plasmalemmal vesicles than those of the main type and light EC (Table 3). In addition to the three types of EC, two relatively small groups of cells with sharply modified morphology were observed in the controls, swollen and necrotic cells. The first cells had translucent cytoplasm and indistinct or disrupted plasma membranes. The second cells showed morphological alterations such as increased electron density of cytoplasm, shrinkage and pyknotic nucleus, disrupted cytoplasmic membrane, and detachment from the basement membrane. They exhibited an altered, degenerated morphology and were designated irreversibly damaged cells (IDE). The number of IDE does not exceed 2% (Table 1). The presence of IDE in the controls is indicative of the natural death of those cells whose life cycle is over.

Ultrastructure of Endothelium under Anesthesia (Group 2, T 5 34 –35°) The specimens obtained from anesthesized dogs differed slightly from those of the controls by general architecture of capillary endothelium. Under anesthesia there were no significant changes in relative proportions of the EC types (Table 1). Semiquantitative

data for qualitative subcellular changes in the EC of the dogs from different experimental groups are summarized in Table 4. As the data show, at the stage of anesthesia there were no conspicuous changes in the ultrastructure of cells of the main and light types compared to the controls. They contained large active GC and unswollen mitochondria (Figs. 2a and 2b). The average thickness of these cells was not altered by the procedure as well (Table 2). The transport vesicles of the main and light EC were directly affected by anesthesia. Morphometric examination of the capillaries of anesthetized animals showed moderate to marked increases in the numerical density of free plasmalemmal vesicles, as well as in the number of vesicles per total volume of cytoplasm of the EC (Table 3). The average thickness of the dark cells decreased gradually from about 0.35 6 0.01 mm in the control dogs to about 0.29 6 0.01 mm in the anesthesized dogs (P , 0.05 vs control value). At the same time the total volume of cytoplasm per average dark EC did not vary significantly against the control (Table 2). Moderate changes were observed in the ultrastructure of cytoplasmic organelles (Table 4). For dark EC the estimates of the numerical density of vesicles in a unit of cell volume and the number of vesicles in the total volume of cytoplasm demonstrated an increase in the

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TABLE 4 Estimation of Ultrastructural Changes in Endothelial Cells of the Capillaries in Different Groups of Total Body Cooling without Perfusion Group 1 (control: 36–37°)

Group 2 (anesthesia: 34–35°)

Group 3 (moderate cooling: 28–30°)

Group 4 (deep cooling: 22–24°)

ME

LE

DE

ME

LE

DE

ME

LE

DE

ME

LE

DE

Clearing of karioplasm Margination of chromatin Clumping of chromatin

— * —

— * —

— * —

— * —

Nucleus — * —

— ** *

— ** *

* * *

— ** **

* ** **

** ** *

— *** ***

Clearing of matrix Fragmentation of cristae Cristolysis

— — —

— — —

— — —

Mitochondria — — — — — —

* * —

* * —

** * *

** ** —

* * —

*** ** ***

*** *** **

* * *

* * *

* * *

** ** **

** ** *

** *** **

*** ** ***

* * *

** * **

** ** **

** * *

*** ** **

*** *** ***

Golgi complex Decrease in number of attached vesicles Dilatation of cisternae Fragmentation of cisternae

— — —

— — —

— — —

— — —

— — —

Rough endoplasmic reticulum Decrease in the number of attached ribosomes Shortening of channels Dilatation of channels

— — —

— — —

— — —

— — —

— — —

* * *

Note. ME, EC of the main type; LE, light EC; DE, dark EC. * slight changes, ** moderate changes, *** massive changes.

vesicle population against the control (Table 3), without simultaneous activation of synthesis organelles. However, the number of vesicles in the total volume of cytoplasm increased against the control by only 15% in dark EC, by 30% in EC of the main type, and by 112% in light cells. There were no detectable effects of anesthesia on the quantitative characteristics of IDE.

Ultrastructure of Endothelium during Moderate Cooling (Group 3, T 5 28 –30°) The total population of the EC consisted of 52.00% of cells of the main type, 11.12% of light cells, and 34.62% of dark cells. The proportion of IDE was small (Table 1). On cooling EC of the main type exhibited an altered morphology, namely a significant decrease in the average thickness to 0.36 6 0.01 mm (P , 0.05 compared to the controls). The total volume of cytoplasm per average capillary cell of the main type became significantly lower compared with the control (Table 2). The majority of EC of the main type showed some signs of inhibition of cellular metabolic activity such as clump-

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ing of nuclear chromatin, slight decrease in mitochondrial matrix density (Fig. 3a), and decrease in the size of the GC and the RER (Table 4). Nevertheless, a few cells of this type retained their initial ultrastructure. Quantitative analysis demonstrated a significant decrease in the number of plasmalemmal vesicles compared with group 2 of anesthesia (Table 3). A moderate degree of ultrastructural changes was observed in the EC of light type at the end of moderate cooling. Endothelial changes of moderate severity included cytoplasmic vacuoles, more or less delicate projections into the vessel lumen, and clumping as well as margination of nuclear chromatin. The slight changes in the ultrastructure of the GC (Fig. 3b) and mitochondria, which were observed in separate light cells in the controls, became more widespread after cooling. They were noted in the majority of light cells (Table 4). The total volume of cytoplasm per average capillary cell of the light type decreased significantly versus the control and anesthesia stage (Table 2). The activity of plasmalemma also decreased in the light cells: the number

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259

FIG. 2. Ultrastructure appearance of heart capillary endothelium after anesthesia (group 2). (a) Note the rich organelles in the cytoplasm of cell of the main type. The nucleus (N) with finely dispersed chromatin are seen in the cytoplasm. There is a Golgi complex (GC), which is composed of relatively long cisternae. A few Weibel–Palade bodies (W-P) are seen lying near the Golgi complex. Original magnification, 22,1003; (b) The cell of the light type contains some mitochondria (M) with normal ultrastructure and random plasmalemmal vesicles (V). Original magnification, 28,6003.

of free vesicles per unit volume of the EC decreased by 1.5 times, on average (Table 3). The profiles of the dark cells were thinner and tortuous as opposed to those of the control cells. At this time point the thickness and total volume values of dark EC cytoplasm remained essentially the same as

during anesthesia (Table 2). Karyoplasm was dense in all cells (Fig. 3c). Two morphological classes of nucleus were identified. Chromatin was represented by a wide marginal band and a single large clump in the center in the first class, while in the second class there was a lesser amount of marginal chromatin, just small

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FIG. 3. Ultrastructure appearance of heart capillary endothelium after moderate cooling (28 –30°, group 3). (a) The cell of the main type shows early evidence of ultrastructural change which manifests itself in mild loss of mitochondrial (M) matrix density (arrow) and reduction of mitochondrial cristae. Original magnification, 32,2503; (b) There are several Golgi complexes (GC) in the light cell. They are composed of relatively long cisternae. The lumen of some cisternae is narrow; that of others is wider at the ends (arrow). Some cisternae appear fragmented. Original magnification, 24,0003; (c) The cell of the dark type appears normal with clearly defined membranes and organelles. The nucleus (N) shows chromatin clumping and margination. Original magnification, 92503.

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clumps scattered almost uniformly over the nuclear area. The ultrastructural changes of other organelles in the dark cells were similar to those seen in the cells of the main type; they were more prominent, though (Table 4). Compared to the stage of anesthesia, the number of plasmalemmal vesicles per unit of cell volume dropped noticeably, although this parameter did not vary significantly against the control (Table 3). On the contrary, the number of vesicles in the total volume of cell cytoplasm was significantly lower against both the control and the anesthetized groups (Table 3). The morphological characteristics of the IDE during moderate hypothermia were the same as at the stage of anesthesia and in the controls.

Ultrastructure of Capillary Endothelium during Deep Cooling (Group 4, T 5 22–24°) The population of the EC was composed of cells of the main type (41.50%), light cells (8.83%), dark cells (47.17%), and irreversibly damaged cells (2.50%). The variations in the general architecture and the state of intracellular organelles were wider in cells of the main type in the course of deep hypothermia rather than during the moderate one. The average thickness of the EC reached a value of 0.38 6 0.03 mm. The total volume of cytoplasm per average EC of the main type was 150.52 6 5.48 mm 3 (Table 2). Nuclear chromatin was more compacted in some cells of the main type and was looser in the other cells. In occasional cells of the main type the cytoplasm contained nuclei resembling those described in the controls. The mitochondria, as a rule, contained a matrix of nonuniform density. Cristae were somewhat disordered with few breaks. The GC was vacuolized. Some channels of the RER were fragmented or dilated (Table 4). No significant differences in the average thickness of light cells between moderate and deep cooling groups were revealed. The light EC were thick (approximately 0.56 6 0.07 mm). The total volume of cytoplasm per average EC of the light type was 159.78 6 4.35 mm 3 (Table 2). Enhanced margination of chromatin in nuclei as well as polygonal profiles of RER with small amount of ribosomes was observed in the light cells. Mitochondria were swollen with pale matrices and

fractured disordered cristae. However, the outer membranes of mitochondria were intact (Fig. 4a). In group 4 of deep cooling the majority of dark cells contained nuclei with large clumps of dense chromatin; the outlines of the nuclei were irregular. At this stage of the experiment the average thickness of dark cells amounted to 0.34 6 0.03 mm and the total volume of cytoplasm per average dark EC equaled to 119.54 6 4.68 mm 3 (Table 2). These values varied insignificantly from those shown for the control group, as well as for the anesthesia and moderate cooling groups. The profiles of dark cells were tortuous: cytoplasmic projections, crater-like gaps, and blebs were commonly observed (Fig. 4b). The nuclear chromatin showed clumping as well as margination. Other organelles demonstrated various degrees of changes, but the pattern of changes was the same as during moderate cooling (Table 4). The number of vesicles in the total volume of cell cytoplasm and numerical densities of free plasmalemmal vesicles was significantly decreased by comparison with group 3 of moderate cooling and the control group (Table 3). The irreversibly damaged cells showed no distinctive characteristics as opposed to group 3. However, the profiles of many swollen EC appeared wider than those in the controls.

DISCUSSION Research demonstrated that without regard to the level of cooling (mild, moderate, or deep) all four morphological types of cells specified in the control remained in endothelium of the left ventricle capillaries. Conflicting explanations have been reported concerning the distribution of the EC with altered phenotype within the vascular wall. There are a great number of papers describing the ultrastructure of light and dark EC in terms of pathological changes of vascular endothelium (Armiger and Gavin, 1975; Schaper and Schaper, 1977; Sherf et al., 1977; Shiokawa et al., 1989). However, in the majority of these works vascular endothelium was studied under the conditions of pathology, e.g., in the case of ischemia or myocardial infarction. For this reason, the authors logically related the appearance of light EC to their osmotic swelling

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FIG. 4. Ultrastructure appearance of heart capillary endothelium after deep cooling (22–24°, group 4). (a) Sharply swollen mitochondria (M) without cristae are seen in the light cell. Original magnification, 20,5003; (b) The capillary lumen (L) is surrounded by dark cells. There are many free and attached vesicles. The abluminal profile of dark cells is tortuous. The cytoplasm of the cell show some projections which protrude into the lumen (arrows). Original magnification, 21,0003.

and dark EC to the extraction of cytosolic protein caused by ischemia. There are other points of view which evolved from studying the ultrastructure of intact organs and tissues. According to one of them the variations of electron density of EC correlate with a life cycle of these cells (Schahlamov, 1971). This

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concept suggest that EC of the light type might represent the young form of the EC with a small number of plasmalemmal vesicles and a low electron density of cytoplasm. Ultrastructural study of the cellular components of the developing myocardial capillary wall indicates that numerical density of plasmalemmal ves-

Studies on Endothelium under Hypothermia

icles demonstrates a gradual and significant increase, as well as opaque density of cytoplasm (Porter and Bankston, 1987). According to a second viewpoint phenotypic heterogeneity of capillary EC is representative of their hypo- or hyperfunction (Chernykh et al., 1975). The dependence between electron density of cytoplasm and specific work of cells has been under study for a fairly long time. The mechanisms of the functioning of dark and light cells have been already established in a variety of organs and tissues, such as skeletal muscles of human beings, mucous membrane of bronchi, and liver and kidneys of dogs, as well as in terminals of neuromuscular synapses (Sarkisov, 1970; Sekamova and Beketova, 1975). Some features of submicroscopic organization of capillary EC of the control animals revealed in the present study also enable us to point to the fact that they have a different functional specialization. According to our results, the cells of the main type contain equally well-developed organelles of the transport and protein-synthesizing system and are probably the principal reserve of the whole endothelial capillary population. Light EC feature a somewhat low activity of all intracellular organelles. The absence of signs of osmotic swelling caused by intracellular swarm of water in light EC is an important factor indicating their viability and functional nature. As for dark EC, the absence of intracellular aggregation of organelles and cytoplasm homogenization may be considered to be such a factor. Dark EC primarily perform a transport function. This is evidenced by the fact that these cells have the highest numerical density of plasmalemmal vesicles in a unit of cell volume and the greatest number of vesicles in the total volume of cell cytoplasm (Table 3). The populational and subcellular changes in capillary endothelium at the stage of anesthesia are adaptive; they tend to enhance transport, the major specific function of the capillaries. At a cellular level, dark EC showed a general trend to an increase in the relative proportion in the total population of the EC due to transformation of some cells of the main type to dark cells. This appears highly probable, if the results obtained by some authors (Lipton et al., 1991) relating to morphological transformation of various cell types are taken into account. The transformation we observed is

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not associated with a decrease in the proportion of cells of the main type because a part of light EC is transformed into cells of the main type. The noted changes reflect a reorganization of the endothelial lining of the myocardial capillaries to a more active state of transendothelial transport. There was an increase not only in the number of dark cells, but also in the number of their free vesicles compared to the control group (the difference being significant at P , 0.05) (Table 3). A similar situation holds with the cells of the main and light types (Table 3). The enhanced transendothelial transport might be caused by the need for mobilization of myocardial reserves under anesthesia. It should be emphasized that conditions of inhalation anesthesia have a depressive action on cardiac performance, and, as a consequence, produce a decrease in blood flow as well as in oxygen supply of the myocardium (Matsukawa et al., 1993; Sonntag, 1980; Terrar and Victory, 1988). The disturbed metabolism may be possibly compensated by an increase in the activity of plasmalemma of the capillary EC. This is a likely explanation, especially when taking into account that alcohols and anesthetic agents first perturb the membrane lipid environment and second fluidize membranes (Weight et al., 1991). There is another explanation. General anesthesia decreases body temperature precipitously. Subsequently, hypothermia results simply from heat loss exceeding metabolic heat production (Saito, 1997). Our observations suggests that a dramatic increase in the numerical densities of plasmalemmal vesicles represents adaptation of capillary EC to spontaneous cooling and may function to prevent myocardium from the heat loss at the stage of anesthesia. Activation of vesicular transport does not produce appreciable changes in the ultrastructure of mitochondria and the protein-synthesizing organelles in the EC of the main type. Reasoning from the morphology of nucleus, mitochondria, Golgi complex, and rough endoplasmic reticulum, the synthetic and energy processes are partially inhibited in dark cells. Nonetheless, these cells retain the power to increase vesicular population under anesthesia, although to a lesser extent compared to light EC and EC of the main type. Based on these data, it may be suggested that in the

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anesthesia phase dark EC are at a breaking point of their functional activity. Based on the results of ultrastructural analysis of changes in capillary endothelium under moderate and deep hypothermia, the following conclusions can be drawn. The adaptation pattern of the capillary endothelium responding to the two levels of active hypothermia is, in general, the same. From our point of view, the composition of cell population varies as follows. Compared to group 2 of anesthesia, the number of cells of the main type decreases as a result of their transformation to dark cells, and, vice versa, the number of dark cells increases. The relative proportion of light cells decreases as a result of the transformation of some light cells into the main type and others into swollen cells. The number of irreversibly damaged cells remains basically the same. Thus, we have obtained ultrastructural evidence that active body cooling does not produce irreversible damages to capillary endothelium. Necrotic cells appearing in the experimental groups represent naturally dying cells irrelevant to the hypothermic procedure as such. Nevertheless, lowering of body temperature down to 22–24° calls for more significant changes in the ultrastructure of capillary endothelium as opposed to moderate hypothermia. At a cellular level this predominantly manifests itself as a more conspicuous increase in the amount of dark cells. Some speculations might be made with regard to the mechanisms lying at the heart of the general darkening of the cellular population in the conditions of active artificial cooling below 30°. Obviously, these mechanisms differ from those observed under spontaneous mild cooling (34 –35°) in the course of anesthesia. (1) Enzyme-catalyzed reactions are sensitive to temperature changes. The function of the plasma-membrane Na/K-ATPase is therefore decreased by hypothermia (Solberg et al., 1987). The transmembrane electrochemical gradients could then possibly force water out of the cells (Gizewski et al., 1997). This is evidenced by a small decrease in the average width of the EC of the main type compared to the controls (Table 2). (2) At low body temperatures, membrane lipids undergo a stabilizing phase change (Axford-Gatley et al.,

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Kazanskaya et al.

1990) which provides reorganization of cell metabolism associated with accumulation of protein products in the cytoplasm. A pattern of ultrastructural changes on the background of inhibited cell metabolism was observed in the EC of the main, dark, and light types under moderate and deep hypothermia (Chiavarelli et al., 1989; McDonagh and Laks, 1982). The pattern was more clear-cut in the dark and light EC rather than in the cells of the main type (Table 4). The subcellular changes were adaptive and reversible regardless of the level of hypothermia. The extent of these changes was dependent on the age of the cell and individual specificity of the dog. Cells of all the types showed a general trend to a decrease in the activity of the organelles of the protein-synthesizing machinery. Concomitantly, they exhibited wide variations in the ultrastructure of the GC, the RER, and the nuclei. Thus, even conditions of hypometabolism cannot completely level off the differences in the functional state between individual cells of each morphological type. It is common knowledge that lowering of body temperature is associated with a sharp decrease in the density of plasmalemmal vesicles in vascular endothelium (Wagner and Casley-Smith, 1981). Judging by the results of the present study, this view holds water under moderate and deep hypothermia. However, the surprising thing is that at a temperature of 28 –30° a statistically meaningful (compared to the control) decrease in the number of plasmalemmal vesicles was noted only in dark cells. In cells of the main type the number of vesicles in the total volume of cytoplasm differed insignificantly from that of the control, while in light cells it was significantly higher compared to that in the control (Table 3). There are many plausible explanations for this conflicting evidence. As is known, generalized surface cooling of intact animals to 28° leads to a decrease in coronary blood flow and produces a decrease in the number of open, functioning capillaries; below 25° coronary blood flow is increased (Black et al., 1976; Sakai et al., 1988). Our data also indicate that during moderate hypothermia there is a significant decrease in the average diameter of blood capillaries compared to the controls (Table 5). The decrease in perfusion capacity of the capillary bed in the myocardium is

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TABLE 5 Electron Microscopic Measurements of Geometry of Capillary Endothelium in Different Groups of Topical Cooling without Perfusion Groups Group 1 (control 36–37°) Group 2 (anesthesia 34–35°) Group 3 (moderate cooling 28–30°) Group 4 (deep cooling 22–24°)

Average diameter of capillaries (mm)

Average length of luminal surface of capillaries (mm)

Average length of abluminal surface of capillaries (mm)

4.8 6 0.1

19.8 6 1.3

19.9 6 0.9

4.6 6 0.1

18.9 6 1.1

18.8 6 0.7

4.3 6 0.1*

15.1 6 0.4*

16.8 6 0.5*

4.9 6 0.1**

19.8 6 0.7**

19.6 6 0.5**

Note. Data are given as mean 6 standard error of the mean; n 5 6 for control group and all experimental groups. * Value significantly different from control value; P , 0.05 (Student’s t test). ** Value significantly different from Group 3 value; P , 0.05.

compensated by retention in a high number of plasmalemmal vesicles in the EC of capillaries which remain patent. There is another explanation. The retention of vesicle abundance might be brought about by a switch from aerobic to anaerobic glycolysis under the effect of hypothermia (Ballinger et al., 1962). Greater amounts of substrate are presumably required for the formation of a sufficient number of ATP molecules under conditions of anaerobic rather than aerobic metabolism. According to the third explanation there may be two populations of plasmalemmal vesicles, one involved in transport of macromolecules and the other in creating reserves for the plasmalemma (Gavrish, 1986; Simionescu, 1983). We speculate that the first population of plasmalemmal vesicles decreases, whereas the second one continues to increase in endothelial cells of the dogs treated with moderate hypothermia. Further evidence that the second population of plasmalemmal vesicles is increased during moderate hypothermia comes from the observation that there are significant differences in the average length of luminal and abluminal plasmalemma of capillaries between the control group and group 3 of moderate cooling (Table 5). Thus, the pattern of changes in the vesicular activity of the EC under moderate hypothermia confirms not only the heterogeneity of the functional specialization of the EC of different morphological types, but also their different adaptive reserve. Lowering of body temperature from 28 –30° to 22–24° produces a decrease in the number of

plasmalemmal vesicles in the EC of all types. This is consistent with the data in the literature indicating that both the formation and the transport of vesicles is inhibited under conditions of deep hypothermia (Chinard and DeFouw, 1981).

CONCLUSION It is concluded that the diversity of structural organization and functional states of the EC underlie their tolerance of hypothermia. This heterogeneity manifests itself as differentiation of the EC according to electron density and as different qualitative and quantitative characteristics of the EC organelles at an ultrastructural level. Total body cooling without perfusion, no matter whether it is moderate or deep, gives rise to a set of adaptive changes in the capillary endothelium. At a populational level adaptation is provided by an increase in the number of dark EC with a predominantly transport function. At a subcellular level, adaptation makes itself evident as a decrease in the activity of the organelles of the protein-synthesizing machinery. Adaptation of the capillary endothelium after hypothermic procedure is related to a decrease in the number of plasmalemmal vesicles in the EC of all morphological types, this decrease being more prominent at deep hypothermia.

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ACKNOWLEDGMENTS The authors thank Mrs. T. Djakonitsa and Mrs. R. Kostina for their methodological contribution to this study and Mr. A. Pokrovsky for his help in preparing this report for publication.

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