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Morphometric study of myocites in the myocardial septum of the diabetic rat fetus

Menezes Honório S.; Martins Cristiano; Belló André; Barra Marinez; Zimmer Lúcia P.; Zielinsky Paulo.

Animal Lab., Research Unit, Institute of Cardiology
University Cardiology Fundation
Porto Alegre, Brasil.

Abstract
Introduction
Material and Methods
Results
Discussion
Conclusions
References

Abstract
The frequent occurence of prenatal hypertrophic ventricular septum in fetuses of diabetic mothers has been widely reported.
This experimental study was carried out to test the hypothesis that the area occupied by nuclei myocites profile of the ventricular septum are greater in fetuses from diabetic mothers than in those from normal pregnancies.
Diabetes was induced in 5 pregnant Wistar rats (30 fetuses), at day 8 after conception, by 50 mg/kg IP of streptozotocin. Five normal pregnant Wistar rats, made up the control group (20 fetuses). The morphometric data were obtained by a computer-assisted method applied to the measurements of myocytes nuclei diameter and nuclei myocytes area. Statistical analysis utilized Student’s t test and Kruskal-Wallis test.
Mean nuclei area of septum myocites was 14.70 m m2 in the "normal" group and 21.43 m2 in the "diabetic" group (p<0.001). The nuclear celular diameter was 2,55 m m in the "normal" group and 4,29 m m in the "diabetic" group (p<0.001)
We conclude that the significant difference between the two groups, for each analized feature, demonstrate the presence of celular heart hypertrophy in fetuses of diabetic mothers.

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Introduction:

Hypertrophic heart from newborn of diabetic mothers appeared in the literature in 1937 1. Miller 2 (1943) and Driscoll 3 (1960) also described heart macrosomia.
The diagnostic of human hypertrophic cardiomyopathy has long been reported using echocardiography 4,5 and septal hypertrophy also has been studied in our institution6.
Experimental studies performed comparing hearts of human and rats embryos stablished relations on stages development 7,8,9.
Cardiac myocites in the rat retain some capacity for proliferation up to about the age of weaning 10, although significant hyperplasia mey cease earlier 11. Average size of myocites also increases in parallel with postnatal body growth 12.
Mitotic activity in mammalian cardiac myocites, wich is high during the earliest stages of histogenesis, declines rapidly during the postnatal period, and cell enlargement becomes the main growth mechanism 13.
The rat has become the major species for diabetes-pregnancy model wich can be made by streptozotocin injection 14.
Recent stereological methods for cell biology has been used in pathological research 15,16,17 and biological morphometry.
The aims of this study were to investigate the rat foetal heart nuclei myocites morphology in experimental diabetic mothers.

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Material and Methods:

Experiments were performed on fetuses of Wistar rats (Animal Quarter House of the Institute of Cardiology, Porto Alegre, Brazil). The pregnants rats were housed in individual cages with free acces to tap water abd standard rat food (Nuvilab, PR). Animals were made diabetic by a single injection of streptozotocin (50 mg/kg IP, sigma Chemical Co.). Streptozotocin was dissolved in citrate buffer (pH 4.5) and injected within 5 minutes18.
Diabetes was induced in 5 pregnat Wistar rat (30 fetuses), at day 8 after conception, by 50 mg/kg IP of streptozotocin. Five normal pregnant Wistar rats made up the control group (20 fetuses). The morphometric data were obtained by a computer-assisted method applied to the measurement of myocites nuclear diameter and nuclei myocites area at a magnification of 40x.
The hearts from fetuses were excised at day 19 or 20 after conception19 and were fixed in formahaldehydro and embedded in wax.
Each fetus heart were sectioned for light microscopic nuclear counting, area and diameter measurement, near atrioventricular line sectioning septum myofibers longitudinally at nominal thicknesses of 4 m m using a microtome. The sections were mounted on glass slides, and stained with hematoxylin to enhance nuclear contrast.
A square tissue area equal to 11519 m m2 at center of interventricular septum and 4 laterally adjacents fields were examined in each heart section. The image was analysed through brightness and color contrast using a LEICA Q500MC computer-assisted microscope.
The protocol was approved by the institutional ethic committee.
Data Analysis
Data are reported as mean ± SD, and Student´s t test and Kruskal-Wallis was used to compare values obtained between groups. Changes were considered signficant at a value of p<0,5 for all tests20.

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Results:

Table 1 shows the nuclear myocites area of the five interventricular septum fields sampling (central, right inferior, left inferior, right superior, left superior).

Table 1
Nuclear myocites area of the five interventricular septum
fields sampling (central, right inferior, left inferior, right
superior, left superior).

table1.gif (3862 bytes)

Table 1 shows results of the celular counting according each microscopic field.

Table 2
Results of the celular counting in each
interventricular septum fields sampling

table2.gif (2629 bytes)
Total cells counted: 18.799.

Figure 1 demonstrates the nuclear profile diameter of each group (normal and diabetic)

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Fig. 1:nuclear profile diameter (µ )

All of the data were pooled, and the values shown should be considered representative of each interventricular septum.

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Discussion:

The embryonic period of the rat is 17.5 day and the fetal period is from 17.5 to 21 days. The stage 23 occurs at 17.5 day of development, at this time the verti-coccix lenght is 16 mm 21. At this age the interventricular septum is relatively thick and located exactly between the ventricules differing from humans. The population of myocites shows 3% of myocites counting, which is less than in the dog (60 to 80% of binucleate cells) 22.
From the present data, the interventricular septum nuclear myocite diameter increase was clearly evident among fetuses of diabetic rat mothers in comparison with fetuses of non diabetic mothers. The 3.17 m m nuclear diameter described by Anversa et al 12 is from newborn not from fetus data. This study demonstrated greater nuclear diameter in the fetus than in newborn previously reported 12. The dimentions of the average increased steadly from 1 to 5 days postnatal results mainly by cytoplasmatic volume and from 5 to 11 days results by increased frequency of binucleate cells 23. Although any of these effects should produce an increase in the measured parameter the results show no indication of such a change.
The interpretation of morphometric determination with respect to tissue properties in the living state involves some variation of relative and absolute volumes during the process of fixation. The fixative utilized here provided excellent structural preservation of fetal myocardium, there was no qualitative indication of either tissue or cell swelling in the tissue measured in the present study. The computer-assisted method identified the binucleate cells counting the real number of myocitesper area been equal to use the disector method 8.
The variation of the area average by the nuclear myocites of the interventricular septum among the square tissue area demonstrated a statistical difference (p<0.001) between the nuclear area from diabetic and non diabetic group, confirming celular hypertrophy in the fetuses from diabetic rat mothers, feature also reported in diferent organs by Naeye 24. The same feature does not occurs in hypertensive cardiomyopathy, in wich the myocite nuclei hyperplasia is the main mechanism of growth 25.
The fetal heart from diabetic mothers in this study seems to enlarge as a mature heart in wich the celular growth is the process to reach the normal size. The cardiac muscle cell increases in size during postnatal growth. As a consequence, the nuclear density decreases (the number of myocardial cell nuclei per cross-sectional area of cell)26.
The celular diameter increases from 5 µm in the newborn to 16 µm in the adult rat27. This ventricular celular diameter increasing occurs in mammalian, which does not occurs in fowl when cell proliferation mechanism follow the body growth26.
Human embryos heart wall is a relatively thick structure from stage 19 through the end of embryonic period7,8. In the rat this process occurs in approximately the same stage9.
The present study was conducted in fetal period when the celular development is more stable and the myocardium is sensitive to diabetes factors28, as seen in the human pregnancy.

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Conclusions:

We conclude that the significant difference (p<0.001) between morphometric data from cardiac muscle cell of fetuses from diabetic and normal pregnant rats demonstrate the presence of celular heart hypertrophy in fetuses of diabetic mothers.

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References

1. Hurwitz D, Irving FC. Diabetes and pregnancy. Am J Med Sci 1937;194:85-92
2. Miller HC, Wilson HM. Macrosomia, cardiac hypertrophy, erytroblastosis, and hyperplasia Ef the islets of Langerhans in infants of diabetic mothers. J Pediatr 1943;23:251-66
3. Driscoll SG, Benirschke K, Curtis GW. Neonatal deaths among infants of diabetic mothers. Am J Dis Child 1960;100:818-35
4. Gutgesell HP, Mullins CE, Gillette PC. Transient hypertrophic subaortic stenosis in infants of diabetic mothers. J Perinatol 1976;89:120-5
5. Wolfe RR, Way GL. Cardiomyopathies in infants of diabetic mothers. Johns Hopkins Medical J 1977;140:177-80
6. Zielinsky P. Role of prenatal echocardiography in the study of hypertrophic cardiomyopathy in the fetus. Echocardiography 6(8):661-668, 1990
7. Mandarim-de-Lacerda CA. A multivariate analysis of cardiac growth in human embryos: Endocardial cushions and ventricular myocardium. Cardiov Res 1991b;25:855-60
8. Mandarim-de-Lacerda CA. Growth allometry of the myocardium in human embryos (from stages 15 to 23). Acta Anat 1991a;141:251-6
9. Xavier-Vidal R, Pimentel-de-Souza RM, Mandarin-de-Lacerda CA. Development of the coronary arteries in staged embryos of rat. Quad Anat Pr 1991;S47:35-41
10. Dowell RT, McManus RE. Pressure-induced cardiac enlargement in neonatal and adult rats. Left ventricular functional characteristics and evidence of cardiac muscle cell proliferation in the neonate. Circ Res 1978;42:303-310
11. Claycomb WC. Biochemical aspects of cardiac muscle differentiation. Deoxyribonucleic acid synthesis and nuclear and cytoplasmic deoxyribonucleic acid polymerase activity. J Biol Chem 1975;250:3229-35
12. Anversa P, Olivetti G, Loud AV. Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. Circ Res 1980;46:495-502
13. Anversa P, Fitzpatrick D, Argani S, Capasso JM. Myocyte miotic division in the aging mammalian rat heart. Circ Res 1991;69:1159-64
14. Hellerstrom C, Swennw I, Erikson UJ. Is there an animal model for gestacional diabetes? Diabetes 1985;34:28-31
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16. Gundersen HJG, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, West MJ. The new stereological tools: Dissector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988b;96:857-81
17. Mandarim-de-Lacerda CA. Métodos quantitativos em morfologia. Rio de Janeiro: UERJ, 1995
18. Maeda CY, Fernandes TG, Timm HB, Irigoyen MC. Autonomic dysfunction in short-term experimental diabetes. Hypertention 1995;26(6):1100-104
19. Calderon IMP, Rudge MVC, Brasil MAM, Henry MACA. Diabete e gravidez experimental em ratas: I - Indução do diabete, obtenção e evolução da prenhez. Acta Cir Bras 1992;7(4):142-6
20. Zar JH. Biostatistical analysis. Engleewood Cliffs: Prentice-Hall, 1984.
21. Butler H, Juurlink BHJ. An Atlas for Staning Mammalian an Chick Embryos. Florida: CRC Press Inc, 1987
22. Webb S, Brown NA, Anderson RH. The structure of the mouse heart in late fetal stages. Anat Embryol 1996;194:37-47.
23. Katzberg AA, Farmer BB, Harris RA. The predominance of binucleation in isolated rat heart myocites. Am J Anat_1977;149:489-500.
24. Naeye RL. Infants of diabetic mothers - a quantitative, morphologic study. Pediatrics 1965;35:980-8.
25. Anversa P, Thomas P, Sonnenblick EH, Olivetti G, Capasso JM. Hypertensive cardiomyopathy. Myocite nuclei hyperplasia in the mammalian rat heart. J Clin Invest 1990;85:994-97
26. Zak R. Cell proliferation during cardiac growth. Am J Cardiol 1973;31:211-19.
27. Rakusan K, Korecky B, Roth S, Poupa O. Development of the ventricular weight of the rat heart with special reference to the early phases of postnatal ontotogenesis. Physiol Bohemoslov 1963;12:518-24.
28. Zielinsky P, Hagemann LL, Daudt LE, Behle I. A pre and postnatal analysis of factors associated with fetal myocardial hypertrophy in diabetic pregnancies. J Mat Fet Invest 1992;(2):163-7.

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Questions, contributions and commentaries to the Authors: send an e-mail message (up to 15 lines, without attachments) to heartfail-pcvc@pcvc.sminter.com.ar , written either in English, Spanish, or Portuguese.

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Update
Dic/02/1999