Leila Maria Meirelles Pereira

Numerical density of cardiomyocytes in chronic nitric oxide synthesis inhibition.

Objective: The myocardial effect on the normalization of arterial blood pressure in animals with hypertension previously induced by nitric oxide synthesis (NOS) inhibition is still unknown. The purpose of this study was to estimate the numerical density of cardiomyocytes that is able to show cardiomyocyte loss as a consequence of NOS inhibition. Methods: Sixty rats were divided into the following groups: control for 25 days (C25), control for 40 days (C40) control for 80 days (C80) and three other groups in which the rats received the NOS inhibitor N(G)-nitro-L-arginine-methyl-ester hydrochloride L-NAME; 50 mg/kg/day for 25 days (L25), 40 days (L40) and 40 days, respectively, the latter group having another 40 days of only water and food ad libitum (without L-NAME; L80 group). The detection of apoptotic cells was performed using a monoclonal antibody. Results: In the L25, L40 and L80 groups, blood pressure was 74.5, 90.2 and 56.3% higher than the respective age-matched control rats, the myocardium had hypertrophy of the cardiomyocytes and scattered areas of fibrosis, and apoptosis occurred in isolated cells. Compared to the controls, the heart mass/body mass ratio was significantly greater in the L-NAME groups L25, L40 and L80, i.e. 31, 26 and 21%, respectively, the numerical density of cardiomyocytes in L-NAME rats was 32.7, 48.8 and 41.7% lower and the mean volume of cardiomyocytes was 33, 53 and 48% greater. Conclusion: The cardiomyocyte loss in NOS inhibition seems to be mainly due to necrosis, although apoptosis is also present.

Effect of antihypertensive drugs on the myocardial microvessels in rats with nitric oxide blockade.

In the present study, myocardial microvessels were investigated by stereology in rats with nitric oxide blockade and concomitant antihypertensive treatment for 40 days. The following five groups (10 rats each) were studied: control; L-NAME; L-NAME + spironolactone; L-NAME + enalapril; L-NAME + verapamil. The blood pressure (BP) increased every week in the L-NAME group; after an initial increase BP decreased in the treated groups and was not different from the control group. Compared to control animals, the myocardium had hypertrophied myocytes and capillary rarefaction; the tunica media and the tunica intima of small arteries were thickened, and an increase in collagen fibrils in L-NAME treated animals was noted. The enalapril, verapamil and spironolactone groups showed uniform myocardium, quite similar to the control group. The volume density of vessels, in comparison with the L-NAME group, was greater in the spironolactone group (57%), in the enalapril group (76%) and in the verapamil group (81%). The length density of vessels was, respectively, 56%, 50%, and 76% greater in the spironolactone, enalapril and verapamil groups than in the L-NAME group. The surface density of the vessels of the L-NAME group was, respectively, 88%, 96%, and 113% lower than in the spironolactone, enalapril and verapamil groups. These results are compatible with the occurrence of angiogenesis in the verapamil rats.

Quantitative Examination of the Cardiac Myocytes in Hypertensive Rats under Chronic Inhibition of Nitric Oxide Synthesis

This study quantitatively examined myocardial changes during the hypertensive process. Four groups (n=10 for each group) of adultRattus norvegicus (Wistar strain) were studied. Animals were sacrificed at 40 and 80 days of experimentation (control and experimental groups in each age). Although animals in the experimental groups received L-NAME (50 mg/kg/day) for 40 days, one group of animals was without L-NAME for the remaining 40 days. In addition, stereology was performed to determine the volume and numerical densities of myocytes (Vv and Nv, respectively), the mean volume and the total number of myocytes (Vol and N, respectively). According to those results, the blood pressure increased following L-NAME administration and remained high even at 40 days after administering L-NAME. The cardiac weight significantly increased in L-NAME animals and also in control of older animals. Moreover, Vv and the Vol increased in older animals. Notably, inhibition of the NO synthase increased Vol while decreasing Nv and N. These indices remained unchanged even after 40 days of the L-NAME intake interruption. Nv and N also decreased in older animals. Furthermore, hypertrophy and loss of myocytes were not entirely reversible after cessation of the chronic inhibition of the NO synthase in an extended follow-up.

The Effects of Spironolactone Monotherapy on Blood Pressure and Myocardial Remodeling in Spontaneously Hypertensive Rats: A Stereological Study

The aim of this study was to evaluate the cardiac structure of spontaneously hypertensive rats (SHRs) treated with different doses of spironolactone. Twenty SHRs were separated into four groups and treated for 13 weeks, as follows: one control group and three spironolactone treatment groups receiving doses of 5, 10 or 30 mg/kg/day. The spironolactone treatment either attenuated or prevented the tendency for increased blood pressure. However, the myocardial structure was not significantly affected by the spironolactone monotherapy treatment (all doses); it showed hypertrophied cardiac myocytes, focal areas of reactive fibrosis, inflammatory infiltrate and a decrease in the density of intramyocardial microvessels. None of the cardiac myocyte stereological parameters in the left ventricular myocardium showed significant differences among the SHR groups. The cardiac myocyte volume density was around 80%, the cardiac myocyte surface density varied from 3.6 to 4.1 x 10(4) mm2/mm3 and the cardiac myocyte mean cross-sectional area varied from 351 to 415 micro m2. The connective tissue volume density of the SHRs treated with the highest dose of spironolactone was 75% lower than in the control SHRs, and this was the only significant difference found for this parameter among SHR groups. The intramyocardial vessels showed some differences when the control SHRs and the other SHRs were compared. The lowest intramyocardial vessel volume density was found in the control group (more than 20% lower than that in the treated SHRs), but no significant difference was detected among the treated SHRs (all doses). The intramyocardial vessel length density (Lv[v]) and surface density (Sv[v]) showed a similar tendency, being significantly greater in the treated SHRs than in the control rats. The Lv[v] was 45% greater in the high-dose spironolactone group than in the control group, and it was 28% greater in the high-dose spironolactone SHRs than in the other treated SHRs. The Sv[v] was 50% greater in the high-dose spironolactone SHRs than in both control and low-dose spironolactone SHRs. Long-term spironolactone monotherapy showed a partial effect in the preservation of intramyocardial vessels and also in the attenuation of interstitial fibrosis.

Renal cortical remodelling by NO-synthesis blockers in rats is prevented by angiotensin-converting enzyme inhibitor and calcium channel blocker

The cortical remodelling was studied when chronically nitric oxide synthesis (NOs) blockade (L‐NAME‐induced) hypertensive rats are simultaneously treated, or not, with angiotensin‐converting enzyme inhibitor or calcium channel blocker. Four groups of eight rats each were studied as follows: Control (C), L‐NAME (L), L‐NAME+Enalapril (L+E) and L‐NAME+Verapamil (L+V). The systolic blood pressure (SBP) was weekly recorded. The cortex of the left kidneys was analysed according to the vertical section design. The volume‐weighted mean glomerular volume (VWGV) was made through the “point‐sampled intercepts” method. Enalapril and verapamil were efficient in reducing the SBP in rats submitted to NOs blockade. Glomeruli had considerable alterations in L group rats (glomerular hypertrophy or sclerosis) and tubular atrophy. The VWGV was 100% greater in L group rats than in the C group rats, while it was 30% smaller in L+E and L+V groups than in L group. The tubular volume was 30–50% greater, while the tubular lenght was 20–30% smaller in L group than in the other groups. The renal cortical region showed glomerular sclerosis/hypertrophy and tubular remodelling in rats with NOs blockade that was efficiently prevented with the simultaneous treatment with enalapril or verapamil.

Myocardial microcirculation stereological changes in rats subjected to nitric oxide synthesis inhibition.

This work aims to study stereological changes in intramyocardial blood vessels in rats submitted to nitric oxide (NO) synthesis inhibition within different periods. NO synthesis inhibition was achieved by administration of L-NAME (50 mg/kg/day); control and L-NAME rats were sacrificed 25 and 40 days after experimentation. Light microscopy and stereology [according to references 7, 13 and 14] were used for analyzing the myocardium. Arterial blood pressure and cardiac weight increased by 74.5% and 57.8% after 25 days and by 90.2% and 34.6% after 40 days, respectively. Comparing the L-NAME rats with corresponding controls revealed that the volume density of the vessels decreased by 31.3% after 40 days, and the length density by 53.5% after 25 days and by 25.7% after 40 days. The mean cross-sectional area of the vessels increased by 154.6% after 25 days. In this study on intramyocardial vessels, we observed an important decrease of the length density in L-NAME animals. Likewise, the volume density also decreased significantly in L-NAME animals. The mean cross sectional area of the vessels, which normally increases during cardiac growth between 25 and 40 days, was precociously increased in L-NAME animals at 25 days, suggesting that these animals suffer from a precocious increase of the heart (including blood vessels) due to pressure overload. Stereology of cardiac microvessels revealed remodeling of these vessels in rats under NO synthesis inhibition. Although these changes may be caused by NO inhibition and not by arterial hypertension, further comparative studies on different models of arterial hypertension are needed to confirm this hypothesis.

Renal cortex remodeling in nitric oxide deficient rats treated with enalapril.

The kidney NO synthase is one of the most important renal controlling systems. This paper aims the quantification of renal cortical components involved in blood pressure regulation under NOs blockade. Spontaneous hypertensive rats (SHRs) are submitted to chronic blockade of NOs by L‐nitro‐arginine‐methyl‐ester (L‐NAME) and an ACE inhibitor (enalapril) in comparison with the normotensive Wistar rats. Twenty SHRs and 5 Wistar rats were divided in 5 groups and observed for 21 days for blood pressure (BP) and serum creatinine: control Wistar (5) (C‐W), control SHR (5) (C‐SHR), L‐SHR (5) ‐ received L‐NAME 30 mg/kg/day, L+E‐SHR (5) ‐ received L‐NAME and Enalapril maleate 15 mg/kg/day, E‐SHR (5) ‐ received Enalapril maleate. A quantitative morphometric study (glomerular density, QA[g1], interstitium volume density, Vv[i], tubular surface and length densities, Sv[t] and Lv[t]) were performed at the end. The BP reached 226±15 mmHg in L‐SHR group. The BP difference between the L‐SHR and the C‐SHR groups was significant from the first week while the E‐SHR group became significant from the second week. At the end of the experiment the BP of the E‐SHR group was similar to the BP in the C‐W group. The QA[g1] was similar among C‐SHR, L‐SHR and L+E‐SHR groups and no difference was found between E‐SHR and C‐W groups. In the L‐SHRs serum creatinine was greatly increased, and microscopy showed thickening of arteriolar tunica media with an increase of the wall‐to‐lumen ratio, perivascular fibrosis, inflammatory infiltrated, tubular atrophy and interstitial fibrosis with focal segmental glomerulosclerosis. The use of enalapril was not completely efficient in reducing BP and morphological injury when the hypertension of SHRs was increased with the NOs blockade suggesting that NO deficiency‐induced hypertension is not entirely mediated by the RAAS.

Enalapril attenuates cardiorenal damage in nitric-oxide-deficient spontaneously hypertensive rats.

Stereological structural alterations of the heart and kidney were studied in four groups (n=5) of spontaneously hypertensive rats (SHRs) treated for 30 days: (i) control, (ii) NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthesis inhibitor] alone, (iii) enalapril alone and (iv) L-NAME plus enalapril. Blood pressure (BP) was elevated significantly in NO-deficient SHRs (rats receiving L-NAME) or significantly lower in enalapril-treated SHRs. Co-administration of L-NAME and enalapril caused a 20% decrease in BP compared with untreated SHRs. NO-deficient SHRs had a decrease in body mass, but this loss of body mass was prevented efficiently in the enalapril-treated group. Enalapril treatment decreased the left ventricular (LV) mass index in SHRs, even in animals with NO synthesis blocked. NO deficiency in SHRs caused a larger decrease in the number of LV cardiomyocyte nuclei, which had a negative correlation with both LV mass index and BP. The volume-weighted glomerular volume (VWGV) separated the SHRs into two groupings: (i) control and NO-deficient SHRs, and (ii) enalapril- and L-NAME plus enalapril-treated SHRs. There was a significant difference between these two groupings, with VWGV being more than 15% smaller in the latter compared with the former grouping. The present findings reinforce the evidence that enalapril efficiently treats genetic hypertension, and demonstrate that this effect is observed even when NO synthesis is inhibited. Enalapril administration also decreases cardiac and renal structural damage caused by genetic hypertension, as well as by the interaction between genetic hypertension and NO deficiency.

Morfologia e estereologia do miocárdio em ratos hipertensos. Correlação com o tempo de inibição da síntese do óxido nítrico

PURPOSE To study structural changes of the myocardium in relation to the time of nitric oxide synthesis inhibition. METHODS Four groups of 10 rats each were studied: 2 control groups and 2 groups with administration of L-Name (50 mg/kg/day), one during 25 days and the other during 40 days. The animals were then sacrificed and the hearts were prepared for study in light microscopy, where sections strained by picro-sirius were studied with and without polarized light for analysis of the cardiac interstitium collagen. Volume densities of myocytes (Vv[m]) and interstitium (Vv[int]), the numerical density of myocytes (Nv[m]) and the mean cross-sectional area of myocytes were (A[m]) also determined. RESULTS The L-Name animals were compared with the respective controls. In the L-Name rats, the tail arterial pressure increased 74.5 and 90.2% in the 25 days group and in the 40 days group, respectively. The heart weight increased 50% in the 25 days group and 28.6% in the 40 days group. The myocardium of the L-Name animals presented myocyte hypertrophy with increased A(m), perivascular and interstitial fibrosis, thickness of the tunica intima and tunica media of the intramyocardial arteries. In the 40 days group the L-Name animals had decreased Vv(m) and Nv(m) and increased Vv(int). CONCLUSIONS Inhibition of nitric oxide synthesis provokes myocardial changes that progress with the time of L-Name administration. The stereology is useful to determine and to evaluate the myocardial changes in this model of arterial hypertension.

Beneficial effect of enalapril in spontaneously hypertensive rats cardiac remodeling with nitric oxide synthesis blockade.

Aims. To study the efficiency of an angiotensin converting enzyme inhibitor on the blood pressure (BP) and the myocardium remodeling when spontaneously hypertensive rats (SHRs) are submitted to nitric oxide synthesis (NOs) blockade (with L‐NAME) and simultaneously treated.