Perinatal changes in amylase and serum corticosterone levels in rats

In rats amylase activity in the pancreas increased greatly from day 15 of gestation to a maximum on day 21. Then it decreased to less than one-tenth of this maximum value on about day 5 after birth. It increased again about 15 days after birth and reached the adult level about 30 days after birth.No amylase activity was in the parotid gland before birth: it appeared about 12 days after birth and reached the adult level, which was higher than that in the pancreas, about 30 days after birth.The serum corticosterone level was as high as the adult level before birth. Then it decreased to less than one-tenth of the adult level 5 days after birth and increased again from 15 to 25 days after birth to the adult level. The developmental change in the serum corticosterone level seemed to influence amylase activity in the pancreas both before and after birth, and that in the parotid gland only after birth.The serum contained both pancreatic and paratoid type isozymes of amylase until 1 day after birth but only the parotid type from 3 days after birth.     

Expression and localization of caveolins during postnatal development in rat heart: implication of thyroid hormone.

Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.     

The development of sympathetic innervation and the functional state of the cardiovascular system in newborn dogs.

The present study in dogs indicates that the peripheral sympathetic fibers develop mostly after birth and reach a full maturity at about 2 months of life. The norepinephrine content of the heart, spleen, intestine, salivary glands, and adrenal glands increased from birth to 56 days of age. In contrast, the content of the stellate ganglia decreased during this period. In most of the organs studied, the uptake of [3H] norepinephrine developed in parallel with the norepinephrine content, except in the right atrium and salivary glands where it was fully developed soon after birth. During development, the systemic blood pressure increased from 40 to 100 mm Hg. Bilateral adrenal vessel clamping failed to induce a fall in blood pressure in growing dogs which indicates that the adrenal medulla or the baroreceptors did not fully compensate for the lack of peripheral sympathetic fibers and for the lower blood pressure in newborn animals. Although cardiac norepinephrine content was still very low in 10-day-old animals, cardiovascular responses to direct and reflex sympathetic stimulation were similar to those observed in 56-day-old animals. These results indicate that the sympathetic nervous system becomes functional before the fibers reach their full maturity.     

Cardiac tolerance to ischemia in neonatal spontaneously hypertensive rats

Hypertension is the risk factor of serious cardiovascular diseases, such as ischemic heart disease and atherosclerosis. The aim of the present study was to analyze the development of cardiac tolerance to ischemia in neonatal spontaneously hypertensive rats (SHR) and possible protective effect of ischemic preconditioning (IP) or adaptation to intermittent high-altitude hypoxia (IHAH). For this purpose we used 1- and 10-day-old pups of SHR and their normotensive control Wistar Kyoto rats (WKY). Isolated hearts were perfused in the Langendorff mode with Krebs-Henseleit solution at constant pressure, temperature and rate. Cardiac tolerance to ischemia was expressed as a percentage of baseline values of developed force (DF) after global ischemia. IP was induced by three 3-min periods of global ischemia, each separated by 5-min periods of reperfusion. IHAH was simulated in barochamber (8 h/day, 5000 m) from postnatal day 1 to 10. Cardiac tolerance to ischemia in 1-day-old SHR was higher than in WKY. In both strains tolerance decreased after birth, and the difference disappeared. The high cardiac resistance in 1- and 10-day-old SHR and WKY could not be further increased by both IP and adaptation to IHAH. It may be concluded that hearts from newborn SHR are more tolerant to ischemia/reperfusion injury as compared to age-matched WKY; cardiac resistance decreased in both strains during the first ten days, similarly as in Wistar rats.     

Perinatal development of hepatic cholesterol synthesis in the rat

Rates of cholesterol synthesis and HMG CoA reductase activity in rat liver, have been reported to be high before and low after birth. The timing of the decline in perinatal rates of cholesterol synthesis, however, is uncertain. These studies, therefore, determined in vivo rates of cholesterol synthesis using [3H]water and hepatic reductase activity in vitro in perinatal rats. The lipid composition of the plasma, liver and its microsomal subfraction were also determined. Reductase activity increased during late gestation, remained high immediately after birth, then decreased with the commencement of suckling. Rates of cholesterol synthesis increased from gestation day 18 to 20, but in contrast to reductase activity, decreased on the day before birth. Plasma cholesterol and triacylglycerol levels increased to gestation day 19, then decreased to term. By the 6th h after birth, plasma and liver cholesterol and triacylglycerol levels had increased markedly. By 48 h after birth, the high hepatic cholesterol content was associated with an increase in the cholesteryl ester fraction. The microsomal cholesterol/phospholipid molar ratio decreased from gestation day 16 until 12 h after birth, then increased markedly from 36 to 48 h. There was an apparent inverse relationship between the change in microsomal cholesterol/phospholipid molar ratio and the fatty acid unsaturation index from gestation day 16 to 36 h after birth. The results suggest that in late gestation and before suckling, the low in vivo rate of hepatic cholesterol synthesis may not be due to low activity of HMG CoA reductase.     

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.     

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.     

Thymidine kinase activity in cardiac muscle during embryonic and postnatal development

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.     

Cholinesterases of rat sympathetic ganglia after immunosympathectomy, decentralization and axotomy

Abstract— Immunosympathectomy was produced in Sprague-Dawley rats by the subcutaneous injection of 300 units of nerve growth factor (NGF)-antiserum (1.56 mg of freeze-dried serum)/g/day for 6 days, the first dose being given 5–8 hr after birth. The immunosympathectomized rats and their control littermates were killed 2½ and 7 months after birth. Ganglionic acetylcholinesterase and pseudocholinesterase activities were measured by an adaption (Kungman , Kungman and Pouszczuk , 1968) of the colorimetric method of Ellman , Courtney , Andres and Featherstone (1961). Following immunosympathectomy the activities of these enzymes decreased significantly in superior cervical, stellate, thoracic chain, cardiac (abdominal), coeliac and superior mesenteric ganglia. The reduction of the acetylcholinesterase activity was greater than expected in a number of sympathetic ganglia, e.g. superior cervical, stellate, coeliac and cardiac ganglia, if one considered that only the postganglionic neurons were affected by immunosympathectomy. The activities of these enzymes were also reduced in the cervical sympathetic trunks from NGF-antiserum-treated rats. By means of decentralization and axotomy it was shown that 45 per cent of the total ganglionic acetylcholinesterase activity was associated with the preganglionic and 55 per cent with the postganglionic elements of the superior cervical ganglion from control rats. It was concluded that immunosympathectomy also affects the preganglionic sympathetic neurons. It is not known whether this is a primary effect of the NGF-antiserum or a secondary effect resulting from the absence of over 90 per cent of the postganglionic sympathetic cell bodies.     

Ribonuclease H activity in developing rat brain

A ribonucleolytic enzyme (RNase H) which degrades the RNA strand of a RNA-DNA double stranded hybrid has been extracted from rat brain and characterized. RNase H activity in the cerebella increased up to around 6th day after birth and then decreased in adult rat cerebella, just as DNA polymerase and DNA ligase. The RNase H activity in non-cerebellar part decreased gradually toward adult after birth. On the other hand, RNase activity did not change during development of brain. The activity of total RNase is high at newborn, and decreased to 12th day after birth. These results suggest that RNase H is implicated in DNA replication in rat brain.     

Developmental changes of cardiac function and mass assessed with MRI in neonatal, juvenile, and adult mice

Cardiovascular transgenic mouse models with an early phenotype or even premature death require noninvasive imaging methods that allow for accurate visualization of cardiac morphology and function. Thus the purpose of our study was to assess the feasibility of magnetic resonance imaging (MRI) to characterize cardiac function and mass in newborn, juvenile, and adult mice. Forty-five C57bl/6 mice from seven age groups (3 days to 4 mo after birth) were studied by MRI under isoflurane anesthesia. Electrocardiogram-gated cine MRI was performed with an in-plane resolution of (78-117 microm)(2). Temporal resolution per cine frame was 8.6 ms. MRI revealed cardiac anatomy in mice from all age groups with high temporal and spatial resolution. There was close correlation between MRI- and autopsy-determined left ventricular (LV) mass (r = 0.95, SE of estimate = 9.5 mg). The increase of LV mass (range 9.6-101.3 mg), cardiac output (range 1.1-14.3 ml/min), and stroke volume (range 3. 2-40.2 microl) with age could be quantified by MRI measurements. Ejection fraction and cardiac index did not change with aging. However, LV mass index decreased with increasing age (P < 0.01). High-resolution MRI allows for accurate in vivo assessment of cardiac function in neonatal, juvenile, and adult mice. This method should be useful when applied in transgenic mouse models.     

Age-related hematological changes in normal F344 rats: during the neonatal period.

In order to clarify age-related changes in hematological values of normal rats after birth, blood samples from neonatal F344 rats of both sexes were examined periodically during the period from 0 to 40 days postpartum. The erythrocyte count (RBC) increased with time after birth as a function of age. In contrast, the reticulocyte count (Retics) continuously decreased with time after birth. Hemoglobin (Hb), hematocrit (Ht), and the mean corpuscular hemoglobin concentration (MCHC) tended to decrease after birth until weaning (about 21 days postpartum), but they began to increase after weaning. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) also gradually decreased after birth until weaning, but they were unchanged thereafter. The platelet count (PLT) gradually increased after birth and reached a plateau at weaning. Microscopic examination of blood smears revealed that erythrocytes at birth had characteristic morphological features such as anisocytosis, polychromasia, basophilic stippling, Howell-Jolly body, and erythroblastosis. These characteristic features, however, disappeared by 30 days after birth. The total leukocyte count (WBC) gradually increased with time after birth, due to an increase in the number of lymphocytes. The lymphocyte count started to rapidly increase within several days after birth and the increase continued thereafter. Other differential leukocyte counts also showed various characteristic patterns of changes during the neonatal period. There were no apparent differences between males and females in these changes in hematological values.     

Glycogen metabolizing enzyme activities in the developing rat liver

1. The glycogen content of rat liver increased prenatally and dramatically decreased after the birth. 2. Glycogen synthase and glycogen phosphorylase activities increased prenatally, declined at 12 hr after birth and then again increased. 3. Phosphorylase kinase activities did not significantly change prenatally but steadily increased after the birth. 4. Protein kinase activities (units/mg liver) did not change prenatally but slightly increased after the birth. 5. Phosphoprotein phosphatase activities using phosphorylase a and histone as substrates dramatically increased at birth and then decreased after 24 hr of the birth.     

LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low‐density lipoprotein receptor‐related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac‐specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin‐dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.     

Plasma and adrenal corticosterone concentrations and liver glycogen content in premature mice at birth and during neonatal development

The concentrations of corticosterone in the plasma and adrenal glands and the content of glycogen in the liver were estimated from birth to day 6 after birth in surviving premature mice removed by Caesarean section on day 19 of pregnancy and submitted to reanimation during 30 min; the neonates were nourished by nursing mothers from 30 min after birth. A group of full-term newborns was removed by Caesarean section on day 20 of pregnancy and killed 30 min after reanimation. Premature mice were characterized by neonatal changes of three parameters used. The plasma corticosterone level reached a peak in the first 6 h after birth, then decreased until day 6. The adrenal corticosterone level did not vary significantly 30 min after birth, then decreased progressively until day 2. The liver glycogen content, very high on day 19 of pregnancy, increased 30 min after birth, then fell sharply until day 2. In full-term newborns removed by Caesarean section and killed 30 min after reanimation the plasma corticosterone level increased, whereas the adrenal corticosterone level and the liver glycogen content did not decrease. The adrenal gland of surviving premature mice was able to respond to the stress induced by the reanimation; the stimulation of glucocorticoid function was similar in both neonates.     

Proliferation of cardiomyocytes and interstitial cells in the cardiac muscle of the mouse during pre- and postnatal development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. The number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. The ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Proliferation of Cardiomyocytes and Interstitial Cells In the Cardiac Muscle of the Mouse During Pre- and Postnatal Development

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. the number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. the ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.     

Consequences of cardiac myocyte-specific ablation of KATP channels in transgenic mice expressing dominant negative Kir6 subunits

Cardiac ATP-sensitive K+ (K(ATP)) channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice that express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to nontransgenic mice, but an increased mortality was noted after the age of 4-5 mo. Transgenic mice lacked cardiac K(ATP) channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed EADs after isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate), whereas in vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The proarrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggest that these features are intrinsic to the myocardium and that K(ATP) channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations.     

Ontogeny of reactive nitrogen species production by blood phagocytes in pigs

The aim of this work was to evaluate ontogeny of reactive nitrogen species (RNS) production by peripheral blood phagocytes in pig. Pig fetuses (55 and 92 days of gestation) and postnatal piglets (1, 3, 8, 17, 31 and 41 days after birth) were used. RNS production was measured by fluorescent probes diaminofluorescein-diacetate (DAF-FMDA) and dichloro-fluorescein-diacetate (H2DCFDA). Levels of nitration of cell proteins were established by immunofluorescent detection of nitrotyrosine. Levels of plasma nitrites/nitrates were detected spectrophotometrically by Griess reaction. Nitric oxide production measured by DAF-FMDA in neutrophils decreased during postnatal life. Spontaneous RNS measured by H2DCFDA decreased from 55th day of gestation to the 41st day of life. Phorbol-12-myristate-13-acetate activated production decreased during postnatal life. Production of NO measured by DAF-FMDA in macrophages decreased from the first to 41st day after birth. RNS production measured by H2DCFDA in monocytes did not show any significant changes during ontogeny. The level of nitrotyrosine significantly decreased from the third to 17th day. Levels of plasma nitrites/nitrates gradually decreased from the 55th day of gestation to the 41st day after birth. A temporary increase in all parameters occurred after weaning, but without any significance. In conclusion, RNS production has a decreasing trend during ontogeny and is transiently upregulated after weaning.