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The influence of age on the wound healing of experimental myocardial infarction in rats
Exp. Path., Bd.H, S.107-114 (1975) Institute of Pathology of the Humboldt-University in Berlin (Director: Prof. Dr. sc. med. I,.-H. KETTLER)
The influence of age on the wound healing of experimental mvocardial infarction in rats "'
(Autoradiographic studies)
By D. KRANZ, A. HECHT and 1. FUHRMANN With 2 figures (Received March 11, 1975) Key-words: wound healing; infarction: myocanlial; cell proliferation: age dependence; mitotic index; granulation tissue; connective tissue; tropocallagen synthesis; collagen precursors; endothelium; autoradiography; 3H-proline; 3H-thymidine; 35S-sulphate; mucopolysaccharides; rat
Summary In G-weeks-old, 5-months-old and 21-months-old rats myocardial infarction was induced by ('oronary artery Iigature. After performing the ligature the animals were administered 3H-thymidine, 3H-proline or 35S-sulphate at different times. The following parameters were determined: number of D~A- and tropocollagen-synthesizing connective tissue cells at the infarction border and at the infardion site; mean silver grain density above the nuclei or cells; duration of a cycle; number of mitoses, and incorporation of radioactive sulphate at the infarction site. In additon, the labelling and mitotic indices as well as the percentage of the standard deviation from the mean values were estimated. The following results were obtained: 1. The rate of gmnulation tissue formation in the neerotic zone is determined by the mitotic activity of the cells. With advancing age the cell cycles are being prolonged which results in retardation of wound healing. 2. The disturbed DNA-replication in old age is not asso('iated with a time shift in the occurrence of the mitotic and labelling peaks. 3. With advancing age the number of fibroblasts synthesizing collagen precursors decreases. There exists no age-dependence of the 3}-f-proline incorporation rate, of the intmeellular transport, of the synthesis of collagen precursor and of the release of labelled tropocollagen. In all age-groups under study these processes last approximately 4 hours. 4. Collagen fibre formation is accompanied by an increased synthesis of acid mucopolysaccharides. In infarction callosities the content of acid mucopolysaccharides most,ly is constant. 5. The proliferating endothelial cells have a pronounced metabolic activity and a markedly short generation time.
In morbidity and mortality statistics ischemic heart disease ranks first among cardiovascular diseases. In recent years there has been a notable shift towards younger age-groups. Approximately 60 per cent of patients with mycocardial infarction do not survive the first 4 weeks, most of them die shortly after the acute episode (BOTHIG et al. 1972). In order to prevent disturbed wound healing which may entail cardiorrhexis, it is important to know the factors influencing granulation tissue formation in the necrotic zone - which takes place analogously to the wound-healing processes in other tissues. The present paper investigates the influence of age on the process of wound healing in experimental myocardial infarction.
Material and 1.11ethods In total 246 male Wistar rats of the Rehbriicke strain were included in the study. 70 rats were G weeks old, 106 rats were 5 months old and 70 rats were 21 months old. Myocardial infarction was produced by ligating the left coronary artery closely below the left auricular appendix. 2 shamoperated animals (thoracotomy and touching of the left coronary artery 12 hours prior to 3H-thy8 Exp. Path. Bd. 11, H. 3/4
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midine administration), and 2 non-operated animals were used for controls in each age-group. The radioisotopes were administered by intraperitoneal injection. To prevent differences in the silver grain density caused by variations in the cytometabolism in the course of the day, the radioactive metabolic precursors were always injected at 8 a. m. The following radioactive isotopes were employed for the study: 1. Thymidine-6- 3H; specific activity 23.3 CijmM (Institute for Research, Production and Application of Radioisotopes, Prague, CSSR). Dose: 2p, Cijg body weight. 2. I,-Prolin-3H(G); specific activity 550 mCijmM; Radiochemical Centre AmershamjEngland. Dose: 3 (.l, Cijg body weight. 3. Na235S0" carrier-free; Central Institute for Nuclear Research, DresdenjRossendorf (GDR). Dose: 5 (.l, Cijg of body weight. The animals were sacrificed by vertebral dislocation. Immediately afterwards the heart was excised and the remaining blood carefully removed. After having been fixed in 10% neutral formalin for 24 hours the hearts were dissected in such a way that the tissue blocs, embedded in paraffin and lining the upper and lower borders of the infarction or of the infarction callosity (which, in most cases, could be clearly distinguished from the surrounding tissue), still contained a macroscopically unchanged muscular layer of about 3 mm. We used 28 sections of 3-5 (.l,m, i. e. 4 sets comprising 7 sections each, from every bloc for preparation of autoradiographs employing K5 ORWO emulsion. The exposition times of the autoradiographs for the study of DNA metabolism were 7, 14 and 21 days; for the study of collagen synthesis 6, 13 and 20 days; and for the study of acid mucopolysaccharide synthesis 10, 15 and 24 days. After having been exposed, developed and fixed under strictly constant conditions, the alltoradiographs were stained with haemalum-eosine. The preparation for the study of acid mucopolysaccharide synthesis remained unstained after 24 days of exposition for the purpose of photometric evaluation. Following 3H-thymidine administration the evaluation was made at the infarction site or at the infarction border by counting the labelled connective-tissue nuclei and the mitoses and by determining the percentage of labelled cells and the silver grain density per nucleus. After 3H_ proline application the labelled connective tissue cells were counted. The percentage of labelled connective tissue cells and the mean silver grain density above these cells were determined. After 35S-sulphate injection the extinction at the infarction site or at the infarction callosity site was determined by means of photometry of the autoradiographs. The individual histological sections from the various sets of each heart were evaluated separately if a sufficient number of labelled cells could be identified. In these cases the labelling indices (x%,) were above 1 'Yo. Most of the postoperative and radioactive test times yielded 8 autoradiographs (2 animals each per experimental group or control group). The total number of cells covered per animal varied between 6,000 and 8,000 cells. If, in the presence of sufficiently big numbers of labelled cells, the various preparations of each animal were evaluated separetely, the number of the labelled cells or of the mitoses in the 8 preparations under study was related to the autoradiograph with t1V~ biggest cell population counted. In all cases autoradiographs were used that had the same eJ\Iiosition time. When tritium-labelled metabolic precursors were used the zero effect was so small as to be negligible. Taking the values obtained as a basis the following parameters were determined: 1. the arithmetic mean value (x); 2. the labelling index (5: 'Yo), i. e. the average number of labelled cells related to the total number of the cell population; 3. the standard deviation as a measure of the dispersion of the indhidual values (s) and 4. the percentage of the standard deviation from the mean value as a measure of the dispersion of the individual values (s (\.); 5. the generation time while taking the thinning of the silver grain density per nucleus as obtained at different radioactive test times as a basis. The corresponding values for the control animals were calculated in the same manner. In determining the incorporation of radioactive sulphate into the acid mucopolysaccharides, the mean extinction value was obtained from 8 to 10 single values (measurements). Dispersion is best expressed in the percentage of the standard deviation and is, therefore gi ven in this way and not in absolute figures. By "infarction border" we understand the tissue section which is directly adjacent to the infarction site and has a width of 12 myocyte nuclei. Its shape is very irregular which renders an evaluation rather difficult. Counting was done by means of oil immersion. To avoid double counting an ocular counting raster (VEB Carl Zeiss JENA) was used. Photometry of the unstained autoradiographs, 'Yhich was employed to study the amount of the incorporation of radioactive sulphate into the acid mucopolysaccharides, was performed at a wavelength of 554 nm. In addition, histological preparations from ervery set with the following stains or histochemical reactions were available
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for evaluation: elastica-van Gieson (connective tissue staining), toluidine blue, aldan blue and astra blue (demonstratiofJ of acid mucopolysaccharides).
Results The quotients obtained from labelled connective tissue cells and mitoses which range between 8.7 and 12.1 clearly show that in all 3 age-groups under study the connective tissue cells at the infarction site and at the infarction border proliferate by mitotic cell division. In the 6-week-old rats a generation cycle lasts 12 hours on the 1st postoperative day. In the 5-months-old animals the connective tissue cells double once in 12 hours on the 2nd postoperative day. The subsequent cell cyeles are prolonged in comparison with the first generation time. In the 21-months-old rats the doubling time of the connective tissue cells at the infarction site and at the infarction border amounts to about 72 hours on the 2nd postoperative day. That means, that the most pronounced time shifts in the generation cyele occur in the senile experimental animals. In all 3 age-groups under study the proliferation peaks of the connective tissue cells occurred on the 2nd postoperative day (figs. 1 and 2). The pereentage of the dividing cells decreases with the advancing age of the experimental animals. In young animals the granulation tissue formation (wound healing) is completed faster than in adult or old animals, and that is why the original number of labelled connective erissue cells sn regain~d much earlier. Aftter an interval of 30 minutes between the application of 3H-proline and sacrification of the animals, the silver grains are mainly found above the cytoplasm directly surrounding the nucleus. After one hour they can be observed chiefly in the periphery of the nucleus while after 4 hours they are predominantly localized in the intercellular space (table 1). There are no age differences in the silver grain density and in the release rate of the newlyformed tropocollagen. Age differences can be established distinctly only in the percentage of the synthesizing-connective tissue cells. On the first two postoperative days the share of the tropocollagen synthesizing connective tissue cells is higher at the infarction border than it is at the infarction site. At the infarction site the synthesis peak for collagen precursors appears on the 4th postoperative day. At the infarction border and at the infarction site the endothelial cells have a considerable capacity for proliferation. On the 4th postoperative day a generation cycle takes about 12 to 24 hours (table 2). DNA synthesis is accompanied by distinct collagen precursor synthesis. The release of tropocollagen takes
%
10
6
14::~....-~~--r~.--....-_-.-_---. "" 3 4 5 6 7 9 11
-.-_-=~~===~f.ostoperative Uberlebenszeit 11.
17
21
(in TOBen)
Fig. 1. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction site in the hearts of 6-week-old (--), 5-months-old (- - - -) and 21-months-old (....) rats in dependence on the postoperative survival time. S'
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Fig. 2. Content of DNA-synthesizing connective tissue nuclei (in %) at the infarction border in the hearts of 6-week-old (--), 5-months-old (- - - -) and 21-months-old (....) rats in dependence on the postoperative survival time. Table 1. !\fean silver grain number per connective tissue cell after 3H-proline application both at the infarction site and the infarction border in the hearts of 5-months-old rats in dependence on the postoperative survival time Postoperative survival time (days)
Radioactive test time (minutes)
Infarction s%
Infarction border s%
x
x
1
30 60 240
8.7 9.1 6.3
26.2 31.4 24.0
lOA
9.0
27.2 29.9 19.6
2
30 60 240
16.0 18.1 7.2
2504 28.9 19.2
14.1 16.8 6.7
25.4 25.9 20.6
4
30 60 240
14.9 1704 9.3
26.2 28.2 10.7
12.7 16.2 904
26.2 24.1 19.8
8
30 60 240
13.3 15.8 9.9
1704 20.1 20.6
11.4 14.2 8.0
16.3 19.2 19.3
9.5
Table 2. Content of DNA-synthesizing endothelial nuclei (in %) and mean silver-grain number per nucleus at the infarction site in the hearts of 5-months-old rats in dependence on the radioactive test time (4th postoperative day) Radioactive test time (honrs)
1 3
6 12 24 48 72
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Labelled endothelial cells
Mean silver grain number per nucleus x s%
2.2 2.2 2.9 4.1 5.6 5.7 5.8
30.6 30.0 22.8 15.9 11.6 10.9 8.8
10.0 10.1 24.9 24.8 23.1 22.7 21.1
Table 3. Extinction after injection of radioaetive sulphate at the infarction site in dependenee on the postoperative survival time. (Unstained autoradiographs: radioaetive test time: 24 hours) Extinction so//0
Period of time after coronary artery ligature was performed (days)
x
2. 3. 4. 5. 6. 7. 9. ll. 14. 17. 2l. 30. 120. 150. 180.
0.499 0.479 0.645 0.795 0.702 0.569 0.509 0.473 0.445 0.440 0.472 0.482 0.434 0.473 0.482
24.9 27.4 27.5 19.6 16.8 25.8 26.1 18.1 25.8 26.0 26.4 25.7 28.1 27.2 26.6
place at a strikingly slow speed. The incorporation of radioactive sulphate into the acid mucopolysaccharides of the connective tissue at the infarction site remains relatively constant over many test times (table 3). The incorporation peak lies between the 4th and 7th postoperative day.
Discussion The proliferation of the cardiac eonneetive-tissue eells always begins direetly at the infaretion border. These findings are in eontrast to the observations made by BLOCK et al. (1963) who after inflieting eut and eauterization wounds on rat skin and tongue epithelium observed that proliferation starts some 200 to 300 basal eells away from the edge of the wound. Also in stab wounds of the·teetum optieum of lebistes retieulatus (KRANZ and RICHTER 1971) proliferation started at a distanee from the puneture ehannel. The cells migrate into the puneture ehannel only after some time has passed. Both studies indicate that the type of the injured tissue plays a great role. Also the degree of severity of the tissue damage (KRANZ et al. 1968, 1971) as well as the sensitivity of the eells to the liberated eytoelastie substanees, which were ealled neerohormones by ALTMANN (1966) and KLIC'iGE (1967) and are probably DNA fission produets, are of great significance. Like GORNAK and LUSNIKOV (1963) and HECHT (1964) we observed in 6-weeks-old and 5-months-old rats a pronounced proliferation of mesenehymal cells at the infarction site as late as 24 hours postoperatively whereas at the infarction border proliferation could be demonstrated already 12 hours after the operation. In the 21-months-old rats the onset of the connective tissue proliferation of the infarction site and at the infarction border is delayed by 12 hours as compared with the 2 other age-groups. The redueed proliferation rate of the eonneetive tissue eells and the prolonged generation times observed in the 21months rats suggest an inhibition of the DNA synthesis. The diminished capability to synthesize DNA is not to be attributed to radiation damage by 3H-thymidine, since the prolonged generation time is only a result of a delay in the proliferation proeesses. The prolongation - due to radiation - of the generation time of diploid hepatic eells, for instance, is conditioned by a prolongation of the postmitotie, presynthetic resting stages (G1-phase). The duration of the synthesis phase, the postsynthetic premitotie resting stage and the mitotie phase, llOwever, are unehanged. This damage oecurs as late as 3 to 5 weeks after 3H-thymidine application (POST and HOFMAN 1967). We did not observe pathological cOll,nective tissue karyokinesis. Also ehanges in the degrees of ploidy (POST and HOFMAN 1961, 1967) ean be exeluded beeause of the ratio between labelled cells and mitoses.
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Our observations that the capacity for proliferation of connective tissue cells in advanced age is decreasing without any time shifts occurring in the proliferation peaks are corroborated by the study of PHILLIPS and LEONG (1967). These authors examined the influence of unilateral nephrectomy on proliferating cortical cells in young and adult rats. They found that, although adult animals hava a lower proliferative activity both groups of animals presented qualitatively similar reactions with regard to the time of the proliferation peak. Differences in the proliferative activity of kidney cells from adult and new-born mice in the presence of compensatory hypertension were described by ANTIPOVA (1966). The influence of ageing 011 tbe labelling index of parenchymal cells of rat liver and kidneys was studied by STOCKER et al. (1964). They discovered a steep decline in the labelling index up to the age of 2-4 months: There is a steady-state growth in these 2 organs. Similar results were obtained by LITVAK and BASERKA (1964) for the renal parenchymal cells in the mouse,'by CRANE et al. (1965) for the pituitary gland, by BERMAN et al. (1966) for the thymus, and by DUBINKO (1966) for the non-striated muscles of the small intestine of the rat. Our autoradiographic findings show that the 3H-proline incorporation, the intracellular transport, the protein synthesis as well as the release of labelled tropocollagen from the fibroblasts take approximately 4 hours. The same duration was observed by KUNZ and BRASELMANN (1967) in the experimental PVC granuloma of the rat. Electron-microscope autoradiographic studies provided evidence of 3H-proline first entering the cisternae of the endoplasmic reticulum (Ross and BENDITT 1965) and being stored in Golgi vacuoles (monomeric tropocollagen molecules). Later these collagen precursors are found near the cell membrane mostly in a filamentous form. Their release into the interstice happens either by a merocrine or an apocrine secretion. The polymerisation of the tropocollagen molecules into collagen takes place outside the fibroblasts (Survey Report,; DAVID 1967). We could not observe an age-dependence of the 3H-proline incorporation rate, the intracellular transport, the synthesis of collagen precursors and of the duration of their release. In the 6-weeks-old, 5-months-old and 21-months-old experimental animals the number of tropocollagen-synthesizing fibroblasts decreases with advancing age, whereas the mean silver grain density per cell is mostly independent of age. This applies to all 3 radioactive test groups. In each case the peak of synthesis is in the 4th postoperative day. The incorporation peak for radioactive sulphate into the acid mucopolysaccharides of the granulation tissue at the infarction site lies between the 4th and 7th postoperative day. The increase of mllcopolysaccharides in the granulation tissue lOPPENHEIMER et al. 1360, KETTLER 196'i) is accompanied by an accelerated catabolism of acid mucopolysaccharides (BOSTROM 1952, 1960, HAUSS and LOSSE 1959, JUNGE-HuLSING 1959, 1965, MEYER 1963, MUIR 1969). The synthesis of mllcopolysaccharides takes place in the Golgi field of the fibroblasts, as was demonstrated by GODMAN and LANE (1964) and LANE et aI. (1964). The main product of this synthesis is chondroitin sulphate (EDWARDS and DDUPA 1957, HARWORD et a.l. 1961, MANCINI et aI.1961). Particularly GRAUMANN (1957, 1961), MEYER (1960, 1969), BRUNS et al. (1964) and GRAUMANN et al. (1966) pointed out the close connection between the synthesis of acid mucopolysaccharides and that of collagen. The acid mucopolysaccharides are the matrix for the polymerisation of the collagen precursors in the extracellular space, and, in addition, they are supposed to be responsible for controlling and regulating the new formation of collagen fibres. Every connective tissue synthesis is accompanied by an increase in the 35S-su lphate incorporation (DRENCKHAHN and MEISSNER 1956, OPPENHEIMER et al. 1960, HARWOOD et al. 1961, HAUSS et al. 1961, 1968). The decrease in the incorporation of radioactive sulphate at later times of survival as well as the constancy of the synthesis of acid mllcopolysaccharides in the cicatrice of the infarction site suggest such a dependency. The processes involved in the new formation of capillaries in wound healing are reported to be similar to those taking place in ontogenesis (SCHOEFL 1963). Endothelial cells are growing into the gaps in the granulation tissue. According to our observations the proliferation peak of the endothelial cells occurs between the 2nd and 4th postoperative day. On the 4th postoperative day a generation cycle of the endothelial cells takes only about 12 hours. Apart from that, the amount of the tropocollagen synthesis suggests an active metabolism of the proliferating endothelial cells. In their studies concerning the development of capillaries in embryonic rat hearts DALBY et al. (1968) and OSTADAL and SCHIEBLER (1971) made similar conclusions.
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Author's address: Dr. D. KRANZ, Pathologisches Institut der Humboldt- Universitat, DDR mannstraBe 20/21.
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104 Berlin, Schu-
The influence of age on the wound healing of experimental mvocardial infarction in rats "'
(Autoradiographic studies)
By D. KRANZ, A. HECHT and 1. FUHRMANN With 2 figures (Received March 11, 1975) Key-words: wound healing; infarction: myocanlial; cell proliferation: age dependence; mitotic index; granulation tissue; connective tissue; tropocallagen synthesis; collagen precursors; endothelium; autoradiography; 3H-proline; 3H-thymidine; 35S-sulphate; mucopolysaccharides; rat
Summary In G-weeks-old, 5-months-old and 21-months-old rats myocardial infarction was induced by ('oronary artery Iigature. After performing the ligature the animals were administered 3H-thymidine, 3H-proline or 35S-sulphate at different times. The following parameters were determined: number of D~A- and tropocollagen-synthesizing connective tissue cells at the infarction border and at the infardion site; mean silver grain density above the nuclei or cells; duration of a cycle; number of mitoses, and incorporation of radioactive sulphate at the infarction site. In additon, the labelling and mitotic indices as well as the percentage of the standard deviation from the mean values were estimated. The following results were obtained: 1. The rate of gmnulation tissue formation in the neerotic zone is determined by the mitotic activity of the cells. With advancing age the cell cycles are being prolonged which results in retardation of wound healing. 2. The disturbed DNA-replication in old age is not asso('iated with a time shift in the occurrence of the mitotic and labelling peaks. 3. With advancing age the number of fibroblasts synthesizing collagen precursors decreases. There exists no age-dependence of the 3}-f-proline incorporation rate, of the intmeellular transport, of the synthesis of collagen precursor and of the release of labelled tropocollagen. In all age-groups under study these processes last approximately 4 hours. 4. Collagen fibre formation is accompanied by an increased synthesis of acid mucopolysaccharides. In infarction callosities the content of acid mucopolysaccharides most,ly is constant. 5. The proliferating endothelial cells have a pronounced metabolic activity and a markedly short generation time.
In morbidity and mortality statistics ischemic heart disease ranks first among cardiovascular diseases. In recent years there has been a notable shift towards younger age-groups. Approximately 60 per cent of patients with mycocardial infarction do not survive the first 4 weeks, most of them die shortly after the acute episode (BOTHIG et al. 1972). In order to prevent disturbed wound healing which may entail cardiorrhexis, it is important to know the factors influencing granulation tissue formation in the necrotic zone - which takes place analogously to the wound-healing processes in other tissues. The present paper investigates the influence of age on the process of wound healing in experimental myocardial infarction.
Material and 1.11ethods In total 246 male Wistar rats of the Rehbriicke strain were included in the study. 70 rats were G weeks old, 106 rats were 5 months old and 70 rats were 21 months old. Myocardial infarction was produced by ligating the left coronary artery closely below the left auricular appendix. 2 shamoperated animals (thoracotomy and touching of the left coronary artery 12 hours prior to 3H-thy8 Exp. Path. Bd. 11, H. 3/4
107
midine administration), and 2 non-operated animals were used for controls in each age-group. The radioisotopes were administered by intraperitoneal injection. To prevent differences in the silver grain density caused by variations in the cytometabolism in the course of the day, the radioactive metabolic precursors were always injected at 8 a. m. The following radioactive isotopes were employed for the study: 1. Thymidine-6- 3H; specific activity 23.3 CijmM (Institute for Research, Production and Application of Radioisotopes, Prague, CSSR). Dose: 2p, Cijg body weight. 2. I,-Prolin-3H(G); specific activity 550 mCijmM; Radiochemical Centre AmershamjEngland. Dose: 3 (.l, Cijg body weight. 3. Na235S0" carrier-free; Central Institute for Nuclear Research, DresdenjRossendorf (GDR). Dose: 5 (.l, Cijg of body weight. The animals were sacrificed by vertebral dislocation. Immediately afterwards the heart was excised and the remaining blood carefully removed. After having been fixed in 10% neutral formalin for 24 hours the hearts were dissected in such a way that the tissue blocs, embedded in paraffin and lining the upper and lower borders of the infarction or of the infarction callosity (which, in most cases, could be clearly distinguished from the surrounding tissue), still contained a macroscopically unchanged muscular layer of about 3 mm. We used 28 sections of 3-5 (.l,m, i. e. 4 sets comprising 7 sections each, from every bloc for preparation of autoradiographs employing K5 ORWO emulsion. The exposition times of the autoradiographs for the study of DNA metabolism were 7, 14 and 21 days; for the study of collagen synthesis 6, 13 and 20 days; and for the study of acid mucopolysaccharide synthesis 10, 15 and 24 days. After having been exposed, developed and fixed under strictly constant conditions, the alltoradiographs were stained with haemalum-eosine. The preparation for the study of acid mucopolysaccharide synthesis remained unstained after 24 days of exposition for the purpose of photometric evaluation. Following 3H-thymidine administration the evaluation was made at the infarction site or at the infarction border by counting the labelled connective-tissue nuclei and the mitoses and by determining the percentage of labelled cells and the silver grain density per nucleus. After 3H_ proline application the labelled connective tissue cells were counted. The percentage of labelled connective tissue cells and the mean silver grain density above these cells were determined. After 35S-sulphate injection the extinction at the infarction site or at the infarction callosity site was determined by means of photometry of the autoradiographs. The individual histological sections from the various sets of each heart were evaluated separately if a sufficient number of labelled cells could be identified. In these cases the labelling indices (x%,) were above 1 'Yo. Most of the postoperative and radioactive test times yielded 8 autoradiographs (2 animals each per experimental group or control group). The total number of cells covered per animal varied between 6,000 and 8,000 cells. If, in the presence of sufficiently big numbers of labelled cells, the various preparations of each animal were evaluated separetely, the number of the labelled cells or of the mitoses in the 8 preparations under study was related to the autoradiograph with t1V~ biggest cell population counted. In all cases autoradiographs were used that had the same eJ\Iiosition time. When tritium-labelled metabolic precursors were used the zero effect was so small as to be negligible. Taking the values obtained as a basis the following parameters were determined: 1. the arithmetic mean value (x); 2. the labelling index (5: 'Yo), i. e. the average number of labelled cells related to the total number of the cell population; 3. the standard deviation as a measure of the dispersion of the indhidual values (s) and 4. the percentage of the standard deviation from the mean value as a measure of the dispersion of the individual values (s (\.); 5. the generation time while taking the thinning of the silver grain density per nucleus as obtained at different radioactive test times as a basis. The corresponding values for the control animals were calculated in the same manner. In determining the incorporation of radioactive sulphate into the acid mucopolysaccharides, the mean extinction value was obtained from 8 to 10 single values (measurements). Dispersion is best expressed in the percentage of the standard deviation and is, therefore gi ven in this way and not in absolute figures. By "infarction border" we understand the tissue section which is directly adjacent to the infarction site and has a width of 12 myocyte nuclei. Its shape is very irregular which renders an evaluation rather difficult. Counting was done by means of oil immersion. To avoid double counting an ocular counting raster (VEB Carl Zeiss JENA) was used. Photometry of the unstained autoradiographs, 'Yhich was employed to study the amount of the incorporation of radioactive sulphate into the acid mucopolysaccharides, was performed at a wavelength of 554 nm. In addition, histological preparations from ervery set with the following stains or histochemical reactions were available
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for evaluation: elastica-van Gieson (connective tissue staining), toluidine blue, aldan blue and astra blue (demonstratiofJ of acid mucopolysaccharides).
Results The quotients obtained from labelled connective tissue cells and mitoses which range between 8.7 and 12.1 clearly show that in all 3 age-groups under study the connective tissue cells at the infarction site and at the infarction border proliferate by mitotic cell division. In the 6-week-old rats a generation cycle lasts 12 hours on the 1st postoperative day. In the 5-months-old animals the connective tissue cells double once in 12 hours on the 2nd postoperative day. The subsequent cell cyeles are prolonged in comparison with the first generation time. In the 21-months-old rats the doubling time of the connective tissue cells at the infarction site and at the infarction border amounts to about 72 hours on the 2nd postoperative day. That means, that the most pronounced time shifts in the generation cyele occur in the senile experimental animals. In all 3 age-groups under study the proliferation peaks of the connective tissue cells occurred on the 2nd postoperative day (figs. 1 and 2). The pereentage of the dividing cells decreases with the advancing age of the experimental animals. In young animals the granulation tissue formation (wound healing) is completed faster than in adult or old animals, and that is why the original number of labelled connective erissue cells sn regain~d much earlier. Aftter an interval of 30 minutes between the application of 3H-proline and sacrification of the animals, the silver grains are mainly found above the cytoplasm directly surrounding the nucleus. After one hour they can be observed chiefly in the periphery of the nucleus while after 4 hours they are predominantly localized in the intercellular space (table 1). There are no age differences in the silver grain density and in the release rate of the newlyformed tropocollagen. Age differences can be established distinctly only in the percentage of the synthesizing-connective tissue cells. On the first two postoperative days the share of the tropocollagen synthesizing connective tissue cells is higher at the infarction border than it is at the infarction site. At the infarction site the synthesis peak for collagen precursors appears on the 4th postoperative day. At the infarction border and at the infarction site the endothelial cells have a considerable capacity for proliferation. On the 4th postoperative day a generation cycle takes about 12 to 24 hours (table 2). DNA synthesis is accompanied by distinct collagen precursor synthesis. The release of tropocollagen takes
%
10
6
14::~....-~~--r~.--....-_-.-_---. "" 3 4 5 6 7 9 11
-.-_-=~~===~f.ostoperative Uberlebenszeit 11.
17
21
(in TOBen)
Fig. 1. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction site in the hearts of 6-week-old (--), 5-months-old (- - - -) and 21-months-old (....) rats in dependence on the postoperative survival time. S'
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Fig. 2. Content of DNA-synthesizing connective tissue nuclei (in %) at the infarction border in the hearts of 6-week-old (--), 5-months-old (- - - -) and 21-months-old (....) rats in dependence on the postoperative survival time. Table 1. !\fean silver grain number per connective tissue cell after 3H-proline application both at the infarction site and the infarction border in the hearts of 5-months-old rats in dependence on the postoperative survival time Postoperative survival time (days)
Radioactive test time (minutes)
Infarction s%
Infarction border s%
x
x
1
30 60 240
8.7 9.1 6.3
26.2 31.4 24.0
lOA
9.0
27.2 29.9 19.6
2
30 60 240
16.0 18.1 7.2
2504 28.9 19.2
14.1 16.8 6.7
25.4 25.9 20.6
4
30 60 240
14.9 1704 9.3
26.2 28.2 10.7
12.7 16.2 904
26.2 24.1 19.8
8
30 60 240
13.3 15.8 9.9
1704 20.1 20.6
11.4 14.2 8.0
16.3 19.2 19.3
9.5
Table 2. Content of DNA-synthesizing endothelial nuclei (in %) and mean silver-grain number per nucleus at the infarction site in the hearts of 5-months-old rats in dependence on the radioactive test time (4th postoperative day) Radioactive test time (honrs)
1 3
6 12 24 48 72
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Labelled endothelial cells
Mean silver grain number per nucleus x s%
2.2 2.2 2.9 4.1 5.6 5.7 5.8
30.6 30.0 22.8 15.9 11.6 10.9 8.8
10.0 10.1 24.9 24.8 23.1 22.7 21.1
Table 3. Extinction after injection of radioaetive sulphate at the infarction site in dependenee on the postoperative survival time. (Unstained autoradiographs: radioaetive test time: 24 hours) Extinction so//0
Period of time after coronary artery ligature was performed (days)
x
2. 3. 4. 5. 6. 7. 9. ll. 14. 17. 2l. 30. 120. 150. 180.
0.499 0.479 0.645 0.795 0.702 0.569 0.509 0.473 0.445 0.440 0.472 0.482 0.434 0.473 0.482
24.9 27.4 27.5 19.6 16.8 25.8 26.1 18.1 25.8 26.0 26.4 25.7 28.1 27.2 26.6
place at a strikingly slow speed. The incorporation of radioactive sulphate into the acid mucopolysaccharides of the connective tissue at the infarction site remains relatively constant over many test times (table 3). The incorporation peak lies between the 4th and 7th postoperative day.
Discussion The proliferation of the cardiac eonneetive-tissue eells always begins direetly at the infaretion border. These findings are in eontrast to the observations made by BLOCK et al. (1963) who after inflieting eut and eauterization wounds on rat skin and tongue epithelium observed that proliferation starts some 200 to 300 basal eells away from the edge of the wound. Also in stab wounds of the·teetum optieum of lebistes retieulatus (KRANZ and RICHTER 1971) proliferation started at a distanee from the puneture ehannel. The cells migrate into the puneture ehannel only after some time has passed. Both studies indicate that the type of the injured tissue plays a great role. Also the degree of severity of the tissue damage (KRANZ et al. 1968, 1971) as well as the sensitivity of the eells to the liberated eytoelastie substanees, which were ealled neerohormones by ALTMANN (1966) and KLIC'iGE (1967) and are probably DNA fission produets, are of great significance. Like GORNAK and LUSNIKOV (1963) and HECHT (1964) we observed in 6-weeks-old and 5-months-old rats a pronounced proliferation of mesenehymal cells at the infarction site as late as 24 hours postoperatively whereas at the infarction border proliferation could be demonstrated already 12 hours after the operation. In the 21-months-old rats the onset of the connective tissue proliferation of the infarction site and at the infarction border is delayed by 12 hours as compared with the 2 other age-groups. The redueed proliferation rate of the eonneetive tissue eells and the prolonged generation times observed in the 21months rats suggest an inhibition of the DNA synthesis. The diminished capability to synthesize DNA is not to be attributed to radiation damage by 3H-thymidine, since the prolonged generation time is only a result of a delay in the proliferation proeesses. The prolongation - due to radiation - of the generation time of diploid hepatic eells, for instance, is conditioned by a prolongation of the postmitotie, presynthetic resting stages (G1-phase). The duration of the synthesis phase, the postsynthetic premitotie resting stage and the mitotie phase, llOwever, are unehanged. This damage oecurs as late as 3 to 5 weeks after 3H-thymidine application (POST and HOFMAN 1967). We did not observe pathological cOll,nective tissue karyokinesis. Also ehanges in the degrees of ploidy (POST and HOFMAN 1961, 1967) ean be exeluded beeause of the ratio between labelled cells and mitoses.
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Our observations that the capacity for proliferation of connective tissue cells in advanced age is decreasing without any time shifts occurring in the proliferation peaks are corroborated by the study of PHILLIPS and LEONG (1967). These authors examined the influence of unilateral nephrectomy on proliferating cortical cells in young and adult rats. They found that, although adult animals hava a lower proliferative activity both groups of animals presented qualitatively similar reactions with regard to the time of the proliferation peak. Differences in the proliferative activity of kidney cells from adult and new-born mice in the presence of compensatory hypertension were described by ANTIPOVA (1966). The influence of ageing 011 tbe labelling index of parenchymal cells of rat liver and kidneys was studied by STOCKER et al. (1964). They discovered a steep decline in the labelling index up to the age of 2-4 months: There is a steady-state growth in these 2 organs. Similar results were obtained by LITVAK and BASERKA (1964) for the renal parenchymal cells in the mouse,'by CRANE et al. (1965) for the pituitary gland, by BERMAN et al. (1966) for the thymus, and by DUBINKO (1966) for the non-striated muscles of the small intestine of the rat. Our autoradiographic findings show that the 3H-proline incorporation, the intracellular transport, the protein synthesis as well as the release of labelled tropocollagen from the fibroblasts take approximately 4 hours. The same duration was observed by KUNZ and BRASELMANN (1967) in the experimental PVC granuloma of the rat. Electron-microscope autoradiographic studies provided evidence of 3H-proline first entering the cisternae of the endoplasmic reticulum (Ross and BENDITT 1965) and being stored in Golgi vacuoles (monomeric tropocollagen molecules). Later these collagen precursors are found near the cell membrane mostly in a filamentous form. Their release into the interstice happens either by a merocrine or an apocrine secretion. The polymerisation of the tropocollagen molecules into collagen takes place outside the fibroblasts (Survey Report,; DAVID 1967). We could not observe an age-dependence of the 3H-proline incorporation rate, the intracellular transport, the synthesis of collagen precursors and of the duration of their release. In the 6-weeks-old, 5-months-old and 21-months-old experimental animals the number of tropocollagen-synthesizing fibroblasts decreases with advancing age, whereas the mean silver grain density per cell is mostly independent of age. This applies to all 3 radioactive test groups. In each case the peak of synthesis is in the 4th postoperative day. The incorporation peak for radioactive sulphate into the acid mucopolysaccharides of the granulation tissue at the infarction site lies between the 4th and 7th postoperative day. The increase of mllcopolysaccharides in the granulation tissue lOPPENHEIMER et al. 1360, KETTLER 196'i) is accompanied by an accelerated catabolism of acid mucopolysaccharides (BOSTROM 1952, 1960, HAUSS and LOSSE 1959, JUNGE-HuLSING 1959, 1965, MEYER 1963, MUIR 1969). The synthesis of mllcopolysaccharides takes place in the Golgi field of the fibroblasts, as was demonstrated by GODMAN and LANE (1964) and LANE et aI. (1964). The main product of this synthesis is chondroitin sulphate (EDWARDS and DDUPA 1957, HARWORD et a.l. 1961, MANCINI et aI.1961). Particularly GRAUMANN (1957, 1961), MEYER (1960, 1969), BRUNS et al. (1964) and GRAUMANN et al. (1966) pointed out the close connection between the synthesis of acid mucopolysaccharides and that of collagen. The acid mucopolysaccharides are the matrix for the polymerisation of the collagen precursors in the extracellular space, and, in addition, they are supposed to be responsible for controlling and regulating the new formation of collagen fibres. Every connective tissue synthesis is accompanied by an increase in the 35S-su lphate incorporation (DRENCKHAHN and MEISSNER 1956, OPPENHEIMER et al. 1960, HARWOOD et al. 1961, HAUSS et al. 1961, 1968). The decrease in the incorporation of radioactive sulphate at later times of survival as well as the constancy of the synthesis of acid mllcopolysaccharides in the cicatrice of the infarction site suggest such a dependency. The processes involved in the new formation of capillaries in wound healing are reported to be similar to those taking place in ontogenesis (SCHOEFL 1963). Endothelial cells are growing into the gaps in the granulation tissue. According to our observations the proliferation peak of the endothelial cells occurs between the 2nd and 4th postoperative day. On the 4th postoperative day a generation cycle of the endothelial cells takes only about 12 hours. Apart from that, the amount of the tropocollagen synthesis suggests an active metabolism of the proliferating endothelial cells. In their studies concerning the development of capillaries in embryonic rat hearts DALBY et al. (1968) and OSTADAL and SCHIEBLER (1971) made similar conclusions.
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Author's address: Dr. D. KRANZ, Pathologisches Institut der Humboldt- Universitat, DDR mannstraBe 20/21.
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104 Berlin, Schu-