- Email: [email protected]
The influence of hyperthyroidism and hypothyroidism on the wound healing of experimental myocardial infarction in the rat
Exp. Path., Bd. 12, S. 129-136 (1976) Humboldt-University in Berlin, Institute of Pathology (Director: Prof. Dr. sc. med. L.-H. KETTLER)
The influence of hyperthyroidism and hypothyroidism on the wound healing of experimental myocardial infarction in the rat (Autoradiographic studies) By D. KRANZ, A. HECHT and 1. FUHRMANNl ) With 4 figures (Received November 11, 1975) Key-words: hyperthyroidism; hypothyroidism; wound healing; myocardial infarction; hormonal imbalance; thyroxine; autoradiography; connective tissue; tropocollagen; mucopolysaccharides; ra.t.
Summary In hyperthyroid and hypothyroid rats myocardial infarction was produced by coronary artery ligature. At different times following the ligature the animals were given 3H-thymidine or 3H-proline. The following parameters were determined: the number of DNA- and tropocollagen-synthesizing connective-tissue cells at the infarction border and at the infarction site; the mean silvergrain density above the nuclei or cells; the duration of one cell cycle; and the number of mitoses. The labelling and mitotic indices as well as the percentage of the standard deviation from the mean values were estimated. The following results were obta,ined: 1. In hyperthyroidism wound healing is accelerated. The number of DNA-synthesizing connective-tissue cells is increased. The cell-cycles are shorter. The release of tropocollagen by the fibroblasts ta,kes place at a strikingly fast rate. 2. In hypothyroidism wound healing is delayed. The 3H-thymidine and mitotic indices are lowered. The release of tropocolla.gen is slowed down. The granulation tissue contains localized accumulations of mucopolysaccharides. Functional changes of the thyroid gland result in disturbed hemodynamics and cardiovascular changes. In Anglo-American literature the relevant findings are summed up under the term of "thyroid heart disease". Insidious (pre-clinical) hypothyroidism represents a risk factor for the development of coronary arteriosclerosis, and thus for the development of myocardial infarction. One third of the patients with myocardial infarction in whom ischemic heart disease has an unfavourable clinical prognosis, are reported to suffer from hyperthyroidism (CEREMUZYNSKI et al. 1971). We studied the wound healing of experimental myocardial infarction in hyperthyroid and hypothyroid rats.
Material and methods Myocardial infarction was produced by ligating the left coronary artery closely below the left auricular appendix. A. Production of hormona,l imbalance Hyp erthyroidism 124 hyperthyroid rats were available for the studies. At the beginning of the experiment the animals were 5 months old. Starting 14 days prior to the inducement of experimental myocardial infarction and until the day the animals were sacrificed, they were daily administered L-thyroxine 1) Dedicated to Prof. KETTLER on the occasion of his 65th birthday. 9 Exp. Path. Rd. 12
129
produced by Messrs. Reanal (Budapest) which was subcutaneously injected with a microsyringe using individual doses of 10 pg dissolved in 25 pI of physiological sa.Iine per 100 g of body weight. To improve the solubility of thyroxine 2 drops of 2n caustic soda solution were added to the preparation which was freshly made every day. Already after the second injection the nutrition and fresh water requirements of the rats increased distinctly. Despite this fa.ct there occurred in the course of the experiment a, considerable loss of weight as compared to the control animals, which amounted to an average of 20 per cent. Control animals were: 1. 2 sham-operated and 2 non-operated hyperthyroid rats; 2. 70 five-month-old rats with normal metabolism in which myocardia.I infarction had been produced. Hypothyroidism 125 hypothyroid rats were studied which, at the beginning of the experiment, were 5 months old. Under ether-oxygen anesthesia the thyroid tissue was excised as completely as possible while sparing the epithelial bodies. In order to remove even the smallest remaining parts of the thyroid gland the animals were intraperitoneally injected ra,dioactive iodine (carrier-free Na 131 in physiological saline; Nuclear Research Institute at Swierk, Poland) in a dose of 1 mCi per 100 g of body weight two da,ys after the operation. Measures were taken to prevent the anima.Is from irradia,ting one another. The rats were kept separately in special cages designed for meta,bolic studies and shielded against each other by lead blocks, until ra,dioactivity subsided. Radioactive feces a,nd urine were constantly removed. 15 months after thyroidectomy and administration of radioactive iodine, experimental myocardial infa.rction was produced. Two thyroidectomized ra,ts of the same age which had been injected radioactive iodine (= nonopera.ted controls), two operated animals of the same age which, in addition, ha,d undergone thoracotomy, (= sham-operated rats), and 70 21-month-old rats with normal metabolism in which myoca,rdia.I infarction had been produced, were used as controls. The complete destruction of the thyroid tissue could be confirmed by histologica.I serial tests. The treatment did not result in any histological changes of the structure of the epithelial bodies.
B. Employed radioactive metabolic precursors, the preparation and evalua,tion of the autoradiographs The radioisotopes were administered by intraperitoneal injection. To prevent differences in the silver-grain density ca,used by variations in the cytometabolism in the course of the day, the radioactive metabolic precursors were always injected at 8 a.m. The following ra,dioactive isotopes were employed for the study: 1. Thymidine-6-3H; specific activity 23.3. Ci/mM; Institute for Research, Production and Application of Radioisotopes, Prague. Dose: 2 pCi/g of body weight. 2. L-Proline-3H(G); specific activity 550 mCi/mM; Radiochemical Centre Amersham/England. Dose: 3 I1Ci/g of body weight. The animals were sacrificed by vertebral dislocation. Immediately thereafter the hearts were excised and the remaining blood carefully removed. After having been fixed for 24 hours in 10-per-cent neutral formalin the hearts were dissected in such a way that the tissue blocks, imbedded in pa,ra,ffin and lining the upper and lower borders of the infarction or of the infa.rction callosity (which, in most cases, stood out distinctly against the surrounding tissue), still contained a macroscopically unchanged muscular la,yer of a,bout 3 mm. We used 28 sections of 3-5 11m, i.e. four sets comprising 7 sections each, from every block to prepare autora,diogra.phs employing K5 ORWO emulsion. The exposition times of the a,utoradiographs for the study of the DNA metabolism were 7, 14 and 21 days, and for the study of the collagen synthesis 6, 13 and 20 days. After having been exposed, developed and fixed under strictly constant conditions, the autoradiographs were stained with hema.Ium-eosin. Following 3 H-thymidine administra.tion the evalua,tion was made at the infa.rction site or at the infarction border by counting the labelled connective-tissue nuclei and the mitoses, and by determining the percentage of labelled cells a,nd the silver-gra.in density per nucleus; a.fter 3H-proline application by counting the labelled connective-tissue cells, and by determining the percentage of labelled connective-tissue cells and the mean silver-grain density above these cells. In all cases autoradiograms were used that had the same exposition times. The zero effect of the autoradiographs was so small as to be negligible. 124 hyperthyroid, 125 hypothyroid rats, 70 five-month-old and 70 21-month-old rats with normal metabolism were a,vailable for the studies. 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 sepa.rately, the number of the labelled cells or of the mitoses in the preparations under study was related to the autoradiogra.m with the biggest cell population counted.
130
Taking the values obtained as a basis the following pa,rameters were determined:
1. the a,rithmetic mean value (x); 2. the labelling index (x 'Yo), i.e. the avera,ge number of la,belled cells rela,ted to the total number of
the cell population;
3. the standard deviation as measure of the dispersion of the individual values (s),
and 4. the percentage of the standard deviation from the mean value as measure of the dispersion of the individual values (s 'Yo); 5. the generation time while taking the thinning of the silvergrain density per nucleus obtained at different radioactive test times as a basis. 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 a,n evaluation rather difficult. Counting was done by means of oil immersion. To avoid double counting an ocular counting raster (VEB Ca,rl Zeiss JENA) was used. In addition, histological prepa,rations from every set with the following stains or histochemical reactions were available for evaluation: Elastica-van-Gieson (connective tissue staining), toluidine blue, alcian blue and astra, blue (demonstra,tion of acid mucopolysaccha,rides).
Results Hyperthyroidism Light-microscope findings On the 1st postoperative day the connective-tissue cells at the infarction border and at the infarction site, are swollen and focally proliferated. On the 2nd postoperative day a fine network of collagenous fibres begins to extend between the proliferated cells interspersed with isolated islands of intact muscle cells. The latter cells soon become atrophic leaving empty sarcolemmic tubes behind. On the 5th postoperative day a collagenous tissue still rich in cells has already become distinctly visible. At the infarction border large sub-infarctions can be remarked. The intima of mediumsize twigs of coronary vessels located at the infarction border is proliferated; quite often the vascular lumina are considerably narrowed. Starting with the 11th postoperative day the connective-tissue cell content clearly decreases. Fine elastic fibres begin to appear between the densely arranged collagenous fibres with the latter slowly increasing in number and strength. Finger-like thick bundles of collagenous fibres are growing into the enlarged interstices of the infarction border thereby producing a firm fusion of the callous tissue and the surrounding muscles. Autoradiographic findings At the infarction site and at the infarction border the proliferation of the connective-tissue cells takes place by mitotic division of the nuclei, as can be seen from the quotient obtained from labelled cells and mitoses its values varying between 7.5 and 13.3. Between the 2nd and 14th postoperative day the percentage of DNA-synthesizing cells at the infarction site and at the infarction border is strikingly high; it sharply decreases on the 17th day following the ligature of the coronary artery (figs. 1 and 2). The mean silver-grain density shows the same tendency. The cell cycle is considerably shorter lasting approximately 12 hours on the 2nd postoperative day. In the ventricular sections remote from the infarction site a great number of labelled interstitial connective-tissue cells were observed. The percentage of DNA-synthesizing cells in the right ventricle clearly decreases on the 11th, and in the left ventricle on the 14th postoperative day. The high mean silver-grain density per connective-tissue nucleus suggests a brisk DNA synthesis. The increase in the percentage of the labelled interstitial connective-tissue nuclei, and the decrease in the mean silver-grain density per nucleus as observed when the radioactive test time is prolonged, indicate karyokinesis. Between the 2nd and 11th postoperative day isolated, pathologically changed muscle cell mitoses can be observed. On the 2nd postoperative day the connective-tissue cells of the infarction border are involved in the tropocollagen synthesis more intensively than those of the infarction site. On the 4th and 8th postoperative day the formation, 9*
131
of collagen precursors is predominant in the connective-tissue cells of the infarction site with the synthesis peak occurring on the 4th postoperative day. The collagen precursors are released very quickly as can be seen from the distinct decrease in the mean silver-grain density which takes place between 60 and 240 minutes of radioactive test time (table 1). Similarly, synthesis of collagen precursors takes place in the interstitial connective-tissue cells of the ventricular sections remote from the infarction site. %
5
postoperative .Lor..~,~2--'3---"~~5~5"---7r--""9-~''''''' ---1~4-----"17----2~1 Uberlebenszeit (in TagenJ
Fig. 1. Content of DNA-synthesizing connective-tissue nuclei (in %) a.t the infarction sites in the hearts of ra.ts with a normal metabolism (---) a,nd of hyperthyroid (---------) rats in dependence on the postoperative survival time. ~
00
"tJt5
.!i'
.s;
:l;!
[
~ I I I
~
..:I:
......./
•
1\
\
\
.-................ .... .....-
.......-., \
-.~
I
I
\ \
, I
I
\
\
I
\
\
I
\
\ \
\
'111 2 3 4 5 6 7
9
11
I
14
• ....
\
i
17
-. i
;>
F:ostoperative
21 Uberlebenszeit
(in TagenJ
Fig. 2. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction borders in the hearts of rats with a normal metabolism (---) and of hyperthyroid (---------) rats in dependence on the postoperative survival time.
132
i-'
I:.>:>
~
Radioactive test time (min.)
30 60 240
30 60 240
30 60 240
Postoperative survival time (days)
2
4
8
13.3 15.8 9.9
14.9 17.4 9.5
16.0 18.1 7.2
17.4 20.1 20.6
26.2 28.2 20.7
25.4 28.9 19.2
11.4 14.2 8.0
11.7 16.2 9.4
14.1 16.8 6.7
x
x
16.3 19.2 19.3
26.2 24.1 19.8
25.4 23.9 20.6
s%
Infarction bord er
Infarction
s%
Rats with normal metabolism
18.8 19.6 5.7
19.6 21.4 4.3
20.3 23.8 4.8
x
Infarction
23.9 21.9 25.2
30.2 25.6 30.5
29.7 23.1 25.9
s%
Hyperthyroidism
8.5 10.4 3.9
15.4 18.6 4.3
16.7 19.9 6.2
x
20.6 19.7 20.2
26.4 28.5 23.2
20.7 21.0 22.3
s%
Infarction border
8.5 9.7 8.2
12.4 13.5 13.1
10.8 12.1 10.3
x
Infarction
24.5 25.0 24.2
23.1 25.2 26.7
27.3 26.8 25.4
s%
Hypothyroidism
7.5 8.1 7.7
10.9 11.8 10.6
8.6 9.9 8.7
x
25.0 20.9 20.1
22.6 21.1 204
26.4 19.7 21.3
s%
Infarction border
Table 1. Mean silver-gra.in number per connective-tissue cell after a,pplication of 3H-proline at the infarction sites and at the infarction borders in the hearts of rats with a normal meta,bolism, of hyperthyroid and hypothyroid rats in dependence on the postoperative survival time
Hypothyroidism Light-microscope findings 21 days following the ligature of the coronary artery, the collagenous tissue at the infarction site is only moderately dense with only a few elastic fibres having been formed. The tissue is strikingly rich in cells and capillaries and interspersed with accumulations of acid mucopolysaccharides in the neighbourhood of which an increasing number of lymphocytes can be identified. Autoradiographic findings There is only a small percentage of DNA-synthesizing connective-tissue cells at the infarction site and at the infarction border (figs. 3 and 4), which, however, is still slightly increased 21 days post operationem.
-
234557
9
11
-
I- -
14
-
-0- -
17
-
-
_....
PostoDeratlve
21 UberL~benszeit (in Tagen)
Fig. 3. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction sites in the hearts of rats with a normal metabolism (---) and of hypothyroid (---------) rats in dependence on the postoperative survival time.
5
-e- - e - - - - e - - __ e • ~~~~..._~~........-......,...---,.---~--~---_..>
121 2 3 4 5 6 7
9
11
14
17
Postoperative
21 Ubertebenszeit CinTagen)
Fig. 4. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction borders in the hearts of rats with a norma.l metabolism (---) and in hypothyroid (---------) rats in dependence on the postoperative survival time.
134
12 hours after the coronary artery ligature has been performed no DNA synthesis can yet be observed in the connective-tissue nuclei. Between the 3rd and the 5th postoperative day there is evidence of a proliferation peak occurring at the infarction site and at the infarction border. Cell proliferation is by mitosis the quotients varying between the values 8.5 and 12.6. On the 2nd postoperative day a generation cycle of the connective-tissue cells at the infarction site lasts approximately 72 hours. Also, on the 2nd postoperative day the cells belonging to the infarction border are involved more intensively in the collagen fibre formation than are those of the infarction site. At the infarction site the synthesis peak for collagen precursors occurs on the 4th postoperative day. In hypothyroid rats the release of tropocollagen by the connective-tissue cells is distinctly inhibited (table 1). Discussion
In hyperthyroid rats the number of DNA-synthesizing connective-tissue cells at the infarction site and at the infarction border Clearly exceeds that of control animals. The synthesis phase is shorter. In animals with hyperthyroidism wound healing after experimental myocardial infarction is clearly accelerated as can be seen from the collagen synthesis after 3H-proline administration. The considerably accelerated release of tropocollagen by the fibroblasts is notable. The increased turnover of radioactive sulphate in hyperthyroidism as observed by DZIEWIATKOWSKI (1957) and BOSTROM (1960) also indicates an enhanced mesenchymal metabolism. The stimulating effect of thyroxine on the metabolism has repeatedly been described by other authors who reported that it activates oxygen consumption, and that the protein synthesis is increased which may even lead to the development of cardiac hypertrophy (VAN LIERE et al. 1969, THYRUM et al. 1970). In our tests, too, the great number of DNA-synthesizing connective-tissue cells in the ventricular sections remote from the infarction site suggests the incipient development of cardiac hypertrophy. In view of the high percentage of tropocollagen-synthesizing connective-tissue cells it seems probable that the supporting fibrous structure is being reinforced. The preferential involvement of one half of the heart as found by GOLBER et al. (1969) and VAN LIERE et al. (1969, 1971) with regard to the right ventricle, has not been observed by us. Like BEZNAK (1962) and COHEN et al. (1966) we have the opinion that it is mainly due to its metabolic effect that thyroxine causes a growth of the cardiac muscle mass, whereas changed hemodynamics play but a secondary role. Due to the proliferation of the intima of medium-size twigs of coronary vessels located at the infarction border there is an insufficient blood supply in this muscular region. The effect of thyroxine which produces an increase in the metabolic rate, very quickly leads to oxygen deficiency in the marginal areas which first affects the more sensitive muscle cells. There so-called sub-infarctions develop which can be demonstrated already on the 5th postoperative day. The development of necroses is preceded by a reduction or decoupling of oxidative phosphorylation and by a decrease in ATP and ADP (MEDOVAR 1964, 1968). Myocardial necroses after thyroxine application were observed by HALL and NELSON (1968). As evidenced by the findings obtained after 3H-proline application, the development of granulation tissue at the infarction site and at the infarction border is reduced in hypothyroid rats as compared with control animals. On the 4th and 8th postoperative day the release of tropocollagen from the fibroblasts is delayed. The number of DNA-synthesizing connective-tissue cells is clearly reduced. Also, the metabolism of the connective-tissue cells in the ventricular sections remote from the infarction site as well as that of all muscle cells is inhibited to varying degrees. Our findings coincide well with the observations made by CHEEK et al. (1965) and v. KNORRING (1970). Apart from a delayed growth of the muscles of the extremities CHEEK et al. (1965) discovered in rats a decrease in the collagen content of these muscles. VON KNORRING (1970) was able to demonstrate a reduction of the hydroxyproline content in the hearts of hyperthyroid rats. The decrease in the succinic dehydrogenase activity as discovered by NANIKAWA et al. (1960) also indicates a reduced metabolic rate.
135
The reduction of the heart weights as observed after thyroidectomy (KOCHAKIAN et aI. 1954) coincides well with the findings by SOKOLOFF et aI. (1963, 1968) and by GARREN et aJ. (1967) who detected in liver cell homogenates a reduced amino acid incorporation. Also, the development of cardiac hypertrophy as a consequence of experimental aortic stenosis can be prevented by thyroidectomy (BEZNAK and HAJDU 1946). The granulous-filamentous mass observed in the granulation tissue of the heart in the presence of hypothyroidism has repeatedly been reported (GUPTA et aI. 1971). It consists of mucopolysaccharides rich in hyaluronic acid (v. KNORRING 1970). A reduced incorporation of radioactive sulphate into the chondroitine-sulphuric acid of cartilage and -skin has been demonstrated by DZIEWIATKOWSKI (1957).
Literature BEZNAK, M., Cardiovascular effects of thyroxine treatment in normal rats. Canad. J. Biochem. 40 1647-1654 (19~f)' - and I. HAJDU, Uber den Einflu6 der Schilddriise auf die Rolle der- Hypophyse in den Veranderungen der Herzmasse. Schweiz. med. Wschr. 76,390-393 (1946). BOSTROM, H., Einige Aspekte zum Metabolismus der Mucopolysaccharide. In: W. H. HAUSS und H. LOSSE, Struktur und Stoffwechsel des Bindegewebes. 2. Sympos. an der Med. Universitats-Klinik Miinster/Westf. Thieme, Stuttgart 1960. CEREMUZYNSKI, L., J. KUCH und K. TYMINSKA, Klinischer Verlauf des Herzinfarktes und Schilddriisenfunktion. Z. ges. inn. Med. 26, 282-284 (1971). CHEEK, D. R, G. K. POWELL and R E. SCOTT, Growth of muscle mass and skeletal collagen in the rat. Bull Johns Hopkins Hosp. 116, 387-395 (1965). COHEN, J., J. M. AROSSTY and H. G. ROSENFELD, Determinants of thyroxine-induced cardiac hypertrophy in mice. Circula,t. Res. 101), 69-74 (1966). DZIEWIATKOWSKI, D. D., Synthesis of sulfomucopolysaccharides in thyroidectomized rats. J. expo Med. 101), 69-74 (1957). GARREN, L. D., A. P. RICHARDSON and R M. CROCCO, Studies on the role of ribosomes in the regulation of protein synthesis in hypophysectomized and thyroidectomized rats. J. bioI. Chern. 242, 650-656 (1967). GOLBER, L. M., K. A. ANANEVA, V. J. KANDROR, 1. V. KRUKOVA and A. V. NEGOWSKAJA, Snatschenije magnija b predupreschoenii metablllitscheskich rasstroistw w miokarde pri tirectoksikose. BjuI!. eksp. Bio!. Med. 3, 23-26 (1969). GUPTA, M. P., S. KIM, J. KANG, L. SHERMAN, H. D. KOLODNY and R 1. HAMBY, Isolated THS deficiency presenting as myxedema, heart disease. JAMA 217, 205-207 (1971). HALL, R J., and W. P. NELSON, Thyroid heart disease. Postgrad. Med. 44, 127-132 (1968). KNORRING, J. VON, Myoca,rdia.I colla,gen in Hyper- and hypothyroid rats and guinea pigs. Ann. Med. fixp. Fenn. 48, 1-7 (1970). - Analysis of myocardial mucopolysaccharides in hyper- and hypothyroid rats a,nd guinea pigs. Ann. expo Fenn. 4 ,8-13 (1970). KOCHAKIAN, C. D., R A. REED and A. l\f. EISCHEID, Changes in tissue weights and enzymes produced by thyroidectomy and testosterone. Am. J. Physiol. 177, 413-417 (1954). LIERE, E. J. VAN, D. A. SIZEMORE and J. HUNNELL, Size of cardiac ventricles in experimental hyperthyroidism in the rat. Proc. Soc. exper. BioI. 132, 663-665 (1969). - and D. A. SIZEMORE, Regression of cardiac hypertrophy following experimental hyperthyroidism in rats. Proc. Soc. exper. BioI. 136, 645-648 (1971). MEDOVAR, E. N., 0 soderzanii ATP i prodnktov so ra.spada v skeletnych i serdecnoj myacach pri gipertireoze. Ukr. biochem. Z. 36, 253-256 (1964). - und E. M. POPOVA, Okisitelnoe fosforilirovanie v mitochondrija,ch serdecnoj myscy krolikov pri eksperienta.lnom gipertireoze. Ukr. biochem. Z. 40, 39-43 (1968). NANIKAWA, R, T. MIKI and H. HAMACKA, Studies on the succinic dehydrogenase of ca,rdia,c muscle. Quantitative changes in normal and abnormal thyroid functions. Nageya med. J.6, 698-707 (1960). SOKOLOFF, L., S. KAUFMAN, P. L. CAMPBELL, C. M. FRANCIS and H. C. GELBEIN, Thyroxine stimulation of amine add incorporation into protein. J. bioI. Chern. 238, 1432-1437 (1963). - P. A. ROBERTS, M. M. JANUSKA and J. E. KLINE, Mechanism of stimulation of protein synthesis by thyroid hormones in vivo. Proc. nat. Acad. Sc. (Wash.) 60, 652-659 (1968). THYRUM, P. T., E. M. KRITCHER and R J. LUCHr, Effect of L-thyroxine on the primary structure of cardiac myosin. Biochim. biophys. Acta (Arnst.) 197, 335-336 (1970). Author's address: Dr. sc. med. D. KRANZ, Pathologisches Institut der Humboldt-Universitat, Bereich Medizin (Charite), DDR - 104 Berlin, Schumannstra6e 20/21.
136
The influence of hyperthyroidism and hypothyroidism on the wound healing of experimental myocardial infarction in the rat (Autoradiographic studies) By D. KRANZ, A. HECHT and 1. FUHRMANNl ) With 4 figures (Received November 11, 1975) Key-words: hyperthyroidism; hypothyroidism; wound healing; myocardial infarction; hormonal imbalance; thyroxine; autoradiography; connective tissue; tropocollagen; mucopolysaccharides; ra.t.
Summary In hyperthyroid and hypothyroid rats myocardial infarction was produced by coronary artery ligature. At different times following the ligature the animals were given 3H-thymidine or 3H-proline. The following parameters were determined: the number of DNA- and tropocollagen-synthesizing connective-tissue cells at the infarction border and at the infarction site; the mean silvergrain density above the nuclei or cells; the duration of one cell cycle; and the number of mitoses. The labelling and mitotic indices as well as the percentage of the standard deviation from the mean values were estimated. The following results were obta,ined: 1. In hyperthyroidism wound healing is accelerated. The number of DNA-synthesizing connective-tissue cells is increased. The cell-cycles are shorter. The release of tropocollagen by the fibroblasts ta,kes place at a strikingly fast rate. 2. In hypothyroidism wound healing is delayed. The 3H-thymidine and mitotic indices are lowered. The release of tropocolla.gen is slowed down. The granulation tissue contains localized accumulations of mucopolysaccharides. Functional changes of the thyroid gland result in disturbed hemodynamics and cardiovascular changes. In Anglo-American literature the relevant findings are summed up under the term of "thyroid heart disease". Insidious (pre-clinical) hypothyroidism represents a risk factor for the development of coronary arteriosclerosis, and thus for the development of myocardial infarction. One third of the patients with myocardial infarction in whom ischemic heart disease has an unfavourable clinical prognosis, are reported to suffer from hyperthyroidism (CEREMUZYNSKI et al. 1971). We studied the wound healing of experimental myocardial infarction in hyperthyroid and hypothyroid rats.
Material and methods Myocardial infarction was produced by ligating the left coronary artery closely below the left auricular appendix. A. Production of hormona,l imbalance Hyp erthyroidism 124 hyperthyroid rats were available for the studies. At the beginning of the experiment the animals were 5 months old. Starting 14 days prior to the inducement of experimental myocardial infarction and until the day the animals were sacrificed, they were daily administered L-thyroxine 1) Dedicated to Prof. KETTLER on the occasion of his 65th birthday. 9 Exp. Path. Rd. 12
129
produced by Messrs. Reanal (Budapest) which was subcutaneously injected with a microsyringe using individual doses of 10 pg dissolved in 25 pI of physiological sa.Iine per 100 g of body weight. To improve the solubility of thyroxine 2 drops of 2n caustic soda solution were added to the preparation which was freshly made every day. Already after the second injection the nutrition and fresh water requirements of the rats increased distinctly. Despite this fa.ct there occurred in the course of the experiment a, considerable loss of weight as compared to the control animals, which amounted to an average of 20 per cent. Control animals were: 1. 2 sham-operated and 2 non-operated hyperthyroid rats; 2. 70 five-month-old rats with normal metabolism in which myocardia.I infarction had been produced. Hypothyroidism 125 hypothyroid rats were studied which, at the beginning of the experiment, were 5 months old. Under ether-oxygen anesthesia the thyroid tissue was excised as completely as possible while sparing the epithelial bodies. In order to remove even the smallest remaining parts of the thyroid gland the animals were intraperitoneally injected ra,dioactive iodine (carrier-free Na 131 in physiological saline; Nuclear Research Institute at Swierk, Poland) in a dose of 1 mCi per 100 g of body weight two da,ys after the operation. Measures were taken to prevent the anima.Is from irradia,ting one another. The rats were kept separately in special cages designed for meta,bolic studies and shielded against each other by lead blocks, until ra,dioactivity subsided. Radioactive feces a,nd urine were constantly removed. 15 months after thyroidectomy and administration of radioactive iodine, experimental myocardial infa.rction was produced. Two thyroidectomized ra,ts of the same age which had been injected radioactive iodine (= nonopera.ted controls), two operated animals of the same age which, in addition, ha,d undergone thoracotomy, (= sham-operated rats), and 70 21-month-old rats with normal metabolism in which myoca,rdia.I infarction had been produced, were used as controls. The complete destruction of the thyroid tissue could be confirmed by histologica.I serial tests. The treatment did not result in any histological changes of the structure of the epithelial bodies.
B. Employed radioactive metabolic precursors, the preparation and evalua,tion of the autoradiographs The radioisotopes were administered by intraperitoneal injection. To prevent differences in the silver-grain density ca,used by variations in the cytometabolism in the course of the day, the radioactive metabolic precursors were always injected at 8 a.m. The following ra,dioactive isotopes were employed for the study: 1. Thymidine-6-3H; specific activity 23.3. Ci/mM; Institute for Research, Production and Application of Radioisotopes, Prague. Dose: 2 pCi/g of body weight. 2. L-Proline-3H(G); specific activity 550 mCi/mM; Radiochemical Centre Amersham/England. Dose: 3 I1Ci/g of body weight. The animals were sacrificed by vertebral dislocation. Immediately thereafter the hearts were excised and the remaining blood carefully removed. After having been fixed for 24 hours in 10-per-cent neutral formalin the hearts were dissected in such a way that the tissue blocks, imbedded in pa,ra,ffin and lining the upper and lower borders of the infarction or of the infa.rction callosity (which, in most cases, stood out distinctly against the surrounding tissue), still contained a macroscopically unchanged muscular la,yer of a,bout 3 mm. We used 28 sections of 3-5 11m, i.e. four sets comprising 7 sections each, from every block to prepare autora,diogra.phs employing K5 ORWO emulsion. The exposition times of the a,utoradiographs for the study of the DNA metabolism were 7, 14 and 21 days, and for the study of the collagen synthesis 6, 13 and 20 days. After having been exposed, developed and fixed under strictly constant conditions, the autoradiographs were stained with hema.Ium-eosin. Following 3 H-thymidine administra.tion the evalua,tion was made at the infa.rction site or at the infarction border by counting the labelled connective-tissue nuclei and the mitoses, and by determining the percentage of labelled cells a,nd the silver-gra.in density per nucleus; a.fter 3H-proline application by counting the labelled connective-tissue cells, and by determining the percentage of labelled connective-tissue cells and the mean silver-grain density above these cells. In all cases autoradiograms were used that had the same exposition times. The zero effect of the autoradiographs was so small as to be negligible. 124 hyperthyroid, 125 hypothyroid rats, 70 five-month-old and 70 21-month-old rats with normal metabolism were a,vailable for the studies. 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 sepa.rately, the number of the labelled cells or of the mitoses in the preparations under study was related to the autoradiogra.m with the biggest cell population counted.
130
Taking the values obtained as a basis the following pa,rameters were determined:
1. the a,rithmetic mean value (x); 2. the labelling index (x 'Yo), i.e. the avera,ge number of la,belled cells rela,ted to the total number of
the cell population;
3. the standard deviation as measure of the dispersion of the individual values (s),
and 4. the percentage of the standard deviation from the mean value as measure of the dispersion of the individual values (s 'Yo); 5. the generation time while taking the thinning of the silvergrain density per nucleus obtained at different radioactive test times as a basis. 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 a,n evaluation rather difficult. Counting was done by means of oil immersion. To avoid double counting an ocular counting raster (VEB Ca,rl Zeiss JENA) was used. In addition, histological prepa,rations from every set with the following stains or histochemical reactions were available for evaluation: Elastica-van-Gieson (connective tissue staining), toluidine blue, alcian blue and astra, blue (demonstra,tion of acid mucopolysaccha,rides).
Results Hyperthyroidism Light-microscope findings On the 1st postoperative day the connective-tissue cells at the infarction border and at the infarction site, are swollen and focally proliferated. On the 2nd postoperative day a fine network of collagenous fibres begins to extend between the proliferated cells interspersed with isolated islands of intact muscle cells. The latter cells soon become atrophic leaving empty sarcolemmic tubes behind. On the 5th postoperative day a collagenous tissue still rich in cells has already become distinctly visible. At the infarction border large sub-infarctions can be remarked. The intima of mediumsize twigs of coronary vessels located at the infarction border is proliferated; quite often the vascular lumina are considerably narrowed. Starting with the 11th postoperative day the connective-tissue cell content clearly decreases. Fine elastic fibres begin to appear between the densely arranged collagenous fibres with the latter slowly increasing in number and strength. Finger-like thick bundles of collagenous fibres are growing into the enlarged interstices of the infarction border thereby producing a firm fusion of the callous tissue and the surrounding muscles. Autoradiographic findings At the infarction site and at the infarction border the proliferation of the connective-tissue cells takes place by mitotic division of the nuclei, as can be seen from the quotient obtained from labelled cells and mitoses its values varying between 7.5 and 13.3. Between the 2nd and 14th postoperative day the percentage of DNA-synthesizing cells at the infarction site and at the infarction border is strikingly high; it sharply decreases on the 17th day following the ligature of the coronary artery (figs. 1 and 2). The mean silver-grain density shows the same tendency. The cell cycle is considerably shorter lasting approximately 12 hours on the 2nd postoperative day. In the ventricular sections remote from the infarction site a great number of labelled interstitial connective-tissue cells were observed. The percentage of DNA-synthesizing cells in the right ventricle clearly decreases on the 11th, and in the left ventricle on the 14th postoperative day. The high mean silver-grain density per connective-tissue nucleus suggests a brisk DNA synthesis. The increase in the percentage of the labelled interstitial connective-tissue nuclei, and the decrease in the mean silver-grain density per nucleus as observed when the radioactive test time is prolonged, indicate karyokinesis. Between the 2nd and 11th postoperative day isolated, pathologically changed muscle cell mitoses can be observed. On the 2nd postoperative day the connective-tissue cells of the infarction border are involved in the tropocollagen synthesis more intensively than those of the infarction site. On the 4th and 8th postoperative day the formation, 9*
131
of collagen precursors is predominant in the connective-tissue cells of the infarction site with the synthesis peak occurring on the 4th postoperative day. The collagen precursors are released very quickly as can be seen from the distinct decrease in the mean silver-grain density which takes place between 60 and 240 minutes of radioactive test time (table 1). Similarly, synthesis of collagen precursors takes place in the interstitial connective-tissue cells of the ventricular sections remote from the infarction site. %
5
postoperative .Lor..~,~2--'3---"~~5~5"---7r--""9-~''''''' ---1~4-----"17----2~1 Uberlebenszeit (in TagenJ
Fig. 1. Content of DNA-synthesizing connective-tissue nuclei (in %) a.t the infarction sites in the hearts of ra.ts with a normal metabolism (---) a,nd of hyperthyroid (---------) rats in dependence on the postoperative survival time. ~
00
"tJt5
.!i'
.s;
:l;!
[
~ I I I
~
..:I:
......./
•
1\
\
\
.-................ .... .....-
.......-., \
-.~
I
I
\ \
, I
I
\
\
I
\
\
I
\
\ \
\
'111 2 3 4 5 6 7
9
11
I
14
• ....
\
i
17
-. i
;>
F:ostoperative
21 Uberlebenszeit
(in TagenJ
Fig. 2. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction borders in the hearts of rats with a normal metabolism (---) and of hyperthyroid (---------) rats in dependence on the postoperative survival time.
132
i-'
I:.>:>
~
Radioactive test time (min.)
30 60 240
30 60 240
30 60 240
Postoperative survival time (days)
2
4
8
13.3 15.8 9.9
14.9 17.4 9.5
16.0 18.1 7.2
17.4 20.1 20.6
26.2 28.2 20.7
25.4 28.9 19.2
11.4 14.2 8.0
11.7 16.2 9.4
14.1 16.8 6.7
x
x
16.3 19.2 19.3
26.2 24.1 19.8
25.4 23.9 20.6
s%
Infarction bord er
Infarction
s%
Rats with normal metabolism
18.8 19.6 5.7
19.6 21.4 4.3
20.3 23.8 4.8
x
Infarction
23.9 21.9 25.2
30.2 25.6 30.5
29.7 23.1 25.9
s%
Hyperthyroidism
8.5 10.4 3.9
15.4 18.6 4.3
16.7 19.9 6.2
x
20.6 19.7 20.2
26.4 28.5 23.2
20.7 21.0 22.3
s%
Infarction border
8.5 9.7 8.2
12.4 13.5 13.1
10.8 12.1 10.3
x
Infarction
24.5 25.0 24.2
23.1 25.2 26.7
27.3 26.8 25.4
s%
Hypothyroidism
7.5 8.1 7.7
10.9 11.8 10.6
8.6 9.9 8.7
x
25.0 20.9 20.1
22.6 21.1 204
26.4 19.7 21.3
s%
Infarction border
Table 1. Mean silver-gra.in number per connective-tissue cell after a,pplication of 3H-proline at the infarction sites and at the infarction borders in the hearts of rats with a normal meta,bolism, of hyperthyroid and hypothyroid rats in dependence on the postoperative survival time
Hypothyroidism Light-microscope findings 21 days following the ligature of the coronary artery, the collagenous tissue at the infarction site is only moderately dense with only a few elastic fibres having been formed. The tissue is strikingly rich in cells and capillaries and interspersed with accumulations of acid mucopolysaccharides in the neighbourhood of which an increasing number of lymphocytes can be identified. Autoradiographic findings There is only a small percentage of DNA-synthesizing connective-tissue cells at the infarction site and at the infarction border (figs. 3 and 4), which, however, is still slightly increased 21 days post operationem.
-
234557
9
11
-
I- -
14
-
-0- -
17
-
-
_....
PostoDeratlve
21 UberL~benszeit (in Tagen)
Fig. 3. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction sites in the hearts of rats with a normal metabolism (---) and of hypothyroid (---------) rats in dependence on the postoperative survival time.
5
-e- - e - - - - e - - __ e • ~~~~..._~~........-......,...---,.---~--~---_..>
121 2 3 4 5 6 7
9
11
14
17
Postoperative
21 Ubertebenszeit CinTagen)
Fig. 4. Content of DNA-synthesizing connective-tissue nuclei (in %) at the infarction borders in the hearts of rats with a norma.l metabolism (---) and in hypothyroid (---------) rats in dependence on the postoperative survival time.
134
12 hours after the coronary artery ligature has been performed no DNA synthesis can yet be observed in the connective-tissue nuclei. Between the 3rd and the 5th postoperative day there is evidence of a proliferation peak occurring at the infarction site and at the infarction border. Cell proliferation is by mitosis the quotients varying between the values 8.5 and 12.6. On the 2nd postoperative day a generation cycle of the connective-tissue cells at the infarction site lasts approximately 72 hours. Also, on the 2nd postoperative day the cells belonging to the infarction border are involved more intensively in the collagen fibre formation than are those of the infarction site. At the infarction site the synthesis peak for collagen precursors occurs on the 4th postoperative day. In hypothyroid rats the release of tropocollagen by the connective-tissue cells is distinctly inhibited (table 1). Discussion
In hyperthyroid rats the number of DNA-synthesizing connective-tissue cells at the infarction site and at the infarction border Clearly exceeds that of control animals. The synthesis phase is shorter. In animals with hyperthyroidism wound healing after experimental myocardial infarction is clearly accelerated as can be seen from the collagen synthesis after 3H-proline administration. The considerably accelerated release of tropocollagen by the fibroblasts is notable. The increased turnover of radioactive sulphate in hyperthyroidism as observed by DZIEWIATKOWSKI (1957) and BOSTROM (1960) also indicates an enhanced mesenchymal metabolism. The stimulating effect of thyroxine on the metabolism has repeatedly been described by other authors who reported that it activates oxygen consumption, and that the protein synthesis is increased which may even lead to the development of cardiac hypertrophy (VAN LIERE et al. 1969, THYRUM et al. 1970). In our tests, too, the great number of DNA-synthesizing connective-tissue cells in the ventricular sections remote from the infarction site suggests the incipient development of cardiac hypertrophy. In view of the high percentage of tropocollagen-synthesizing connective-tissue cells it seems probable that the supporting fibrous structure is being reinforced. The preferential involvement of one half of the heart as found by GOLBER et al. (1969) and VAN LIERE et al. (1969, 1971) with regard to the right ventricle, has not been observed by us. Like BEZNAK (1962) and COHEN et al. (1966) we have the opinion that it is mainly due to its metabolic effect that thyroxine causes a growth of the cardiac muscle mass, whereas changed hemodynamics play but a secondary role. Due to the proliferation of the intima of medium-size twigs of coronary vessels located at the infarction border there is an insufficient blood supply in this muscular region. The effect of thyroxine which produces an increase in the metabolic rate, very quickly leads to oxygen deficiency in the marginal areas which first affects the more sensitive muscle cells. There so-called sub-infarctions develop which can be demonstrated already on the 5th postoperative day. The development of necroses is preceded by a reduction or decoupling of oxidative phosphorylation and by a decrease in ATP and ADP (MEDOVAR 1964, 1968). Myocardial necroses after thyroxine application were observed by HALL and NELSON (1968). As evidenced by the findings obtained after 3H-proline application, the development of granulation tissue at the infarction site and at the infarction border is reduced in hypothyroid rats as compared with control animals. On the 4th and 8th postoperative day the release of tropocollagen from the fibroblasts is delayed. The number of DNA-synthesizing connective-tissue cells is clearly reduced. Also, the metabolism of the connective-tissue cells in the ventricular sections remote from the infarction site as well as that of all muscle cells is inhibited to varying degrees. Our findings coincide well with the observations made by CHEEK et al. (1965) and v. KNORRING (1970). Apart from a delayed growth of the muscles of the extremities CHEEK et al. (1965) discovered in rats a decrease in the collagen content of these muscles. VON KNORRING (1970) was able to demonstrate a reduction of the hydroxyproline content in the hearts of hyperthyroid rats. The decrease in the succinic dehydrogenase activity as discovered by NANIKAWA et al. (1960) also indicates a reduced metabolic rate.
135
The reduction of the heart weights as observed after thyroidectomy (KOCHAKIAN et aI. 1954) coincides well with the findings by SOKOLOFF et aI. (1963, 1968) and by GARREN et aJ. (1967) who detected in liver cell homogenates a reduced amino acid incorporation. Also, the development of cardiac hypertrophy as a consequence of experimental aortic stenosis can be prevented by thyroidectomy (BEZNAK and HAJDU 1946). The granulous-filamentous mass observed in the granulation tissue of the heart in the presence of hypothyroidism has repeatedly been reported (GUPTA et aI. 1971). It consists of mucopolysaccharides rich in hyaluronic acid (v. KNORRING 1970). A reduced incorporation of radioactive sulphate into the chondroitine-sulphuric acid of cartilage and -skin has been demonstrated by DZIEWIATKOWSKI (1957).
Literature BEZNAK, M., Cardiovascular effects of thyroxine treatment in normal rats. Canad. J. Biochem. 40 1647-1654 (19~f)' - and I. HAJDU, Uber den Einflu6 der Schilddriise auf die Rolle der- Hypophyse in den Veranderungen der Herzmasse. Schweiz. med. Wschr. 76,390-393 (1946). BOSTROM, H., Einige Aspekte zum Metabolismus der Mucopolysaccharide. In: W. H. HAUSS und H. LOSSE, Struktur und Stoffwechsel des Bindegewebes. 2. Sympos. an der Med. Universitats-Klinik Miinster/Westf. Thieme, Stuttgart 1960. CEREMUZYNSKI, L., J. KUCH und K. TYMINSKA, Klinischer Verlauf des Herzinfarktes und Schilddriisenfunktion. Z. ges. inn. Med. 26, 282-284 (1971). CHEEK, D. R, G. K. POWELL and R E. SCOTT, Growth of muscle mass and skeletal collagen in the rat. Bull Johns Hopkins Hosp. 116, 387-395 (1965). COHEN, J., J. M. AROSSTY and H. G. ROSENFELD, Determinants of thyroxine-induced cardiac hypertrophy in mice. Circula,t. Res. 101), 69-74 (1966). DZIEWIATKOWSKI, D. D., Synthesis of sulfomucopolysaccharides in thyroidectomized rats. J. expo Med. 101), 69-74 (1957). GARREN, L. D., A. P. RICHARDSON and R M. CROCCO, Studies on the role of ribosomes in the regulation of protein synthesis in hypophysectomized and thyroidectomized rats. J. bioI. Chern. 242, 650-656 (1967). GOLBER, L. M., K. A. ANANEVA, V. J. KANDROR, 1. V. KRUKOVA and A. V. NEGOWSKAJA, Snatschenije magnija b predupreschoenii metablllitscheskich rasstroistw w miokarde pri tirectoksikose. BjuI!. eksp. Bio!. Med. 3, 23-26 (1969). GUPTA, M. P., S. KIM, J. KANG, L. SHERMAN, H. D. KOLODNY and R 1. HAMBY, Isolated THS deficiency presenting as myxedema, heart disease. JAMA 217, 205-207 (1971). HALL, R J., and W. P. NELSON, Thyroid heart disease. Postgrad. Med. 44, 127-132 (1968). KNORRING, J. VON, Myoca,rdia.I colla,gen in Hyper- and hypothyroid rats and guinea pigs. Ann. Med. fixp. Fenn. 48, 1-7 (1970). - Analysis of myocardial mucopolysaccharides in hyper- and hypothyroid rats a,nd guinea pigs. Ann. expo Fenn. 4 ,8-13 (1970). KOCHAKIAN, C. D., R A. REED and A. l\f. EISCHEID, Changes in tissue weights and enzymes produced by thyroidectomy and testosterone. Am. J. Physiol. 177, 413-417 (1954). LIERE, E. J. VAN, D. A. SIZEMORE and J. HUNNELL, Size of cardiac ventricles in experimental hyperthyroidism in the rat. Proc. Soc. exper. BioI. 132, 663-665 (1969). - and D. A. SIZEMORE, Regression of cardiac hypertrophy following experimental hyperthyroidism in rats. Proc. Soc. exper. BioI. 136, 645-648 (1971). MEDOVAR, E. N., 0 soderzanii ATP i prodnktov so ra.spada v skeletnych i serdecnoj myacach pri gipertireoze. Ukr. biochem. Z. 36, 253-256 (1964). - und E. M. POPOVA, Okisitelnoe fosforilirovanie v mitochondrija,ch serdecnoj myscy krolikov pri eksperienta.lnom gipertireoze. Ukr. biochem. Z. 40, 39-43 (1968). NANIKAWA, R, T. MIKI and H. HAMACKA, Studies on the succinic dehydrogenase of ca,rdia,c muscle. Quantitative changes in normal and abnormal thyroid functions. Nageya med. J.6, 698-707 (1960). SOKOLOFF, L., S. KAUFMAN, P. L. CAMPBELL, C. M. FRANCIS and H. C. GELBEIN, Thyroxine stimulation of amine add incorporation into protein. J. bioI. Chern. 238, 1432-1437 (1963). - P. A. ROBERTS, M. M. JANUSKA and J. E. KLINE, Mechanism of stimulation of protein synthesis by thyroid hormones in vivo. Proc. nat. Acad. Sc. (Wash.) 60, 652-659 (1968). THYRUM, P. T., E. M. KRITCHER and R J. LUCHr, Effect of L-thyroxine on the primary structure of cardiac myosin. Biochim. biophys. Acta (Arnst.) 197, 335-336 (1970). Author's address: Dr. sc. med. D. KRANZ, Pathologisches Institut der Humboldt-Universitat, Bereich Medizin (Charite), DDR - 104 Berlin, Schumannstra6e 20/21.
136