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Ultrastructural aspects of cardiac lymphatic capillaries in experimental cardiac hypertrophy
MICROVASCULAR
RESEARCH
10,
1-7 (1975)
Ultrastructural Aspects in Experimental
of Cardiac Lymphatic Cardiac Hypertrophy’
A. LJUNGQVIST, E. MANDACHE,’
Capillaries
AND G. UNGE
Departments of Pathology and Thoracic Medicine, Karolinska sjukhuset, Stockholm, Sweden Received April 11,1974 The lymphatic capillaries in normal rat hearts and hearts rendered hypertrophic by aortic stenosis and swimming exercise were studied in the electron microscope. No differences were found between the normal and hypertrophied hearts with respect to the ultrastructural aspects of the lymphatic capillaries. It was concluded that lymph stasis and cell proliferation in lymphatic capillaries do not contribute to the heart enlargement resulting from aortic stenosis and swimming exercise.
INTRODUCTION In previous works we found a significant neoformation of myocardial blood capillaries in cardiac hypertrophy secondary to swimming exercise, but not in hypertrophy secondary to renal hypertension and aortic stenosis (Ljungqvist and Unge, 1972; Mandache et al., 1972; Ljungqvist and Unge, 1973; Mandache et al., 1973). In the electron microscope, lymphatic capillaries were also seen, but these were not further analyzed. Since only light microscopical investigations on cardiac lymphatics were found in the literature (Johnson and Blake, 1966; Kline, 1969; Kluge and Ullal, 1972) we performed the present study of the ultrastructural details of lymphatic capillaries in normal hearts and in hearts rendered hypertrophic by swimming exercise and aortic stenosis. MATERIALS
AND
METHODS
Sixteen female Sprague-Dawley rats were used for the experiments. The rats were housed in cages, three to four rats in each cage, and were fed a standard laboratory chow containing 0.4 % sodium chloride and tap water ad libitum. The production of cardiac hypertrophy by aortic stenosis and swimming exercise has been described previously (Ljungqvist and Unge, 1972). As in previous experiments aortic stenosis was produced by the application of an 0.5 mm wide silver clip on the immediate subdiaphragmatic portion of the aorta, and swimming exercise was performed by swimming I hr each day, 6 days per wk. The rats were divided into the following groups : Group I.
Four rats which were killed 5 days after the production and with a terminal weight of 250 f 5 g (mean f SD).
of aortic stenosis
’ This study was supported by the Swedish Medical Research Council (Project No. B72-12X-71607A) and the Research Funds of Karolinska Institutet. * WHO Fellowship No. 70T ROOM ooO8. Copyright 0 1975 by Academic Press, Inc. AI1 rights of reproduction in any form reserved. Printed in Great Britain
2
LJUNGQVIST,
MANDACHE
AND
UNGE
FIG. 1. Electron micrograph showing a cardiac lymphatic capillary with an open lumen above (L) and a collapsed lumen below. The content is finely granular. Similar material is seen in the interstitium (I). Mitochondria (m). Vesicles containing electrone dense material (V). Pinocytotic vesicles (Pv). Discontinuous basement membrane (BM). x21.600.
CARDIAC
LYMPHATIC
CAPILLARIES
FIG. 2A. Electron micrograph showing cardiac lymphatic capillary (L)and adjacent blood capillary (BC). Patent junction (Pj). Tight junctions with dense cytoplasmic accumulations (arrows). x27.000.
4
LJUNGQVIST,
FIG.
MANDACHE
AND
UNGE
2B. Higher magnification of tight junction area in Fig. 2A. x69.300.
Four rats which were killed 2 wks after the production of aortic stenosis and with a terminal weight of 250 + 5 g. Group III Four rats which were killed after 2 wks of exercise and with a terminal weight of 250 5 5 g. Group IV’. Four normal rats which were killed at a weight of 250 + 0 g; these rats served as controls of Groups I-III. Group II.
Blood pressure and body-weight of the rats were recorded at regular intervals and immediately before killing. The blood pressure was determined by the tail pletysmographic method. At the end of the experimental periods the rats were anaesthetized by intraperitoneal injection of Nembutal. The aorta was exposed and a catheter inserted and directed towards the heart. Fixation of the heart was then performed by perfusion via the catheter with 1.5 % glutaraldehyde solution buffered by Na-cacodylate to pH 7.4. After perfusion the hearts were removed and weighed for determination of the heart/body-weight ratios. For practical reasons the ratios were multiplied by 1000. From each heart pieces were taken for electron microscopy from the left ventricle wall (lateral wall, septum and apex). All pieces of heart tissue were postfixed in buffered 0~0, and embedded in Epon. The material was studied in a Siemens Elmiskop I. RESULTS There was a significant increase in the heart/body-weight ratio in the experimental animals. This is consistent with our findings in previous experiments (Ljungqvist and Unge, 1972; Mandache et al., 1972). In no instance was a hypertensive blood pressure level recorded. In the present investigation the interest was focused on the interstitial tissue, particularly the lymphatic capillaries. Such vessels appeared to be relatively few in number and unevenly distributed. No differences were found between the experimental animals and controls with respect to the number and distribution of the lymphatic capillaries and their ultrastructure.
CARDIAC
LYMPHATIC
CAPILLARIES
FIG. 3. Electron micrograph showing cardiac lymphatic capillary (L) with an irregular endothelial, outlining, and adjacent blood capillary (BC) with a smooth endothelial outlining. Microtubules (mt, small arrows). Interdigitated junctions (j). Filaments (large arrows). x22.400.
6
LJUNGQVIST, MANDACHE AND UNGE
The content of the lymphatic capillaries and the interstitium was always finely granular, whereas the perfused blood capillaries appeared “empty” (Figs. 1,2A and 3). The luminal diameter of the lymphatic capillaries was variable. Some lumina were circular, others collapsed (Fig. 1). The diameter of the circular lymphatic capillaries did not exceed that of blood capillaries. The endothelium was of about equal thickness in the lymphatic and blood capillaries, but its outlining appeared more irregular in the lymphatics (Fig. 3). The cytoplasmic organelles were possibly more sparse in the lymphatic endothelial cells than in those of the blood capillaries. Mitochondria were found particularly in the perinuclear area (Fig. 1). Pinocytotic vesicles were always present on the luminal and the abluminal sides (Fig. 1). When compared with the blood capillaries the pinocytotic vesicles were slightly bigger but less numerous. In addition big vesicles containing electron-dense material were observed (Fig. 1). The endothelium of the lymphatic capillaries also contained some filaments accumulated on the abluminal side and microtubules orientated parallel to the long axis of the lymphatic capillary (Fig. 3). The intercellular junctions were found to bepatentjunctions (Fig. 2A),interdigitations (Fig. 3) and tight junctions (Fig. 2B) as is normal, but the tight junctions seemed to predominate. No mitotic figures were found in the lymphatic capillary endothelium. DISCUSSION The general architecture of the lymphatic capillaries of the heart is very similar to that described by Leak (1970) for the dermal lymphatic capillaries. It appeared to be quite easy to differentiate between blood and lymphatic capillaries in the perfusion-fixed material of the present study. All blood capillaries were washed clean and their lumina appeared empty, whereas the lymphatic capillaries contained finely granular material. No differences were found between the experimental groups and controls with respect to the ultrastructure of the cardiac lymphatics. The fact that cardiac enlargement is present already after 5 days of aortic stenosis and swimming exercise and that the ultrastructure and diameter of lymphatic capillaries are the same in experimental and control animals, indicates that no significant lymphatic stasis is present in cardiac hypertrophy secondary to aortic stenosis and swimming exercise. This is further supported by Nair’s et al. (1969) observation of an unchanged water content in the hypertrophied heart secondary to aortic stenosis. The cardiac enlargement is thus probably solely due to cardiac muscle cell hypertrophy and blood capillary proliferation (Ljungqvist and Unge, 1972; Mandache et al., 1972; Mandache et al., 1973; Ljungqvist and Unge, 1973; Unge, 1973). REFERENCES JOHNSON, A. R., AND BLAKE, M. T. (1966). Lymphatics of the heart. Circulation 33,137-142. KLINE, K. I. (1969). Lymphatic pathways. Arch. Puzhol. 88, 638-644. KLUGE, T., AND ULLAL, R. (1972). Pathology of the heart following chronic cardiac lymphatic
obstruction. Acta Pathol. Microbial. &and. Section A. 80,150-158. LEAK, V. L. (1970). Electron microscopic observations on lymphatic capillaries and structural components of the connective tissue lymph interface. Microvasc. Res. 2, 361-391. LJUNGQVIST, A., AND UNGE, G. (1972). The finer intramyocardial vasculature in various forms of experimental cardiac hypertrophy. Acta Pathol. Microbial. Stand. Section A. 80, 329-340.
CARDIAC
LYMPHATIC
CAPILLARIES
7
A., AND UNGE, G. (1973). The proliferative activity of the myocardial tissue in various forms of experimental cardiac hypertrophy. Acta Pathol. Microbial. Scund. Section A. 81,233-240. MANDACHE, E., UNGE, G., AND LJUNGQVIST, A. (1972). Myocardial blood capillary reaction in various forms of cardiac hypertrophy. An electron microscopical investigation in the rat. Virchows Arch. Abt. B. Zellpath. 11, 97-l 10. MANDACHE, E., UNGE, G., APPELGREN, L-E., AND LJUNGQVIST, A. (1973). The proliferative activity of the heart tissues in various forms of experimental cardiac hypertrophy studied by electron microscope autoradiography. Virchow Arch. Abt. B. Zellpath. 12, 112-122. NAIR, G. K., CUTILLETTA, A. I., ZAK, R., KOIDE, T., AND RABINOWITZ, M. (1968). Biochemical correlates of cardiac hypertrophy. I. Experimental model; changes in heart weight, RNA content and nuclear RNA polymerase activity. Circ. Res. 23, 451-462. UNGE, G. (1973). Experimental cardiac hypertrophy. An autoradiographical study after in uito injections of 3H-5-uridine. Acta Puthol. Microbial. Sand. Section A. 81, 806-812.
LJUNGQVIST,
RESEARCH
10,
1-7 (1975)
Ultrastructural Aspects in Experimental
of Cardiac Lymphatic Cardiac Hypertrophy’
A. LJUNGQVIST, E. MANDACHE,’
Capillaries
AND G. UNGE
Departments of Pathology and Thoracic Medicine, Karolinska sjukhuset, Stockholm, Sweden Received April 11,1974 The lymphatic capillaries in normal rat hearts and hearts rendered hypertrophic by aortic stenosis and swimming exercise were studied in the electron microscope. No differences were found between the normal and hypertrophied hearts with respect to the ultrastructural aspects of the lymphatic capillaries. It was concluded that lymph stasis and cell proliferation in lymphatic capillaries do not contribute to the heart enlargement resulting from aortic stenosis and swimming exercise.
INTRODUCTION In previous works we found a significant neoformation of myocardial blood capillaries in cardiac hypertrophy secondary to swimming exercise, but not in hypertrophy secondary to renal hypertension and aortic stenosis (Ljungqvist and Unge, 1972; Mandache et al., 1972; Ljungqvist and Unge, 1973; Mandache et al., 1973). In the electron microscope, lymphatic capillaries were also seen, but these were not further analyzed. Since only light microscopical investigations on cardiac lymphatics were found in the literature (Johnson and Blake, 1966; Kline, 1969; Kluge and Ullal, 1972) we performed the present study of the ultrastructural details of lymphatic capillaries in normal hearts and in hearts rendered hypertrophic by swimming exercise and aortic stenosis. MATERIALS
AND
METHODS
Sixteen female Sprague-Dawley rats were used for the experiments. The rats were housed in cages, three to four rats in each cage, and were fed a standard laboratory chow containing 0.4 % sodium chloride and tap water ad libitum. The production of cardiac hypertrophy by aortic stenosis and swimming exercise has been described previously (Ljungqvist and Unge, 1972). As in previous experiments aortic stenosis was produced by the application of an 0.5 mm wide silver clip on the immediate subdiaphragmatic portion of the aorta, and swimming exercise was performed by swimming I hr each day, 6 days per wk. The rats were divided into the following groups : Group I.
Four rats which were killed 5 days after the production and with a terminal weight of 250 f 5 g (mean f SD).
of aortic stenosis
’ This study was supported by the Swedish Medical Research Council (Project No. B72-12X-71607A) and the Research Funds of Karolinska Institutet. * WHO Fellowship No. 70T ROOM ooO8. Copyright 0 1975 by Academic Press, Inc. AI1 rights of reproduction in any form reserved. Printed in Great Britain
2
LJUNGQVIST,
MANDACHE
AND
UNGE
FIG. 1. Electron micrograph showing a cardiac lymphatic capillary with an open lumen above (L) and a collapsed lumen below. The content is finely granular. Similar material is seen in the interstitium (I). Mitochondria (m). Vesicles containing electrone dense material (V). Pinocytotic vesicles (Pv). Discontinuous basement membrane (BM). x21.600.
CARDIAC
LYMPHATIC
CAPILLARIES
FIG. 2A. Electron micrograph showing cardiac lymphatic capillary (L)and adjacent blood capillary (BC). Patent junction (Pj). Tight junctions with dense cytoplasmic accumulations (arrows). x27.000.
4
LJUNGQVIST,
FIG.
MANDACHE
AND
UNGE
2B. Higher magnification of tight junction area in Fig. 2A. x69.300.
Four rats which were killed 2 wks after the production of aortic stenosis and with a terminal weight of 250 + 5 g. Group III Four rats which were killed after 2 wks of exercise and with a terminal weight of 250 5 5 g. Group IV’. Four normal rats which were killed at a weight of 250 + 0 g; these rats served as controls of Groups I-III. Group II.
Blood pressure and body-weight of the rats were recorded at regular intervals and immediately before killing. The blood pressure was determined by the tail pletysmographic method. At the end of the experimental periods the rats were anaesthetized by intraperitoneal injection of Nembutal. The aorta was exposed and a catheter inserted and directed towards the heart. Fixation of the heart was then performed by perfusion via the catheter with 1.5 % glutaraldehyde solution buffered by Na-cacodylate to pH 7.4. After perfusion the hearts were removed and weighed for determination of the heart/body-weight ratios. For practical reasons the ratios were multiplied by 1000. From each heart pieces were taken for electron microscopy from the left ventricle wall (lateral wall, septum and apex). All pieces of heart tissue were postfixed in buffered 0~0, and embedded in Epon. The material was studied in a Siemens Elmiskop I. RESULTS There was a significant increase in the heart/body-weight ratio in the experimental animals. This is consistent with our findings in previous experiments (Ljungqvist and Unge, 1972; Mandache et al., 1972). In no instance was a hypertensive blood pressure level recorded. In the present investigation the interest was focused on the interstitial tissue, particularly the lymphatic capillaries. Such vessels appeared to be relatively few in number and unevenly distributed. No differences were found between the experimental animals and controls with respect to the number and distribution of the lymphatic capillaries and their ultrastructure.
CARDIAC
LYMPHATIC
CAPILLARIES
FIG. 3. Electron micrograph showing cardiac lymphatic capillary (L) with an irregular endothelial, outlining, and adjacent blood capillary (BC) with a smooth endothelial outlining. Microtubules (mt, small arrows). Interdigitated junctions (j). Filaments (large arrows). x22.400.
6
LJUNGQVIST, MANDACHE AND UNGE
The content of the lymphatic capillaries and the interstitium was always finely granular, whereas the perfused blood capillaries appeared “empty” (Figs. 1,2A and 3). The luminal diameter of the lymphatic capillaries was variable. Some lumina were circular, others collapsed (Fig. 1). The diameter of the circular lymphatic capillaries did not exceed that of blood capillaries. The endothelium was of about equal thickness in the lymphatic and blood capillaries, but its outlining appeared more irregular in the lymphatics (Fig. 3). The cytoplasmic organelles were possibly more sparse in the lymphatic endothelial cells than in those of the blood capillaries. Mitochondria were found particularly in the perinuclear area (Fig. 1). Pinocytotic vesicles were always present on the luminal and the abluminal sides (Fig. 1). When compared with the blood capillaries the pinocytotic vesicles were slightly bigger but less numerous. In addition big vesicles containing electron-dense material were observed (Fig. 1). The endothelium of the lymphatic capillaries also contained some filaments accumulated on the abluminal side and microtubules orientated parallel to the long axis of the lymphatic capillary (Fig. 3). The intercellular junctions were found to bepatentjunctions (Fig. 2A),interdigitations (Fig. 3) and tight junctions (Fig. 2B) as is normal, but the tight junctions seemed to predominate. No mitotic figures were found in the lymphatic capillary endothelium. DISCUSSION The general architecture of the lymphatic capillaries of the heart is very similar to that described by Leak (1970) for the dermal lymphatic capillaries. It appeared to be quite easy to differentiate between blood and lymphatic capillaries in the perfusion-fixed material of the present study. All blood capillaries were washed clean and their lumina appeared empty, whereas the lymphatic capillaries contained finely granular material. No differences were found between the experimental groups and controls with respect to the ultrastructure of the cardiac lymphatics. The fact that cardiac enlargement is present already after 5 days of aortic stenosis and swimming exercise and that the ultrastructure and diameter of lymphatic capillaries are the same in experimental and control animals, indicates that no significant lymphatic stasis is present in cardiac hypertrophy secondary to aortic stenosis and swimming exercise. This is further supported by Nair’s et al. (1969) observation of an unchanged water content in the hypertrophied heart secondary to aortic stenosis. The cardiac enlargement is thus probably solely due to cardiac muscle cell hypertrophy and blood capillary proliferation (Ljungqvist and Unge, 1972; Mandache et al., 1972; Mandache et al., 1973; Ljungqvist and Unge, 1973; Unge, 1973). REFERENCES JOHNSON, A. R., AND BLAKE, M. T. (1966). Lymphatics of the heart. Circulation 33,137-142. KLINE, K. I. (1969). Lymphatic pathways. Arch. Puzhol. 88, 638-644. KLUGE, T., AND ULLAL, R. (1972). Pathology of the heart following chronic cardiac lymphatic
obstruction. Acta Pathol. Microbial. &and. Section A. 80,150-158. LEAK, V. L. (1970). Electron microscopic observations on lymphatic capillaries and structural components of the connective tissue lymph interface. Microvasc. Res. 2, 361-391. LJUNGQVIST, A., AND UNGE, G. (1972). The finer intramyocardial vasculature in various forms of experimental cardiac hypertrophy. Acta Pathol. Microbial. Stand. Section A. 80, 329-340.
CARDIAC
LYMPHATIC
CAPILLARIES
7
A., AND UNGE, G. (1973). The proliferative activity of the myocardial tissue in various forms of experimental cardiac hypertrophy. Acta Pathol. Microbial. Scund. Section A. 81,233-240. MANDACHE, E., UNGE, G., AND LJUNGQVIST, A. (1972). Myocardial blood capillary reaction in various forms of cardiac hypertrophy. An electron microscopical investigation in the rat. Virchows Arch. Abt. B. Zellpath. 11, 97-l 10. MANDACHE, E., UNGE, G., APPELGREN, L-E., AND LJUNGQVIST, A. (1973). The proliferative activity of the heart tissues in various forms of experimental cardiac hypertrophy studied by electron microscope autoradiography. Virchow Arch. Abt. B. Zellpath. 12, 112-122. NAIR, G. K., CUTILLETTA, A. I., ZAK, R., KOIDE, T., AND RABINOWITZ, M. (1968). Biochemical correlates of cardiac hypertrophy. I. Experimental model; changes in heart weight, RNA content and nuclear RNA polymerase activity. Circ. Res. 23, 451-462. UNGE, G. (1973). Experimental cardiac hypertrophy. An autoradiographical study after in uito injections of 3H-5-uridine. Acta Puthol. Microbial. Sand. Section A. 81, 806-812.
LJUNGQVIST,