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Revista chilena de anatomía

versión impresa ISSN 0716-9868

Rev. chil. anat. v.19 n.3 Temuco dic. 2001

http://dx.doi.org/10.4067/S0716-98682001000300014 

CAN DIFFERENT LIPIDS DIETETICS AFFECT THE MYOCARDIAL
STRUCTURE AND THE BLOOD PRESSURE INFLUENCING THE AGING
PROCESS? EXPERIMENTAL STUDY IN RATS

¿PUEDEN DIFERENTES LÍPIDOS DIETÉTICOS AFECTAR LA ESTRUCTURA DEL
MIOCARDIO Y LA PRESIÓN SANGUÍNEA, INFLUENCIANDO EL PROCESP DE

ENVEJECIMIENTO? ESTUDIO EXPERIMENTAL EN RATAS

*,** Marcia Barbosa Aguila & *Carlos Alberto Mandarim-de-Lacerda

* Laboratory of Morphometry & Cardiovascular Morphology, State University of Rio de Janeiro, Brazil.
** Department of Applied Nutrition, Federal University of the Rio de Janeiro State, Brazil.

SUMMARY: We studied sixty-seven male rats (Wistar strain) from birth until three different ages: 6-mo-old, 12-mo-old and 18-mo-old. The animals were separated according to the different experimental diets, i.e., soybean oil (S), canola oil (CA), lard and egg yolk (LE) and canola oil+lard and egg yolk (CA+LE). The systolic blood pressure (SBP) was verified weekly. The determination of the numerical density of the left ventricular myocyte nuclei (Nv[cm]) used the "optical disector" method. Considering all ages, the CA group rats had the Nv[cm] higher since 6-mo-old, whereas the other groups had almost the some values. The 18-mo-old, the CA group rats had a slight decrease of Nv[cm] but smaller than the other groups. The 18-mo-old rats had higher SBP and lower Nv[cm] than the younger ones, but in the CA group the SBP did not alter with the age and the reduction of the Nv[cm] was significantly lower than that of the other groups. Therefore, the variation in the SBP and the Nv[cm] was age-dependent but each experimental group had a different tendency. The elevation of the SBP and the reduction of the Nv[cm] was marked in the LE group, and intermediary in the other groups, suggesting that the density of the cardiac myocytes was influenced by the different dietetic lipids. In conclusion, the present study verified that the edible oil type in diet concerns the blood pressure and the numerical density of cardiac myocytes in rats during the aging process. The long-term canola oil diet produces beneficial structural myocardial changes and the attenuation of the blood pressure than other experimental diets, mainly the diet composing of lard and egg yolk.

KEY WORDS: 1. Myocardium; 2. Aging process; 3. Stereology, 4. Canola oil; 5. Soybean oil.

INTRODUCTION

Cardiovascular diseases are the first cause of death, disability, and economic loss in most developed countries. Epidemiological analysis of the relationship between nutrition and cardiovascular diseases shows that societies becoming more affluent are adopting dietary habits similar to those of the industrialized nations, leading to an increase in the prevalence of cardiovascular diseases. The improvement of nutrition habits and other life-style factors would make possible to avoid this situation (Biosca et al., 1992). The incidence of ischemic strokes and stroke- induced mortality increases with aging. Elderly individuals are more frequently affected by ischemic stroke with a prevalence of 15 per 100 in people over 75 years of age (Albucher, 1996). Structural modifications affect in turn overall heart adaptability to a variety of stimuli that might differ between younger and older rats (Capasso et al., 1986). A major aging-dependent myocardial structural alteration is the left ventricular hypertrophy associated with both cell loss and increase in collagen deposition and fibrosis (Hunter & Chien, 1999).

Physicians and scientists are becoming increasingly aware that the amount, source and composition of fat in the diet may alter the development and progression of human disease (Goodnight, 1989). It has become apparent that manipulation of dietary fats may not only alter levels of serum and other lipids, resulting in sub endothelial lipid deposition, but may also lead to changes in cell membrane composition and function that resemble those associated with aging (Lakatta, 1992).

Linoleic acid is a component of many vegetables oils such as corn, sunflower, and safflower oils, whereas alpha-linolenic acid is a component of green leaves and some seed oils. Once consumed, these fatty acids are converted to the longer chain, more unsaturated derivatives arachidonic acid (AA) and eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, respectively. EPA and DHA are found in abundance in some marine fish oils. Alpha-linolenic acid, EPA and DHA are members of the n-3 family of polyunsaturated fatty acids (PUFAs), whereas linolenic and arachidonic acids are n-6 PUFAs; the n-6 and n-3 PUFA families are not interconvertible in mammals. The key physiologic roles of PUFAs are as precursors of eicosanoids (Calder, 1997).  After n-3 fatty acids are consumed in the diet, they compete with linoleic acid and arachidonic acid in membrane phospholipids where they are able to participate in cellular functions (Goodnight, 1989) as such membrane stability, membrane fluidity, cell mobility and formation of eicosanoids (Goodnight, 1988). In previous studies, we have analyzed the influence of the canola oil on myocardial structure of the rats (Aguila et al., 1998a and b; Aguila & Mandarim-de-Lacerda, 1999, 2000 and 2001).

The aim of this study is to investigate the possible influence of the four high-fat different experimental diets (with soybean oil, canola oil, lard and egg yolk) on blood pressure and on the number of cardiomyocytes during the aging process in rats.

MATERIAL AND METHOD

Rats and diets. We studied sixty seven male rats (Wistar strain) obtained from colonies maintained at our Laboratory of Morphometry & Cardiovascular Morphology (www2.uerj.br/~lmmc) in Rio de Janeiro from birth until three different ages: 6, 12 and 18-mo-old. The animal care complied with the Guide for the Laboratory Rat (Sharp & La Regina, 1998). State University of Rio de Janeiro standing committee on animal research had approved the protocols. The investigation conforms to "Guide for the Care Use of laboratory Animals" published by the US National Institutes of Health (NIH Publication No.85-23, revised 1985).

Rats were housed in polypropylene cage in-group of five animals. They kept in a temperature-controlled (21 ± 30ºC) and humidity-controlled (55 to 65%) room submits at 12-hour light and dark cycle (artificial lights, 7a.m. - 7p.m.) and to air exhaustion cycle (15 minutes/hour). The two groups received experimental diets where the basal diet included cornstarch, casein, maize, egg white and mineral and vitamin mixtures. The animals were separated according to the different experimental diets, i.e., soybean oil (S), canola oil (CA), lard and egg yolk (LE) and canola oil+lard and egg yolk (CA+LE). The diets contained, as percentage of energy, 47% carbohydrate, 29% fat and 23% protein. The diets were weekly prepared in the laboratory, stored at +4ºC, and established to have energy values not significantly different (Aguila & Mandarim-de-Lacerda, 2000, 2001).

Methods. The four groups were studied in three ages: 6, 12 and 18 months. The animals received ad libitum the different diets and free access to water, the diet intake was daily monitored. The systolic blood pressure (SBP) was verified using the non-invasive method of the tail-cuff plethysmography (RTBP1007, Kent Scientific Co., Litchfield, CT, USA).

At the end of the experiment, the animals were deeply anesthetized, the heart was stopped in diastole through a cardiac injection of 3ml 10% KCl. The heart was excised by cutting the vessels shortly and then dissected under stereoscopic microscopy, by separating the atria from the ventricles and the right ventricle from the left ventricle including the interventricular septum. The cardiac volume and the volume of the ventricles, of the right ventricle and of the left ventricle + septum were determined (according to the submersion method of Scherle, 1970 - in which the water displacement due to organ volume is recorded by weighing). As the specific gravity (g) of isotonic saline is 1.0048 the respective volumes were obtained by V[structure]=W/g or simply V=W(Weibel, 1979).

The myocardial random and isotropic fragments were obtained by cutting the heart using the orientator method (Mattfeldt et al., 1990). These fragments were place 48h at room temperature in fixative (freshly prepared 4% w/v formaldehyde in 0.1M phosphate buffer pH 7.2), embedded in Paraplast plus® and sectioned at 3 and 10µm thick. Sections were stained by hematoxylin-eosin, Masson's trichrome and picro-sirius red. The analysis used a video-microscopic system composed of a Leica DMRBE microscope (x100 objective, NA 1.25) and a Kappa video camera.

The determination of the numerical density of the left ventricular myocyte nuclei (Nv[cm]) used the "optical disector" method defined with two parallel sections separated by a distance of 3 µm (automatically controlled with the motorized stage of the Leica microscope) (Sterio, 1984; Cruz-Orive & Weibel, 1990). The Nv[cm] was determined as follows:

  Q-A  
Nv[cm]=

/mm3 , Q-A was the number
  t.AT  

of the left ventricular myocyte nuclei seen in focus only in the look-up plane when they were partly or totally inside the frame and did not intersect the left or inferior exclusion edges or their extension (Gundersen, 1977); t was the thickness of the disector and AT was the test-area (1600 µm2). The Fig. 1 represents the "optical disector" in a schematic drawing showing three cardiac myocytes inside the frame and Fig. 2 are the photomicrographs of the "look-up" and the "look-down" planes of the "optical disector" having three cardiac myocytes too. It is interesting to compare these two figures to understanding the use of the "optical disector".


Fig. 1. Schematic drawing representing an "optical disector" having three different cells and nuclei. In the look-up plane one nucleus was sectioned, in the look-down plane two nuclei were cut. AT is the area test; t is the thickness of the section.

Five random disector pairs were analyzed for each specimen. The total number of the left ventricular myocyte nuclei (N[cm]) was determined as N[cm]=Nv[cm] · V[lvent] (Gundersen et al., 1988); V[lvent] is the volume of the left ventricle including the interventricular septum.


Fig. 2. Photomicrographs of the myocardium for demonstration of an "optical disector": (a) is the look-up plane and (b) is the look-down plane. Like the Fig. 1, one nucleus was cut in the look-up plane and two nuclei were cut in the look-down plane.

Data analysis. The differences of the biometrical parameters among the groups were tested by the analysis of variance and the multiple comparison Newman-Keuls test. The differences of the stereology were tested using the non-parametric Kruskal-Wallis analysis of variance and the Mann-Whitney test. In all cases, the significant level of 0.05 was considered for significant statistics (Zar, 1999).

RESULTS

Results are shown in Figures 3 and 4.


Fig. 3. Bar graph showing the blood pressure variation during the 18 months of the experimentation. Groups: S - soybean oil, CA - canola oil, LE- lard and egg yolk. Different small letters over the bars mark statistical difference among the groups of the same age; different capital letters over the bars mark statistical difference in the same group in the different ages (p<0.05).

The SBP was higher in the LE group at all ages. At the 6-mo-old, the LE group rats had the SBP 10% higher than the other groups (p=0.007). At the 12-mo-old, the SBP of the LE group rats was around 20% higher then the other groups (p=0.008). At the 18-mo-old, the SBP of the LE group rats was around 40% higher than the S and CA groups (p=0.0002 and p=0.0003, respectively), and it was 22% higher than the CA+LE group (p=0.0006). The SBP of the CA+LE group rats was 11% higher than the S group rats (p=0.02) and 18% higher than the SBP of the CA group rats (p=0.003). Considering all ages, the LE group rats had a SBP increase since 6-mo-old, whereas the S and CA+LE groups had a slight SBP increase only at 18-mo-old. The CA group rats had not significant SBP increase in any time of the experimentation (Fig. 3).

The Nv[cm] was higher in the CA group at all ages. At the 6-mo-old, the CA group rats had the Nv[cm] 61% higher than S group (p=0.008), 105% higher than LE group (p=0.007) and 72% higher than CA+LE group (p=0.006). At the 12-mo-old, the CA group rats had the Nv[cm] 96% higher than S group (p=0.008), 114% higher than LE group (p=0.005) and 99% higher than CA+LE group (p=0.008). At 18-mo-old, the N[cm] of the CA group rats was 76% higher than the S group rats, 118% higher than the LE group rats and 58% higher than the CA+LE group rats.

Considering all ages, the CA group rats had the Nv[cm] higher since 6-mo-old, whereas the other groups had almost the some values. A 18-mo-old, the CA group rats a slight decrease of Nv[cm] but smaller than the other groups (Fig. 4).


Fig. 4. Bar graph showing the numerical density of cardiac myocytes variation during the 18 months of the experimentation. Groups: S- soybean oil, CA - canola oil, LE - lard and egg yolk. Different small letters over the bars mark statistical difference among groups of the same age; different capital letters over the bars mark statistical difference in the same group in the different ages (p<0.05).

DISCUSSION

Stereology determines quantitative three-dimensional information based on sectional information that is two-dimensional information. The researcher can study the three-dimensional organ on two-dimensional images. This quantitative information like number, size, distribution of cardiomyocytes, and other components contain important information about the function and organization of the heart being studied (Mandarim-de-Lacerda, 1999; Benes & Lange, 2001). A loss of cardiomyocytes has been reported by previous stereological analyses in rats (Aguila & Mandarim-de-Lacerda, 1998b; 1999; 2000 and 2001).

The 18-mo-old rats had higher SBP and lower Nv[cm] than the younger ones, but in the CA group the SBP did not alter with the age and the reduction of the Nv[cm] was significantly lower than that of the other groups. Therefore, the variation in the SBP and the Nv[cm] was age-dependent but each experimental group had a different tendency. The elevation of the SBP and the reduction of the Nv[cm] was marked in the LE group, and intermediary in the other groups, suggesting that the density of the cardiac myocytes was influenced by the different dietetic lipids.

The present study pointed out that canola oil was more benefic to the cardiovascular system of rats by reducing the SBP and attenuating the loss of cardiac myocytes (quantifying through the determination of Nv[cm]) that normally occur with age. The eicosanoids made from PUFAs has different effects that are important in blood pressure regulation, and it is often assumed that changes seen with altered dietary PUFA intake come about through changes in the endogenous production of eicosanoids. The n-3 fatty acids influence the types and amounts of eicosanoids formed in vivo (thromboxanes, prostacyclins, leukotrienes, etc) and exert moderate antiplatelet effects (Dupont et al., 1996).

Some previous studies demonstrate a marked link among the types of fat in the diet, the age of the animals, and the cardiac function, including arrhythmogenesis following experimentally induced disturbances in coronary flow (McLennan et al., 1988 and 1989). Experimental researches have showed that ventricular fibrillation (the usually fatal form of cardiac arrhythmia) occurs more frequently when a saturated fatty acids rich diet has been consumed; arrhythmias can be limited by the consumption of a diet that is rich in n-3 PUFAs (Hock et al., 1990; McLennan et al., 1985; 1988 and 1992).

The present study observed an association of the lard and egg yolk diet with high levels of SBP and low left ventrcular cardiac myocytes density since the age of 6-mo. It may represent an association between saturated fatty acids and these adverse cardiovascular indices. The long term diet administration of a canola oil and lard and egg yolk mixture resulted in a balance of the SBP and Nv[cm] in rats, suggesting that the canola oil was helpful to reduce the cardiovascular injury due to lard and egg yolk administration. The long-term administration of soybean oil diet resulted in no significant alteration of the SBP but decreased the left ventricular cardiac myocytes density in rats.

An important aspect in this research was the observation of the early increase of the SBP in the LE group rats contrarily to the S and CA+LE group that had the SBP increasing later and in small amount. Instead, the SBP in the CA group rats was maintained low all the time. This agrees with the notion that dietary intervention with saturated fatty acid and cholesterol high diet result in a significant SBP increase (Salonen, 1991; Beegom & Singh, 1997).

A significant loss of myocytes is normal in aging process of the heart, in the ventricular myocardium this phenomenon persists longer in the left than in the right cardiac side (Anversa et al., 1990). Cell loss decreases the cardiac functional reserve and may constitute a critical variable in the development of ventricular dysfunction and failure in the elderly. The chronic death of myocytes may also form the basis for the depressed capacity of the old heart to sustain acute and chronic increases in pressure and volume loads on the myocardium (Wei, 1992).

After 6, 12 and 18 months of experimentation, the Nv[cm] was greatest in animals feed canola oil diet and smallest in animals feed lard and egg yolk diet. The Fig. 5 shows a morphological and quantitative interpretation of the cardiac myocytes during the aging process based in the present results. In a previous study, aged rats feed with canola oil diet shown the best myocardium vascularization characterized by increased stereological estimate length density of blood vessels as well as high HDL-C and low LDL-C serum (Aguila et al., 1998a).


Fig. 5. Cardiac myocyte size and density in rats of the canola oil group (left) and the lard and egg yolk group (right) referred to one cubic millimeter of total left ventricular myocardium.

Dietary modification of myocardial lipids is associated with changes in the myocardial function. Alterations in the fatty acid composition of dietary lipids can alter the response of the heart to ischemic stress. Changes in saturation, chain length, and number of double bonds in the fatty acyl chains of membrane phospholipids can greatly alter the physicochemical properties of the membrane (i.e., fluidity) and thereby membrane function (Hock et al., 1990). This agrees with the present observations in the canola oil rats where the myocardial structure was better preserved than in the other groups.

In conclusion, the present study verified that the edible oil type in diet concerns the blood pressure and the numerical density of cardiac myocytes in rats during the aging process. The long-term canola oil diet produces beneficial structural myocardial changes and the attenuation of the blood pressure than other experimental diets, mainly the diet composing of lard and egg yolk.

ACKNOWLEDGMENTS:This work was partially supported by Brazilian agencies CNPq and Faperj.

RESUMEN: Estudiamos 67 ratas machos (Wistar) desde el nacimiento hasta: 6, 12 y 18 meses. Los animales fueron separados de acuerdo a las diferentes dietas experimentales: aceite de soya, aceite de canola (AC), manteca de cerdo y yema de huevo (CH) y aceite de canola + manteca de cerdo y yema de huevo (AC+CH). La presión arterial (PA) fue medida semanalmente. Para determinar la densidad numérica de núcleos de miocitos del ventrículo izquierdo (Nv[cm]), se utilizó el método del disector óptico. Considerando las edades de las ratas, las del grupo CA tuvieron el Nv[cm] mayor desde los 6 meses de edad, mientras que los otros grupos tuvieron, prácticamente, los mismos valores. A los 18 meses, el grupo de ratas AC tuvo una pequeña disminución del Nv[cm], menor que en los otros grupos. Las ratas de 18 meses presentaron los mayores valores de PA y menores valores de Nv[cm] en comparación con las ratas más jóvenes. En el grupo AC, la PA no se alteró con la edad y la reducción de Nv[cm] fue significativamente menor que en los demás grupos. Se observó que las variacioones de la PA y del Nv[cm] dependían de la edad, pero en cada grupo experimental, estos parámetros tuvieron una tendencia diferente. La elevación de la PA y la reducción del Nv[cm] fue grande en el grupo CH, e intermedia en los otros grupos, sugiriendo que la densidad de los miocitos cardíacos estaba influenciada por los lípidos dietéticos. En conclusión, el estudio comprobó que el tipo de aceite en la dieta influye en las variables citadas, durante el envejecimiento. La ingestión prolongada de dietas que contienen aceite de canola, tienen efecto benéfico sobre la estructura miocárdica y presión arteria,l que los demás aceites dietéticos estudiados, principalmente, las dietas que contiene aceite de cerdo y yema de huevo.

PALABRAS CLAVE: 1. Miocardio; 2. Proceso de envejecimiento; 3. Estereología; 4. Aceite de canola; 5. Aceite de soya.

Address for correspondence:
Prof. Dra. Marcia B. Aguila
Laboratório de Morfometria e Morfologia Cardiovascular,
Universidade do Estado do Rio de Janeiro,
Centro Biomédico, Instituto de Biologia,
Av. 28 de Setembro, 87 (fds)
20551-030 Rio de Janeiro, Brasil

Phone/Fax: [+55.21] 2587-6416
Email: mandarim@uerj.br

Recibido : 24-09-2001
Aceptado: 27-10-2001

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