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Stereology of the myocardium in human foetuses
Early Human Development 48 (1997) 249–259
Stereology of the myocardium in human foetuses a, Carlos Alberto Mandarim-de-Lacerda *, Marcelo Barbosa dos Santos a , Patrice Le Floch-Prigent b , c , Franc¸oise Narcy c a
Laboratory of Morphometry and Cardiovascular Morphology, Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil b ´ ` , U.F.R. de Medecine ´ Institut d’ Anatomie, UFR Biomedicale des Saints-Peres Paris-Ouest, Paris, France c ˆ Service Central d’ Anatomie Pathologique, Hopital Cochin, Paris, France Received 9 May 1996; revised 7 October 1996; accepted 19 November 1996
Abstract The rate of cellular proliferation and hypertrophy of the cardiac myocytes in the human perinatal period is still controversial. This work uses stereology to evaluate the prenatal quantitative changes of the myocardium. The hearts of 36 human foetuses, ranging from the 2nd trimester to the 3rd trimester, were studied. Fifteen random microscopic fields were analyzed in each heart. The following stereological parameters were determined: Vv[myocyte] and Vv[interstitium] (the volume densities of the cardiac myocyte and interstitium, respectively) and the Nv[myocyte] (the numerical density of the cardiac myocytes). The total number of myocytes (N[myocyte] ) and the mean myocyte volume (V[myocyte] ) were also determined. All differences between the second and the third trimester of gestation, tested with the Mann-Whitney test, were statistically significant (P , 0.05). The Vv[myocyte] decreased 8.69% and the Vv[interstitium] increased 49.83% in this period. Simultaneously, the Nv[myocyte] decreased 16.64%, the V[myocyte] increased 16.39%, the cardiac weight increased 366.67% and the N[myocyte] increased 272.06%. In conclusion, during the last two gestational trimesters the human heart increases in weight more than 4.5 times, the volume density of myocytes decreases while the volume density of the cardiac interstitium increases. The numerical density of myocytes per myocardium volume decreases but the myocytes became greater in mean volume (more than 16%). 1997 Elsevier Science Ireland Ltd. Keywords: Myocardium; Human foetuses; Growth; Stereology *Corresponding author, Universidade do Estado do Rio de Janeiro, Centro Biomedico, ´ Instituto de ´ Biologia, Laboratorio de Morfometria e de Morfologia Cardiovascular, Av. 28 de Setembro, 87 (fundos), 20551-030 Rio de Janeiro, RJ, Brasil. Fax: 155 21 5876416 or 5673927; e-mail: [email protected]. 0378-3782 / 97 / $17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0378-3782( 96 )01863-4
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1. Introduction Cardiac myocytes are the largest cells found in the myocardium and occupy 75% of its structural space but constitute only one third of the cell population of the mature myocardium. All remaining cells are in the cardiac interstitium, which consists predominantly of fibroblasts and mesenchymal cells, but also includes endothelial and smooth muscle cells, macrophages and mast cells [1,2]. During the embryonic period, mitotic activity is unequal along the myocardium wall, being more active in the peripheral compact layer than in the inner trabecular layer [3]. In the early post-somitic period the human myocardium has a relatively small number of small myocytes, in the late post-somitic period it is composed of large and relatively abundant myocytes. The conspicuous increase in the ventricular myocardial volume at the end of the post-somitic period seems not to be related to the increase in the interstitial portion of the myocardium. These arguments suggest both the enlargement and the division of the myocytes during the post-somitic period of the human embryonic period proper [4]. The enlargement of the embryonic heart in mammals is largely dependent on an increase in myocyte number. This continues until shortly after birth, when cardiac myocytes lose their proliferative capacity and acquire the terminally differentiated phenotype of adult cardiac muscle cells [5,6]. Soon after birth, the myocytes no longer undergo hyperplasia and further muscle growth is by cellular hypertrophy [7,8]. However, a similar quantitative study in rats suggests a high mitotic activity in the myocardium during prenatal life and after birth [9]. The effort to establish quantitative parameters of the developing heart is recognised to be of medical and biological importance [4,5,7,10–16]. The purpose of the present work was to investigate possible quantitative differences in human myocardium composition in the last two trimesters of gestation.
2. Materials and methods
2.1. Sample We studied a total of 36 hearts of sex pooled human foetuses. Twenty-six foetuses ranged from 12–23 weeks of gestation (second gestational trimester) and 10 foetuses ranged from 25–36 weeks of gestation (third gestational trimester). The distribution of the foetuses by gestational age group is shown in Table 1. Foetuses were analyzed and the hearts were dissected in the Department of ˆ Pathology (Hopital Cochin, Paris). Only foetuses without apparent growth retardation and hearts considered normal were studied. The foetuses had a normal karyotype. The left ventricular wall and interventricular septum are regions of the heart closely associated with cardiac pump function. So, several fragments of these regions were cut from the endocardium to the pericardium and fixed for 24–36 h by immersion in buffered formaldehyde at 4% at room temperature. The samples were dehydrated in ethanol, embedded in paraplast and cut into sections 5 m m thick. Deparaffinised
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Table 1 Distribution of the foetuses by age group (weeks of gestation) Age group (weeks) 12.1 to 18.1 to 24.1 to 30.1 to Total
18 24 30 36
Frequency
%
13 13 5 5 36
36 36 14 14 100
sections were stained by the haematoxylin and eosin and Gomori’s trichrome methods.
2.2. Stereology The myocardial quantification was performed manually with light microscopy (Laboratory of Morphometry and Cardiovascular Morphology, Rio de Janeiro). We used the optical combination of a Nikon CFW eyepiece ( 3 10) and oil planachromatic immersion objective ( 3 100) in a Nikon Optiphot microscope. Unbiased stereological estimates were obtained from isotropic uniform random sections of the myocardium. To generate these sections the heart was sectioned in an isotropic uniform random set of three perpendicular sections for each heart analyzed (an orthogonal triplet probe, ortrip) [17]. We used the disector method to obtain the volume and numerical densities of the cardiac myocytes [18]. The optical disector was defined with two parallel fields in the same section separated by a distance of 5 m m: the look-up and look-down planes. A test lattice mounted in the eyepiece established a known surface test area [19–21]. So, we sampled all myocyte nuclei seen in focus only in the look-up plane. They should be partly or totally inside the frame provided they did not in any way intersect the left or inferior exclusion edges or their extensions [22]. The displacement of the microscope stage in the direction of the optical axis defined the upper and lower faces of the section, the look-up and look-down planes [23,24]. The volume density (relative volume) of the myocardial parts was determined by counting test points. For stereological quantification the myocardium was considered to consist of cardiac myocytes and the cardiac interstitium. The volume density of the myocyte (the relative volume of the myocyte in the myocardium, Vv[myocyte] ) was taken to be the cardiac cell including the cardiac myocyte nucleus. The volume density of the cardiac interstitium (Vv[interstitium] ) was considered to include the connective tissue, non-myocyte cells, vessels and nerves. Fifteen random look-up fields were studied in each heart. A point count was made microscopically using a 64-points eyepiece graticule (PT 5 64). According to Delesse’s principle, it was sufficient to count the number of the test-points lying inside structures (P[structure] ) in order to estimate the volume density (Vv) [25,26]. P[structure] Vv[structure] 5 ]]] PT
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The numerical density of the cardiac myocytes (number of myocytes per volume of myocardium, Nv[myocyte] 1 / mm 3 ) was determined with 15 random disector pairs for each specimen [19]. For reasons of efficiency, one nucleus was considered to represent one myocyte. Q 2 A is the number of the myocyte nuclei in the test-area appearing in an unbiased counting frame of 1600 m m 2 on the one-slice plane; disector is the volume of the disector (V[disector] ), that is, the product of the test-area by the disector thickness. Q2 A Nv 5 ]]] V[disector] The total number of cardiac myocytes (N[myocyte] ) was calculated as the product of Nv[myocyte] and the cardiac volume (V[heart] ). The V[heart] was considered as the cardiac weight (W[heart] ) since specific gravity ( g) of isotonic saline is about 1.0048 and the W[heart] was measured using Scherle’s method [25,27,28]. W[heart] V[heart] 5 ]] g
and
N[myocytes] 5 Nv[myocytes]V[heart]
2.3. Statistical analysis The coefficient of error (CE) for the stereological estimates was calculated as the ratio between the standard error and the mean [26]: Quantitative differences of the stereological parameters were analyzed with the non-parametric two-sided Mann-Whitney test with the significant level a of 0.05 [29]. Foetuses were compared by age group, i.e. second vs. third gestational trimester.
3. Results Table 2 shows the results of the stereology. All differences between the second and the third trimester of gestation were statistically significant (P,0.05). In this period the Vv[myocyte] decreased from 85.18 to 77.78% (28.69%) and the Vv[interstitium] Table 2 Mean6standard error of the mean of the stereological parameters of the foetal myocardium Stereology
Second trimester
Third trimester
P
Cardiac weight (g) Vv[myocyte] (%) Vv[interstitium] (%) Nv[myocyte] (10 4 / mm 3 ) V[myocyte] (mm 3 ) N[myocyte] (10 9 )
1.9560.16 85.1860.92 14.8360.92 68.8662.40 1214.386134.23 1.3660.18
9.1060.87 77.7860.93 22.2260.93 57.4062.39 1412.316151.21 5.0660.61
,0.001 0.001 0.001 0.006 0.03 ,0.001
P is the probability of the difference between foetuses of the second and third trimesters to be different from zero (Mann-Whitney test). Vv is the volume density, Nv is the numerical density, V is the mean volume, N is the total number.
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increased from 14.83 to 22.22% (149.83%). Fig. 1 compares the changes in the composition of the foetal myocardium in the last two gestational trimesters. The Nv[myocyte] decreased from 68.86 to 57.40 (10 4 ) / mm 3 (216.64%) and the V[myocyte] increased from 1213.38 to 1412.31 m m 3 (116.39%). Fig. 2 shows the convergence of the trends of these estimates. From the second to the third trimester the Nv[myocyte] decrease while the V[myocyte] increase. In human foetuses of the last two trimesters of gestation the cardiac weight increased from 1.95 to 9.10 g (1366.67%) and the N[myocyte] increased from 1.36 to 5.06 (310 9 ) myocytes (1272.06%). Fig. 3 shows that the pattern of growth of the trends of these estimates is divergent. Coefficients of error (CE) can be calculated from data in Table 2. The CE for the Vv[myocyte] estimates ranged from 1.08 (2nd trimester) to 1.20% (3rd trimester), and for the Vv[interstitium] estimates ranged from 4.19 (3rd trimester) to 6.20% (2nd trimester). For the Nv[myocyte] estimates the CE ranged from 3.49 (2nd trimester) to 4.16% (3rd trimester), and for the N[myocyte] ranged from 12.06 (3rd trimester) to
Fig. 1. Bar graph comparing the volume densities (Vv) of the human foetal cardiac interstitium and myocytes. From the second to the third trimester of gestation the proportion of the myocytes decreased while the proportion of the interstitium increased.
Fig. 2. Quantitative analysis of the human foetal myocardium: (a) trend of the numerical density of cardiac myocytes (Nv[myocyte] ) and (b) trend of the mean myocyte volume (V[myocyte] ) in the last two trimesters of gestation. These trends are convergent in this period.
254 C. A. Mandarim-de-Lacerda et al. / Early Human Development 48 (1997) 249 – 259
Fig. 3. Quantitative analysis of the human foetal myocardium: (a) trend of the cardiac weight and (b) trend of the total number of myocytes (N[myocyte] ) in the last two trimesters of gestation. These trends are divergent in this period.
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13.24% (2nd trimester). The V[myocyte] had CE ranged from 10.71 (3rd trimester) to 11.05% (2nd trimester).
4. Discussion Some cardiac myocytes can be binucleated during prenatal development [10]. Binucleation of the cardiac myocytes could lead to inconsistencies in the determination of Nv[myocyte] if the rate of binucleation was high. However, the study of the mitotic activity of cultured cardiac muscle cells demonstrated that only 10–15% of cells fail to complete cytokinesis and thus to form binucleated cells. The remaining 85–90% of the mitotic cardiac cells complete cytokinesis and produce two daughter cells [30]. The optical disector seems to be an accurate method for determining the number of nuclei in the cardiac myocytes. It permits the definition within the thickness of the section avoiding the over-counting of binucleated cells [19]. Austin et al. [16] commented that values measured on fixed and processed tissue are biased in consideration of fresh material. However, like these authors, we found small CE for estimates (,15%), suggesting that the intrasubject error introduced by systematic sample was low, and the relatively high accuracy of estimates. Tokuyasu [3] reported that mitotic activity is unequal along the myocardium wall during the embryonic period (more activity in the peripheral compact layer than in the inner trabecular layer). In the human this pattern of growth was never observed in the foetal period and this study has not considered different parts of the myocardium in the selection of the sample for analysis. The growth of the heart, until |2 weeks of age, is due to the increase in cell number in early neonates of the rat and in the human foetal and postnatal period. Human cardiac growth after 2 weeks of age is primarily caused by an increase in myocyte size rather than number [7,8]. The present study demonstrated that the tendency of the Nv[myocyte] decrease with the simultaneous V[myocyte] increase can be observed in the last two gestational trimesters in man. In human foetuses of the last two trimesters of gestation the cardiac weight increased from 1.95 to 9.10 g (1366.67%) and the N[myocyte] increased from 1.36 to 5.06 (310 9 ) myocytes (1272.06%). Fig. 3 shows that the pattern of growth of the trends of these estimates is divergent. Austin et al. [16] used a stereological model and established a protocol that can be employed to test for patterns of cardiac growth in normal and pathological human specimens with archival material. This is similar to the present study and our previous studies about myocardial quantification in primates [31] and cardiac growth [4,14]. In the left ventricle of two normal hearts from neonates, Austin et al. [16] found a mean absolute number of myocytes of 5.0 (10 8 ) / cm 3 , in these neonates the ventricular weight was 14.4 and 18.7 g (mean 16.6 g). These figures are greater than the ones found in the present study of foetuses. Foetuses of the third trimester (present study) and neonates [16] are not completely comparable because of some methodological
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differences, for example, cardiac weight vs. ventricular weight. However, Austin et al. [16] estimated the mean myocyte volume varying between 1220.0 and 2630.0 m m 3 that is of the same magnitude as our estimates (1412.316151.21 m m 3 in foetuses of the third trimester). Villar and Mandarim-de-Lacerda [32] observed an important catch-up of growth in both total length and volume of the capillaries between rat foetuses of age 18.5 and 20.5 days (equivalent to the third trimester in man). In addition, there was an increase in the total length in late neonates while the volume had significantly decreased immediately after birth but improved its size in neonates of 1 week. Absolute quantities of the myocardial capillaries have a tendency to increase during the foetal and neonatal periods because of the cardiac growth. The diameter of the capillaries, however, was uniform during the foetal and neonatal periods. This agrees with the present study that considered the vessels included in the cardiac interstitium had increased almost 50% in the last two gestational trimesters. Mandarim-de-Lacerda [11] and Mandarim-de-Lacerda and Souza [14] studied the growth of the myocardium in human and rat embryos. They noted different trends of growth with the breakpoint around Carnegie stage 20. After this stage, that corresponds to late embryos, the growth in volume of the myocardium is greater than in early embryos. Mandarim-de-Lacerda and Pessanha [9] added that, in rat, the major difference in Nv[myocyte] occurs between embryos and foetuses-neonates while the major difference in Vv[myocyte] and Vv[interstitium] occurs between neonates and embryos-foetuses. On the other hand, the increase of the N[myocyte] is greater than the increase in cardiac weight comparing foetuses and neonates, indicating that great cell proliferation takes place in this period. In the present study the myocardium was studied stereologically. We have taken two groups of foetuses (2nd vs. 3rd trimester) and compared quantification of myocytes and interstitium. Of course, these groups are broad and this can prejudice the comparison between them. The choice to study only two groups is due to the small sample of the foetuses in the 3rd trimester and forbids a greater division into this period. However, the present results are statistically consistent enough to allow an analysis of the trends in growth in the last two human gestational trimesters. Considering the cardiac myocyte, the decrease of the cellular proliferation with the increase of the cellular volume is illustrated in Fig. 2 and Fig. 3. These figures show the trends of the developmental changes of the stereological estimates. From the second to the third trimester of gestation the foetal myocardium grows with convergence of the numerical density of myocytes and the mean myocyte volume. In the same period, the trends of the cardiac weight and the total number of myocytes are divergent. So, the increase of the cardiac weight is not totally explained by the increase in the number of myocytes but the increase of the mean myocyte volume. In conclusion, during the last two gestational trimesters the human heart increases in weight more than 4.5 times, the volume density of myocytes decreases while the volume density of the cardiac interstitium increases. The numerical density of myocytes per myocardium volume decreases but the myocytes became greater in mean volume (more than 16%).
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Acknowledgments The authors are grateful to Mrs. Ledroux for her technical assistance. This work was funded in part by a grant of the National Council of Research of Brazil (CNPq Proc. no. 52.23.73 / 95.0) and Faperj (E-26 / 170.315 / 95).
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[20] Cruz-Orive, L.M. and Weibel, E.R. (1990): Recent stereological methods for cell biology: a brief survey. Am. J. Physiol., 258, L148–L156. [21] Mayhew, T.M. (1991): The new stereological methods for interpreting functional morphology from slices of cells and organs. Exp. Physiol., 76, 639–665. [22] Gundersen, H.J.G. (1977): Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J. Microsc., 111, 219–227. [23] Konigsmark, B.W. (1970): Methods for the counting of neurons. In: Contemporary Research Methods in Neuroanatomy, pp. 315–340. Editors: W.J.H. Nauta and S.O.E. Ebbesson. Springer-Verlag, New York. [24] Royet, J-P. (1991): Stereology: a method for analyzing images. Prog. Neurobiol., 37, 433–474. [25] Weibel, E.R. (1979): Stereological methods. Practical methods for biological morphometry, vol. 1. Academic Press, London, 415 p. [26] Gundersen, H.J.G., Bendtsen, T.F., Korbo, L., Marcussen, N., Møller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, G., Sørensen, F.B., Vesterby, A. and West, M.J. (1988): Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. Acta Pathol. Microbiol. Immunol. Scand., 96, 379–394. [27] Scherle, W. (1970): A simple method for volumetry of organs in quantitative stereology. Mikroskopie, 26, 57–63. [28] Aherne, W.A. and Dunnill, M.S. (1982): Morphometry. Arnold, London, 205 p. [29] Zar, J.H. (1984): Biostatistical Analysis. Prentice-Hall, Englewood, 718 p. [30] Chacko, S. (1973): DNA synthesis, mitosis, and differentiation in cardiac myogenesis. Dev. Biol., 35, 1–18. [31] Mandarim-de-Lacerda, C.A. and Costa, W.S. (1993): An update of the stereology of the myocyte of the baboon’s heart: analysis of the crista terminalis, interatrial and interventricular septa, and atrioventricular bundle. Ann. Anat., 175, 65–70. [32] Villar, V.C. and Mandarim-de-Lacerda, C.A. (1995): Myocardial microvasculature in foetuses and neonates of rat. Stereological study. Ital. J. Anat. Embryol., 100, 211–218.
Stereology of the myocardium in human foetuses a, Carlos Alberto Mandarim-de-Lacerda *, Marcelo Barbosa dos Santos a , Patrice Le Floch-Prigent b , c , Franc¸oise Narcy c a
Laboratory of Morphometry and Cardiovascular Morphology, Biomedical Center, State University of Rio de Janeiro, Rio de Janeiro, Brazil b ´ ` , U.F.R. de Medecine ´ Institut d’ Anatomie, UFR Biomedicale des Saints-Peres Paris-Ouest, Paris, France c ˆ Service Central d’ Anatomie Pathologique, Hopital Cochin, Paris, France Received 9 May 1996; revised 7 October 1996; accepted 19 November 1996
Abstract The rate of cellular proliferation and hypertrophy of the cardiac myocytes in the human perinatal period is still controversial. This work uses stereology to evaluate the prenatal quantitative changes of the myocardium. The hearts of 36 human foetuses, ranging from the 2nd trimester to the 3rd trimester, were studied. Fifteen random microscopic fields were analyzed in each heart. The following stereological parameters were determined: Vv[myocyte] and Vv[interstitium] (the volume densities of the cardiac myocyte and interstitium, respectively) and the Nv[myocyte] (the numerical density of the cardiac myocytes). The total number of myocytes (N[myocyte] ) and the mean myocyte volume (V[myocyte] ) were also determined. All differences between the second and the third trimester of gestation, tested with the Mann-Whitney test, were statistically significant (P , 0.05). The Vv[myocyte] decreased 8.69% and the Vv[interstitium] increased 49.83% in this period. Simultaneously, the Nv[myocyte] decreased 16.64%, the V[myocyte] increased 16.39%, the cardiac weight increased 366.67% and the N[myocyte] increased 272.06%. In conclusion, during the last two gestational trimesters the human heart increases in weight more than 4.5 times, the volume density of myocytes decreases while the volume density of the cardiac interstitium increases. The numerical density of myocytes per myocardium volume decreases but the myocytes became greater in mean volume (more than 16%). 1997 Elsevier Science Ireland Ltd. Keywords: Myocardium; Human foetuses; Growth; Stereology *Corresponding author, Universidade do Estado do Rio de Janeiro, Centro Biomedico, ´ Instituto de ´ Biologia, Laboratorio de Morfometria e de Morfologia Cardiovascular, Av. 28 de Setembro, 87 (fundos), 20551-030 Rio de Janeiro, RJ, Brasil. Fax: 155 21 5876416 or 5673927; e-mail: [email protected]. 0378-3782 / 97 / $17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0378-3782( 96 )01863-4
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1. Introduction Cardiac myocytes are the largest cells found in the myocardium and occupy 75% of its structural space but constitute only one third of the cell population of the mature myocardium. All remaining cells are in the cardiac interstitium, which consists predominantly of fibroblasts and mesenchymal cells, but also includes endothelial and smooth muscle cells, macrophages and mast cells [1,2]. During the embryonic period, mitotic activity is unequal along the myocardium wall, being more active in the peripheral compact layer than in the inner trabecular layer [3]. In the early post-somitic period the human myocardium has a relatively small number of small myocytes, in the late post-somitic period it is composed of large and relatively abundant myocytes. The conspicuous increase in the ventricular myocardial volume at the end of the post-somitic period seems not to be related to the increase in the interstitial portion of the myocardium. These arguments suggest both the enlargement and the division of the myocytes during the post-somitic period of the human embryonic period proper [4]. The enlargement of the embryonic heart in mammals is largely dependent on an increase in myocyte number. This continues until shortly after birth, when cardiac myocytes lose their proliferative capacity and acquire the terminally differentiated phenotype of adult cardiac muscle cells [5,6]. Soon after birth, the myocytes no longer undergo hyperplasia and further muscle growth is by cellular hypertrophy [7,8]. However, a similar quantitative study in rats suggests a high mitotic activity in the myocardium during prenatal life and after birth [9]. The effort to establish quantitative parameters of the developing heart is recognised to be of medical and biological importance [4,5,7,10–16]. The purpose of the present work was to investigate possible quantitative differences in human myocardium composition in the last two trimesters of gestation.
2. Materials and methods
2.1. Sample We studied a total of 36 hearts of sex pooled human foetuses. Twenty-six foetuses ranged from 12–23 weeks of gestation (second gestational trimester) and 10 foetuses ranged from 25–36 weeks of gestation (third gestational trimester). The distribution of the foetuses by gestational age group is shown in Table 1. Foetuses were analyzed and the hearts were dissected in the Department of ˆ Pathology (Hopital Cochin, Paris). Only foetuses without apparent growth retardation and hearts considered normal were studied. The foetuses had a normal karyotype. The left ventricular wall and interventricular septum are regions of the heart closely associated with cardiac pump function. So, several fragments of these regions were cut from the endocardium to the pericardium and fixed for 24–36 h by immersion in buffered formaldehyde at 4% at room temperature. The samples were dehydrated in ethanol, embedded in paraplast and cut into sections 5 m m thick. Deparaffinised
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Table 1 Distribution of the foetuses by age group (weeks of gestation) Age group (weeks) 12.1 to 18.1 to 24.1 to 30.1 to Total
18 24 30 36
Frequency
%
13 13 5 5 36
36 36 14 14 100
sections were stained by the haematoxylin and eosin and Gomori’s trichrome methods.
2.2. Stereology The myocardial quantification was performed manually with light microscopy (Laboratory of Morphometry and Cardiovascular Morphology, Rio de Janeiro). We used the optical combination of a Nikon CFW eyepiece ( 3 10) and oil planachromatic immersion objective ( 3 100) in a Nikon Optiphot microscope. Unbiased stereological estimates were obtained from isotropic uniform random sections of the myocardium. To generate these sections the heart was sectioned in an isotropic uniform random set of three perpendicular sections for each heart analyzed (an orthogonal triplet probe, ortrip) [17]. We used the disector method to obtain the volume and numerical densities of the cardiac myocytes [18]. The optical disector was defined with two parallel fields in the same section separated by a distance of 5 m m: the look-up and look-down planes. A test lattice mounted in the eyepiece established a known surface test area [19–21]. So, we sampled all myocyte nuclei seen in focus only in the look-up plane. They should be partly or totally inside the frame provided they did not in any way intersect the left or inferior exclusion edges or their extensions [22]. The displacement of the microscope stage in the direction of the optical axis defined the upper and lower faces of the section, the look-up and look-down planes [23,24]. The volume density (relative volume) of the myocardial parts was determined by counting test points. For stereological quantification the myocardium was considered to consist of cardiac myocytes and the cardiac interstitium. The volume density of the myocyte (the relative volume of the myocyte in the myocardium, Vv[myocyte] ) was taken to be the cardiac cell including the cardiac myocyte nucleus. The volume density of the cardiac interstitium (Vv[interstitium] ) was considered to include the connective tissue, non-myocyte cells, vessels and nerves. Fifteen random look-up fields were studied in each heart. A point count was made microscopically using a 64-points eyepiece graticule (PT 5 64). According to Delesse’s principle, it was sufficient to count the number of the test-points lying inside structures (P[structure] ) in order to estimate the volume density (Vv) [25,26]. P[structure] Vv[structure] 5 ]]] PT
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The numerical density of the cardiac myocytes (number of myocytes per volume of myocardium, Nv[myocyte] 1 / mm 3 ) was determined with 15 random disector pairs for each specimen [19]. For reasons of efficiency, one nucleus was considered to represent one myocyte. Q 2 A is the number of the myocyte nuclei in the test-area appearing in an unbiased counting frame of 1600 m m 2 on the one-slice plane; disector is the volume of the disector (V[disector] ), that is, the product of the test-area by the disector thickness. Q2 A Nv 5 ]]] V[disector] The total number of cardiac myocytes (N[myocyte] ) was calculated as the product of Nv[myocyte] and the cardiac volume (V[heart] ). The V[heart] was considered as the cardiac weight (W[heart] ) since specific gravity ( g) of isotonic saline is about 1.0048 and the W[heart] was measured using Scherle’s method [25,27,28]. W[heart] V[heart] 5 ]] g
and
N[myocytes] 5 Nv[myocytes]V[heart]
2.3. Statistical analysis The coefficient of error (CE) for the stereological estimates was calculated as the ratio between the standard error and the mean [26]: Quantitative differences of the stereological parameters were analyzed with the non-parametric two-sided Mann-Whitney test with the significant level a of 0.05 [29]. Foetuses were compared by age group, i.e. second vs. third gestational trimester.
3. Results Table 2 shows the results of the stereology. All differences between the second and the third trimester of gestation were statistically significant (P,0.05). In this period the Vv[myocyte] decreased from 85.18 to 77.78% (28.69%) and the Vv[interstitium] Table 2 Mean6standard error of the mean of the stereological parameters of the foetal myocardium Stereology
Second trimester
Third trimester
P
Cardiac weight (g) Vv[myocyte] (%) Vv[interstitium] (%) Nv[myocyte] (10 4 / mm 3 ) V[myocyte] (mm 3 ) N[myocyte] (10 9 )
1.9560.16 85.1860.92 14.8360.92 68.8662.40 1214.386134.23 1.3660.18
9.1060.87 77.7860.93 22.2260.93 57.4062.39 1412.316151.21 5.0660.61
,0.001 0.001 0.001 0.006 0.03 ,0.001
P is the probability of the difference between foetuses of the second and third trimesters to be different from zero (Mann-Whitney test). Vv is the volume density, Nv is the numerical density, V is the mean volume, N is the total number.
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increased from 14.83 to 22.22% (149.83%). Fig. 1 compares the changes in the composition of the foetal myocardium in the last two gestational trimesters. The Nv[myocyte] decreased from 68.86 to 57.40 (10 4 ) / mm 3 (216.64%) and the V[myocyte] increased from 1213.38 to 1412.31 m m 3 (116.39%). Fig. 2 shows the convergence of the trends of these estimates. From the second to the third trimester the Nv[myocyte] decrease while the V[myocyte] increase. In human foetuses of the last two trimesters of gestation the cardiac weight increased from 1.95 to 9.10 g (1366.67%) and the N[myocyte] increased from 1.36 to 5.06 (310 9 ) myocytes (1272.06%). Fig. 3 shows that the pattern of growth of the trends of these estimates is divergent. Coefficients of error (CE) can be calculated from data in Table 2. The CE for the Vv[myocyte] estimates ranged from 1.08 (2nd trimester) to 1.20% (3rd trimester), and for the Vv[interstitium] estimates ranged from 4.19 (3rd trimester) to 6.20% (2nd trimester). For the Nv[myocyte] estimates the CE ranged from 3.49 (2nd trimester) to 4.16% (3rd trimester), and for the N[myocyte] ranged from 12.06 (3rd trimester) to
Fig. 1. Bar graph comparing the volume densities (Vv) of the human foetal cardiac interstitium and myocytes. From the second to the third trimester of gestation the proportion of the myocytes decreased while the proportion of the interstitium increased.
Fig. 2. Quantitative analysis of the human foetal myocardium: (a) trend of the numerical density of cardiac myocytes (Nv[myocyte] ) and (b) trend of the mean myocyte volume (V[myocyte] ) in the last two trimesters of gestation. These trends are convergent in this period.
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Fig. 3. Quantitative analysis of the human foetal myocardium: (a) trend of the cardiac weight and (b) trend of the total number of myocytes (N[myocyte] ) in the last two trimesters of gestation. These trends are divergent in this period.
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13.24% (2nd trimester). The V[myocyte] had CE ranged from 10.71 (3rd trimester) to 11.05% (2nd trimester).
4. Discussion Some cardiac myocytes can be binucleated during prenatal development [10]. Binucleation of the cardiac myocytes could lead to inconsistencies in the determination of Nv[myocyte] if the rate of binucleation was high. However, the study of the mitotic activity of cultured cardiac muscle cells demonstrated that only 10–15% of cells fail to complete cytokinesis and thus to form binucleated cells. The remaining 85–90% of the mitotic cardiac cells complete cytokinesis and produce two daughter cells [30]. The optical disector seems to be an accurate method for determining the number of nuclei in the cardiac myocytes. It permits the definition within the thickness of the section avoiding the over-counting of binucleated cells [19]. Austin et al. [16] commented that values measured on fixed and processed tissue are biased in consideration of fresh material. However, like these authors, we found small CE for estimates (,15%), suggesting that the intrasubject error introduced by systematic sample was low, and the relatively high accuracy of estimates. Tokuyasu [3] reported that mitotic activity is unequal along the myocardium wall during the embryonic period (more activity in the peripheral compact layer than in the inner trabecular layer). In the human this pattern of growth was never observed in the foetal period and this study has not considered different parts of the myocardium in the selection of the sample for analysis. The growth of the heart, until |2 weeks of age, is due to the increase in cell number in early neonates of the rat and in the human foetal and postnatal period. Human cardiac growth after 2 weeks of age is primarily caused by an increase in myocyte size rather than number [7,8]. The present study demonstrated that the tendency of the Nv[myocyte] decrease with the simultaneous V[myocyte] increase can be observed in the last two gestational trimesters in man. In human foetuses of the last two trimesters of gestation the cardiac weight increased from 1.95 to 9.10 g (1366.67%) and the N[myocyte] increased from 1.36 to 5.06 (310 9 ) myocytes (1272.06%). Fig. 3 shows that the pattern of growth of the trends of these estimates is divergent. Austin et al. [16] used a stereological model and established a protocol that can be employed to test for patterns of cardiac growth in normal and pathological human specimens with archival material. This is similar to the present study and our previous studies about myocardial quantification in primates [31] and cardiac growth [4,14]. In the left ventricle of two normal hearts from neonates, Austin et al. [16] found a mean absolute number of myocytes of 5.0 (10 8 ) / cm 3 , in these neonates the ventricular weight was 14.4 and 18.7 g (mean 16.6 g). These figures are greater than the ones found in the present study of foetuses. Foetuses of the third trimester (present study) and neonates [16] are not completely comparable because of some methodological
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differences, for example, cardiac weight vs. ventricular weight. However, Austin et al. [16] estimated the mean myocyte volume varying between 1220.0 and 2630.0 m m 3 that is of the same magnitude as our estimates (1412.316151.21 m m 3 in foetuses of the third trimester). Villar and Mandarim-de-Lacerda [32] observed an important catch-up of growth in both total length and volume of the capillaries between rat foetuses of age 18.5 and 20.5 days (equivalent to the third trimester in man). In addition, there was an increase in the total length in late neonates while the volume had significantly decreased immediately after birth but improved its size in neonates of 1 week. Absolute quantities of the myocardial capillaries have a tendency to increase during the foetal and neonatal periods because of the cardiac growth. The diameter of the capillaries, however, was uniform during the foetal and neonatal periods. This agrees with the present study that considered the vessels included in the cardiac interstitium had increased almost 50% in the last two gestational trimesters. Mandarim-de-Lacerda [11] and Mandarim-de-Lacerda and Souza [14] studied the growth of the myocardium in human and rat embryos. They noted different trends of growth with the breakpoint around Carnegie stage 20. After this stage, that corresponds to late embryos, the growth in volume of the myocardium is greater than in early embryos. Mandarim-de-Lacerda and Pessanha [9] added that, in rat, the major difference in Nv[myocyte] occurs between embryos and foetuses-neonates while the major difference in Vv[myocyte] and Vv[interstitium] occurs between neonates and embryos-foetuses. On the other hand, the increase of the N[myocyte] is greater than the increase in cardiac weight comparing foetuses and neonates, indicating that great cell proliferation takes place in this period. In the present study the myocardium was studied stereologically. We have taken two groups of foetuses (2nd vs. 3rd trimester) and compared quantification of myocytes and interstitium. Of course, these groups are broad and this can prejudice the comparison between them. The choice to study only two groups is due to the small sample of the foetuses in the 3rd trimester and forbids a greater division into this period. However, the present results are statistically consistent enough to allow an analysis of the trends in growth in the last two human gestational trimesters. Considering the cardiac myocyte, the decrease of the cellular proliferation with the increase of the cellular volume is illustrated in Fig. 2 and Fig. 3. These figures show the trends of the developmental changes of the stereological estimates. From the second to the third trimester of gestation the foetal myocardium grows with convergence of the numerical density of myocytes and the mean myocyte volume. In the same period, the trends of the cardiac weight and the total number of myocytes are divergent. So, the increase of the cardiac weight is not totally explained by the increase in the number of myocytes but the increase of the mean myocyte volume. In conclusion, during the last two gestational trimesters the human heart increases in weight more than 4.5 times, the volume density of myocytes decreases while the volume density of the cardiac interstitium increases. The numerical density of myocytes per myocardium volume decreases but the myocytes became greater in mean volume (more than 16%).
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Acknowledgments The authors are grateful to Mrs. Ledroux for her technical assistance. This work was funded in part by a grant of the National Council of Research of Brazil (CNPq Proc. no. 52.23.73 / 95.0) and Faperj (E-26 / 170.315 / 95).
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