Neonatal Glucocorticoid Treatment and Predisposition to Cardiovascular Disease in Rats

Abstract

The present thesis describes the issue of “neonatal glucocorticoid treatment and predisposition to cardiovascular disease in rats”. Glucocorticoid treatment, in particular dexamethasone, is widely used to treat or prevent chronic lung disease in premature infants. However, short and long-term side effects have been reported both in animal and human studies. Especially, the long-term neurological side effects such as cerebral palsy and delayed and abnormal neurological development have been studied. With regard to the cardiovascular system, the short-term side effects are well documented, but the long-term effects are still largely unknown. However, recent studies in adult rats indicated negative long-term effects of neonatal dexamethasone treatment on the heart and a severely reduced life expectancy. In this thesis we have therefore investigated the functional, histopathological and biochemical consequences of neonatal dexamethasone treatment on the heart during life span in rats. An earlier study from our group showed that neonatal dexamethasone treatment in rat pups caused a decrease in heart weight, probably due to inhibition of myocyte mitotic activity during treatment. Moreover this treatment resulted in permanent histopathological changes, in particular cellular hypertrophy, in the adult heart. We investigated the early effects of neonatal dexamethasone treatment in rats on cell proliferation after birth. We tested the hypothesis that a lower number of cardiomyocytes later in life was caused by a dexamethasone-induced, diminished cardiomyocyte proliferation and/or early cell death. This is a plausible assumption in view of the fact that GCs are administered during a critical period of cardiac development. In an experimental but established rat model, histopathological and immunohistochemical studies were performed to look at signs of apoptosis and/or differences in proliferation capacity of cardiomyocytes before, during and after dexamethasone treatment. Male rat pups were injected intraperitoneally with dexamethasone on day 1, 2, and 3 (0.5, 0.3 and 0.1 μg/g) of life. Control pups received saline in equal volumes. The rats were sacrificed at day 0, 2, 4, 7 and 21, and hearts were harvested. We found that dexamethasone treatment caused temporary suppression of cardiomyocyte hyperplasia which will lead to a reduced number of cardiomyocytes during life. To investigate the long-term cardiovascular side effects, cardiac function was determined after neonatal treatment with dexamethasone in 4-, 8-, 50- and 80-week-old rats, representing pre-pubertal, young adult, middle aged and elderly stages. The animals were anesthetized, intubated and ventilated, and a miniature pressure-conductance catheter was introduced into the left ventricle to measure pressure-volume loops. Cardiac function was measured and ventricular pressure-volume relations were determined to quantify intrinsic systolic and diastolic function, virtually independent of loading conditions. Subsequently, hearts were excised for histological examination. Our results showed reduced ventricular weights in 4-, 8- and 80-week-old dexamethasone-treated rats, but not in 50-week old rats. We showed a reduced systolic function in the 4-week-old dexamethasone-treated rats (end-systolic elastance: 1.24±0.43 vs. 2.50±1.39mmHg/μL, p=0.028) accompanied with a maintained cardiac output and increased end-diastolic volume (8423 vs. 5919μL, p=0.012), indicating a state of compensatory dilatation. These relatively early alterations in cardiac function were not followed by more severe dysfunction at 8-weeks. In fact at 8-weeks no difference in systolic function was detected between the dexamethasone-and Sal-treated rats. In the 50- and 80-week-old rats however, systolic function was depressed as evidenced by reduced ejection fractions and rightward shifts of the end-systolic pressure-volume relationships. Histopathologically, we found that dexamethasone treatment affects normal growth of the heart resulting in reduced heart weight, cellular hypertrophy, and increases in collagen deposition in the adult rat heart. The increase in cardiomyocyte cell volume induced by neonatal dexamethasone treatment was associated with long-lasting changes in the ventricular expression of contractile proteins (α-actin, MHC and β-MHC) without changes in the expression of structural proteins such as desmin and tubulin. It is conceivable that the increases in contractile proteins contribute to maintain cardiac function as an adaptive or compensatory response to the reduced cardiac cell number previously found. The studies described in this thesis support our hypothesis that neonatal exposure to dexamethasone in rats has serious life-long adverse consequences on the heart and consequently question the use of early neonatal GC treatment in the human setting. If our findings are confirmed in humans, this may have consequences for a potentially large patient population that has been treated with dexamethasone during the neonatal period in the early nineties up to now.