Ontogenetic changes of lipofuscin-like pigments in the rat heart

Increased generation of reactive oxygen species results in the formation of fluorescent end-products of lipid peroxidation - lipofuscin-like pigments (LFP). LFP increased up to six-fold from the fetal value in the rat heart immediately after birth. In the experimental design of this study the fetuses were sampled 1 day before birth, and then the samples were collected on postnatal days 1, 4, 7, 10, 15, 30, and 60. Males and females were compared on day 30 and 60 when the difference between right and left ventricle was studied as well. Four LFP fluorophores were analyzed: F355/440, F310/470, F350/450, F315/450 (excitation/emission, nm). All fluorophores decreased on day 4 relative to day 1, subsequent transient increases ended in a significant decrease on day 60. However, the LFP levels on day 60 are still about threefold higher than those in fetuses. Differences between male and female hearts were observed on day 30. The corresponding male ventricles contained by one third higher concentration of LFP than the female counterparts. The increase in LFP concentration in male ventricles on day 30 was only transient, no difference between corresponding male and female ventricles was found on day 60. The most distinguished feature in the male heart was a sharp LFP decrease in the right ventricle on day 60.     

Early postnatal development of myocardial stroma in the rat

By means of transmissive electron microscopy and morphometry methods volumetric parts and number of cells in various components of the myocardial stroma have been studied in male Wistar rats at the age up to 45 days. Two age periods have been determined--before and after 10 days. Synchronization of processes in development of the stromal elements is observed.     

Distribution of estramustine-binding protein during postnatal development of rat brain

Estramustine-binding protein (EMBP) is expressed in several types of brain tumors, such as astrocytoma, ependymoma, and meningioma. It binds the cytotoxic drug estramustine with high affinity and is suggested to cause accumulation of the drug in EMBP-expressing tumor cells. In this study, the spatial distribution of EMBP in normal rat brain was studied with immunohistochemistry. Brains from male and female rats of different ages were used. EMBP was found in the cytoplasm of ependymal cells, in the leptomeninges, mainly the arachnoid, and in scattered neurons. Moreover, staining was seen in nuclei of choroid plexus cells, in the granular cell layer in the cerebellum, and in a few scattered endothelial cells. The nuclear staining was more frequent in younger animals. No obvious difference in EMBP expression between male and female rats was observed. The expression of EMBP in rat brain was confirmed with nested RT-PCR. Future studies are justified to elucidate the role of EMBP-like proteins in CNS and in brain tumors.     

Polysomes during early postnatal development of brain in the rat

We have attempted to show eventual modifications in the brain protein synthesis apparatus of rat during the first three weeks after birth. Through this time we noted a steady decrease (about 60%) in the free polysomes, when expressed relative to tissue weight. This decrease does not correlate with changes in the polysome profile, indicating that no loss in the efficiency of protein synthesis was involved. Translation in a reticulocyte lysate also failed to reveal differences.     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Changes in tubulin heterogeneity during postnatal development of rat brain.

Tubulin isolated from rat brain at various stages of postnatal development was subjected to isoelectric focusing on polyacrylamide gels. Multiple bands, indicative of the heterogeneity of the protein, were apparent at all developmental ages. When isoelectric focusing patterns of tubulin from brains of increasing developmental age were compared, changes in the distribution and relative intensities of the bands were observed. These changes were most pronounced between 8–12 days of age and were seen whether the tubulin was isolated by DEAE-cellulose chromatography or by successive cycles of assembly-disassembly. The isoelectric focusing pattern of tubulin isolated from the 22-day-old animal was indistinguishable from that of the protein obtained from 30-day-old rat brain. These developmental changes in tubulin heterogeneity may relate to changes in the assembly properties of the microtubule protein or may reflect age-dependent changes in the relative contributions of mitotic spindles, axons, dendrites, and glia to the total pool of tubulin in brain.     

Changes of hippocampal protein levels during postnatal brain development in the rat

Information on postnatal brain protein expression is very limited, and we therefore compared hippocampal protein levels in rat hippocampus at different developmental time points using two-dimensional gel electrophoresis followed by mass spectrometrical protein identification and specific software for quantification. Proteins from several cascades as e.g., antioxidant, metabolic, cytoskeleton, proteasomal, and chaperone pathways were developmentally regulated, which is relevant for design and interpretation of protein chemical studies in the mammalian brain.     

Early murine T-lymphocyte activation is accompanied by a switch from N-Glycolyl- to N-acetyl-neuraminic acid and generation of ligands for siglec-E

It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor.     

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.Dedicated to Professor Helmut Leonhardt on the occasion of his 75th birthday     

Fetal and postnatal development of cyclic GMP phosphodiesterase activity in the rat brain

Cyclic GMP concentration and cyclic GMP phosphodiesterase activity were studied in rat mothers and fetuses at 17, 19 and 21 days of intrauterine life and 0, 1, 4, 10, 15,20, 30 and 45 days after birth. During this developmental period, the increase in cyclic GMP concentration was discrete and the value in 15-day-old rats was already similar to the adult level. Cyclic GMP phosphodiesterase activity increased from 17- to 19-day fetuses and was significantly reduced in 21-day fetuses, neonates, and 1-day-old rats. This reduction may be a result of fetal endocrine preparation for parturition. During postnatal development, cyclic GMP phosphodiesterase activity increased in a parallel way in the limbic system, corpora striata, cerebral hemispheres, and diencephalon, reaching maximal level between 20 and 30 days after birth, and then decreasing to the adult value. The highest activity was found in corpora striata and the lowest in diencephalon. Cerebellar cyclic GMP phosphodiesterase activity was very high in the 4-day-old rat (257% of adult value) and diminished significantly in the 10-day-old rat with no subsequent changes. Kinetic analysis of the enzyme during postnatal forebrain development showed an increase in both the V_max and the apparent K_m. A decrease in the enzyme's V_max was observed only in the cerebellum.The importance of cyclic GMP phosphodiesterase regulation of cyclic GMP concentrations in the brain during development is discussed.     

Rheb1 is required for mTORC1 and myelination in postnatal brain development

mTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in?vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells?abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.     

The acetoacetyl-coenzyme A thiolases of rat brain and their relative activities during postnatal development

1. The apparent 3-oxoacyl-CoA thiolase activity of rat brain extracts is due to two different acetoacetyl-CoA thiolases, one cytoplasmic and the other mitochondrial. By the methods developed in the preceding paper (Middleton, 1973), the changes in activities of these two enzymes were determined during postnatal development. 2. Although the total brain acetoacetyl-CoA thiolase activity changes not more than 2-fold from birth to adulthood this masks large changes in the relative proportions of the two types of thiolase present. 3. Cytoplasmic acetoacetyl-CoA thiolase activity declines slowly from 4 units/g fresh wt. at birth to an adult value of 1.3 units/g fresh wt. 4. The mitochondrial acetoacetyl-CoA thiolase (activated by K(+)) rises rapidly in activity from 1 unit/g fresh wt. at birth to a peak value of 5 units/g fresh wt. at 20 days. After weaning the activity declines to 2.3 units/g fresh wt. in the adult. 5. These different developmental patterns are discussed in terms of the probable metabolic roles of the two brain acetoacetyl-CoA thiolases.     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

the present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.     

Early effects of lipopolysaccharide-induced inflammation on foetal brain development in rat

Studies in humans and animal models link maternal infection and imbalanced levels of inflammatory mediators in the foetal brain to the aetiology of neuropsychiatric disorders. In a number of animal models, it was shown that exposure to viral or bacterial agents during a period that corresponds to the second trimester in human gestation triggers brain and behavioural abnormalities in the offspring. However, little is known about the early cellular and molecular events elicited by inflammation in the foetal brain shortly after maternal infection has occurred. In this study, maternal infection was mimicked by two consecutive intraperitoneal injections of 200 μg of LPS (lipopolysaccharide)/kg to timed-pregnant rats at GD15 (gestational day 15) and GD16. Increased thickness of the CP (cortical plate) and hippocampus together with abnormal distribution of immature neuronal markers and decreased expression of markers for neural progenitors were observed in the LPS-exposed foetal forebrains at GD18. Such effects were accompanied by decreased levels of reelin and the radial glial marker GLAST (glial glutamate transporter), and elevated levels of pro-inflammatory cytokines in maternal serum and foetal forebrains. Foetal inflammation elicited by maternal injections of LPS has discrete detrimental effects on brain development. The early biochemical and morphological changes described in this work begin to explain the sequelae of early events that underlie the neurobehavioural deficits reported in humans and animals exposed to prenatal insults.     

Changes of lipofuscin-like pigments in erythrocytes and spleen after whole-body gamma irradiation of rats

Whole-body gamma irradiation of rats induced the formation of lipofuscin-like pigments in erythrocytes. Erythrocytes that were damaged by oxidation were scavenged in the spleen, and lipofuscin-like pigments were transferred from erythrocytes to the spleen during this process. The time course of lipofuscin-like pigments in erythrocytes and spleen indicates that the pigments were not induced by the action of free radicals produced by ionizing radiation but rather were a sequela of postirradiation metabolic changes.     

Neonatal monosodium glutamate treatment modifies glutamic acid decarboxylase activity during rat brain postnatal development

Monosodium glutamate (MSG) produces neurodegeneration in several brain regions when it is administered to neonatal rats. From an early embryonic age to adulthood, GABA neurons appear to have functional glutamatergic receptors, which could convert them in an important target for excitotoxic neurodegeneration. Changes in the activity of the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), have been shown after different neuronal insults. Therefore, this work evaluates the effect of neonatal MSG treatment on GAD activity and kinetics in the cerebral cortex, striatum, hippocampus and cerebellum of the rat brain during postnatal development. Neonatal MSG treatment decreased GAD activity in the cerebral cortex at 21 and 60 postnatal days (PD), mainly due to a reduction in the enzyme affinity (K(m)). In striatum, the GAD activity and the enzyme maximum velocity (V(max)) were increased at PD 60 after neonatal MSG treatment. Finally, in the hippocampus and cerebellum, the GAD activity and V(max) were increased, but the K(m) was found to be lower in the experimental group. The results could be related to compensatory mechanisms from the surviving GABAergic neurons, and suggest a putative adjustment in the GAD isoform expression throughout the development of the postnatal brain, since this enzyme is regulated by the synaptic activity under physiological and/or pathophysiological conditions.