PURPOSE: To develop a stereological comparison between right (RV) and left ventricle (LV) myocardium during the third human gestational trimester. METHODS: Five human fetal hearts of the third trimester provided representative samples of 5 RV myocardium and 4 LV myocardium. The material was fixed in 10% buffered formaldehyde, and processed through routine methods. Fifteen microscopic fields were randomly chosen and counted in each ventricular myocardium using an "M-42" test system. The following stereological parameters were assessed: Vv (%), Lv (µm²), Sv (µm²/µm³), Vp (µm³), Nv (1/mm³) and total N. RESULTS: No significant difference between the stereological parameters of the myocardial structures assessed was evidenced, when comparing RV and LV. CONCLUSION: Right and left human ventricular myocardium are very similar during the fetal period at least in regard to their structural aspects.
Original Article
Ricardo Xavier-Vidal, Kalil Madi
Although reasonable knowledge about intrauterine cardiovascular development currently exists, certain aspects of this process are still causing much scientific discussion. Some details of intrauterine cardiovascular development in the human and other species have been clarified recently 1-10, but these still have yet to be confirmed. It is also important to assess interspecific differences to avoid rough interpretational errors in the results obtained 9-11. Even though the idea that differences exist between RV and LV myocardial structures in the same individual during development is controversial, few studies have focused on this subject 12,13.
A significant benefit of the cardiovascular assessment of anatomicopathological studies is that the cause of death can be more rapidly clarified during the perinatal period when death is due to gross malformations. However, difficulties increase when the events are mainly related to microscopic structures 14. For example, even though myocardial changes during sudden infant death have been described, only incipient knowledge in this area is available 14,15. In industrialized countries, sudden death accounts for 300,000 to 400,000 deaths per year, of which 80% to 90% are due to coronary heart diseases. It is known that 50% of the deaths due to coronary heart disease are sudden, and congenital anomalies of the coronary arteries are a very important cause of such deaths 16. These coronary artery anomalies may lead to changes in the capillary bed and in the general structure of the myocardium, which may be significant in perinatal mortality. For example, even though sudden cardiopulmonary arrest in children is very rare (with not always very successful results of resuscitation) and ventricular fibrillation occurs in less than 10% during that event, these occurrences are more common in children with congenital heart disease 17. Cardiopulmonary arrest associated or not with congenital anomalies leads, in many cases, to some type of cardiac alteration, which may cause some degree of structural reorganization of the myocardium. Sudden death following myocardial infarction is usually due to abnormalities in the cellular electrophysiology caused by ischemia. However, sudden death seems to be related to the postinfarction scarring process, because the subsequent and persistent emerging arrhythmogenic potential is not primarily dependent on the effect of ischemia on cellular electrophysiology, but on the alteration of the stability of the injured and recovering tissular structure 18. Therefore, it is of scientific importance to evaluate the structural organization of the myocardial tissue, defining a normal standard because infants with abnormal hearts and/or succumbing to sudden death seem to have a smaller number of nuclei of the myocytes (Nv) per unit of volume in the myocardium than normal 13. Another important tissular alteration in cases of sudden death syndrome in infants is the occurrence of reactive fibrosis (interstitial fibrosis) 15, where the involvement of the nitric oxide debit is possible 19. This fibrosis can also be evaluated by the stereological method.
The objective of this study was to develop a stereological comparison between the normal myocardial structure of the RV and LV during the third human gestational trimester.
Methods
Five human fetal hearts of the third gestational trimester provided representative samples of five RV myocardia and four LV myocardia obtained from the free cardiac wall. The fetuses were obtained from spontaneous abortions. Maternal age and sexes of the fetuses could not be considered for the approach here developed. The fetuses were dated according to the length of the largest foot 20-23. The material was fixed in buffered 10% formaldehyde, processed according to routine methods, sliced into 5µm fragments, and stained with hematoxylin-eosin.
Holmes effect, the overestimation error 24, is considered nonexistent with the use of the disector method 25-29. Fifteen microscopic fields randomly chosen in each right or left ventricular myocardium were counted for stereological evaluation. The countings were performed with an "M-42" test system mounted in a Nikon CFW eyepiece (10X) using immersion objective (100X). The numbers of structures and points and the intersections overlapping the structures considered were counted. Statistical analysis was performed using Student's
Vv% - volumetric density32.
Where: PP = counted points, PT = total of points of the test-system;
Sv (µm2/µm3) - surface density 32. Sv = 2 x I1, where I1 = intersections of the structures with the test lines;
Nv (1/mm3) - numerical density 25-27.
Where: V(dis) = t x At; V(dis) = volume of the disector; t = thickness of the microtome section; At = area of the test; S Q- = sum of the structures counted in one of the faces of the histological section; SV(dis) = sum of the volume of the disector;
Vp (µm3) - ponderal volume28. Vp = Vv/Nv;
Lv (µm2) - density of length 32; Lv = 2 x Qa, Qa (µm-2) - density of profiles in the area of the test.
Where: S profiles = number of structures in the area of the test; At = area of the test;
N - total number of structures in the organ27. N = Nv (mm3) x cardiac weight (mg).
Results
Our results showed no significant difference between the stereological parameters of the myocardial structures evaluated (
Discussion
Stereological and allometric quantitative studies of the human fetal myocardium recently developed are scant 33-38. Comparison between human and animal data is important to avoid interpretational distortions that may happen in experimental studies 9,11. Morphometrical models are important to determine physiological and morbid variations that occur in biological tissues26,27,39-43.
The degree of proliferation/hypertrophy of the cardiomyocytes during the perinatal period is still controversial 5,44,45. The degree of myocardial regeneration during this period varies in a directly proportional manner with the potential of cellular proliferation 5. It was suggested that differences in the capillary bed could cause the difference in the pattern of growth of RV and LV 12. Other authors 13, however, have identified a similar pattern of growth of the Nv of myocytes (per volume unit in the myocardium), in both ventricles, during the perinatal period. Our results showed no significant differences between the stereological parameters of the blood vessels, when the RV and LV were compared. However, an increase in the number of fields to be counted - and an increase in the number of hearts taken into account - may provide significant differences in the evaluation of myocardial revascularization. A reasonable number of fields to be counted for the evaluation of myocytes is around 10 fields per ventricle; for blood vessels, however, the number of fields should be greater, around 142 fields per ventricular myocardium 46,47. Another example occurs in the adult rat, whose consistent evaluation of myocardial blood vessels requires a much larger number of fields than the evaluation of myocytes and connective tissue 43. In this case, the number of fields to be counted should be around 422 fields per ventricular myocardium 46,47.
Studies of the myocyte nuclei are important, for example, for the identification of the orientation of the bundles of cardiomyocytes in the myocardium, which can be performed by means of the confocal scanning laser microscopy 48. Disorganization in the orientation of the bundles of cardiomyocytes has been described in certain cardiomyopathies 49-52. It is known that these diseases can affect human beings, even the very young 14. The most emphatic difference between the myocardium of an infant and that of an adult is the diameter of myocytes, which is much smaller in the former. Yet in the infant, the nucleus is proportionally larger than in the adult cell. Another important characteristic is that, in the infant, both the endocardium and the epicardium are thinner, and the latter has less adipose tissue than the adult 15.
In stereological analysis using the disector method, we verified a reduction in the Nv (1/mm3) of the LV myocyte nuclei from 703,948 (EP = 70490) to 397.304 (EP = 27362), from the second to the third gestational trimester, respectively. The mean (second + third trimesters) was around 550,626/mm3. However, the total number of nuclei (N) in the heart significantly increased from 2,101,152,700 (EP = 449634529) in the second trimester to 11,147,569,300 (EP = 2222847632) in the third trimester, which resulted from the increase in the heart volume throughout time (p<0.06; Mann-Whitney Rank Sum Test). In human beings, Vv% of the nucleus significantly decreases from the second to the third trimester during fetal development 36. Components of the ventricular wall, such as myocytes, connective tissue and vessels, however, maintain a proportional relation from the second to the third gestational trimester 33. In the rat, during the fetal and perinatal periods, heart growth results mainly from the increase in the number of myocardial cells, which ceases after approximately two weeks of age. After this period, the growth results from hypertrophy rather than an increase in the number of myocytes, which end up by ceasing their process of division 1,53.
Volumetric density (Vv%) relates to the proportion of structures evaluated in the organ or tissue studied 32. In a previous article 33 we reported that we identified in the LV in the third gestational trimester a Vv% of myocytes, connective tissue (except for blood vessels), and blood vessels of 67.67, 25.94, and 6.39, respectively, while for the RV the values in the present study were 64.64, 29.14, and 6.22, respectively. Testing the samples, we did not identify any significant difference between the two ventricular walls (p>0.05). In a former article 36, assessing Nv nuclei and its volume (Vp) in LV during the third gestational trimester, we found the following values: Nv = 397,304 (1/mm3) and Vp = 97.66 µm3. In the present study, the values of RV for Nv and Vp were, respectively, 399,675.48 (1/mm3) and 174.06 µm3. Testing the samples, we did not identify significant differences between the two ventricular walls (p>0.05). The ponderal volume (Vp) is the volume of the cell (or structure) indirectly obtained as described (Vp = Vv/Nv) 28. To obtain the direct volume, it is necessary to use the Cavalieri method (with serial or alternate sections), which is also a morphometric method 47. The numerical density (Nv) is the number of structures per a certain volume (for example, mm3) 25-27. In the case, the number of cells is not always very close to the number of their nuclei in a certain volume. In the present study, while the number of myocytes in the RV is 547,116.74, the Nv is 399,675.48; therefore, hypertrophy is clearly evident in a diseased heart when compared with a healthy heart. The larger the cells, the rarer it is to visualize the cell nuclei inside the counting grid. In the same way, it is more common to find more cardiomyocyte nuclei of human fetuses inside the counting grid than it is to find cardiomyocyte nuclei of adults.
Porter and Bankston 3, studying fetuses of rats, found a mean Vv% for cardiac myocytes of 85.6%, for connective tissue (vessels excluded) of 2.8%, and for vessels of 11.6%. Zak 45 and Weber 54 estimate that the ventricular myocardial myocytes in the adult occupy 75% of the structural space, while the cardiac interstitium (intercellular substance including the vessels) occupies the remaining 25%. Santos and Mandarim-de-Lacerda 38, also developing a stereological study during the third gestational trimester, obtained results partially close to ours. The mean values found were: Vv% of 77.8% for the cardiac myocytes and 22.2% for the interstitium. Nv of the cardiac myocytes (calculated based on the number of their nuclei) was 574,000/mm3. The volume of the myocyte was 1412.3µm3. The mean total "N" of myocytes per heart was 5.06E+09.
The results of this study did not show a significant difference between any of the stereological parameters of the myocardial structures evaluated in the comparison of right and left ventricular myocardium. This suggests that, at least in regard to the structural question, human right and left ventricular myocardium are very similar during the fetal period. However, at least in the adult rat, there may be differences in regard to some biochemical details, such as, for example, the fact that RV tends to be notably more sensitive to injury due to systemic blockade of nitric oxide (L-NAME model) than the LV. However, this greater intensity of damage may have occurred as a result of the greater demand for the LV during the hypertensive process in this model 19.
Other stereological parameters here studied such as surface density (Sv, 1/µm2) and density of length (Lv, µm2) were used only for blood vessels. The density of length (Lv, µm2) refers to the variation in the length of the evaluated structures. The surface density (Sv, 1/µm2) relates to the variation in the surface of contact with the external medium and it is sensitive to the variation of the volume of the structure evaluated. Evaluation of cellular hypertrophy is a clear manner to understand the sensibility of the surface density. In a former study 43, evaluating the hypertensive process and the cardiac hypertrophy through the systemic blockade of the nitric oxide (L-NAME model), the mean surface density (Sv) of the healthy cardiac myocytes (control) was 0.152 (1/µm2), while in the hypertrophied cardiac myocytes, the increase in the mean volume was found as 30% with reduction in the mean surface density to 0.0812 (1/µm2) (p<0.001).
Currently, the association between microscope and equipment, such as the confocal scanning laser microscope, that uses software to handle imaging, makes it possible to observe the deep planes of the tissues, either fixed or in vivo. These analyses use fine and precise focus on one single plane. These planes can be deepened in the tissue so that sequential images can be obtained along the structures. This way, it is possible to stipulate the depth of the focal plane of visibility as, for example, in 2 or 7µm of depth, which makes the observations according to the disector 55 method much easier. The focus on a single plane, as in the confocal device, nullifies the Holmes effect 24. Analyses by tridimensional reconstruction are also possible in this type of device 55. The confocal scanning laser microscope has a striking versatility, being useful either for studies of identification of structures of adhesion, such as gap junctions 56 and the appearance of the adhesion molecules during the embryonic period 57, or for the study of the orientation of bundles of muscle cells in the cardiac tissue 55.
Acknowledgements
To the professors Drs. W.S. Costa, C.A. Mandarim-de-Lacerda, Vera Aires, M. Costa-Neves, W.N. Viana, V.C. Villar, L.R. Mendes Barboza, M. Guimarães and Dempson Chilinque, for their incentive. Supported by CAPES, CNPq and CIC-HUCFF/UFRJ, AXV.
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Hospital Universitário Clementino Fraga Filho - UFRJ - Rio de Janeiro.
Mailing address: Prof. Ricardo Xavier-Vidal - ASSOCIAÇÃO XAVIER-VIDAL para Direitos Humanos, Ciência, Tecnologia e Desenvolvimento, Caixa Postal 100.768, Niterói, RJ, Brazil, CEP 24001-970.
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