Early Neonatal Stage Research Articles

Hyperbaric oxygenation prevented brain injury induced by hypoxia–ischemia in a neonatal rat model

The occurrence of hypoxia–ischemia (HI) during early fetal or neonatal stages of an individual leads to the damaging of immature neurons resulting in behavioral and psychological dysfunctions, such as motor or learning disabilities, cerebral palsy, epilepsy or even death. No effective treatment is currently available and this study is the first to use hyperbaric oxygen (HBO) as a treatment for neonatal HI. Herein, we sought out to determine if HBO is able to offer neuroprotectivity against an HI insult. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O 2 at 37 °C). HBO treatment was administered by placing pups in a chamber (3 ATA for 1 h) 1 h after hypoxia exposure. Brain injury was assessed based on ipsilateral hemispheric weight divided by contralateral hemispheric weight, light microscopy, and EM. Sensorimotor functional tests were administered at 5 weeks after hypoxia exposure. After HI, the ipsilateral hemisphere was 52.65 and 57.64% ( P<0.001) of the contralateral hemisphere at 2 and 6 weeks, respectively. In HBO treated groups, the ipsilateral hemisphere was 77.77 and 84.19% ( P<0.001) at 2 and 6 weeks. There was much less atrophy and apoptosis in HBO treated animals under light or electron microscopy. Sensorimotor function was also improved by HBO at 5 weeks after hypoxia exposure (Chi-square, P<0.050). The results suggest that HBO is able to attenuate the effects of HI on the neonatal brain by reducing the progression of neuronal injury and increasing sensorimotor function.

Gene expression of SERCA2a and L- and T-type Ca channels during human heart development.

In this study we report, for the first time, on the gene expression of human cardiac SERCA2a, L-type (alpha(1C)) and T-type (alpha(1H)) Ca channels during development, using RNase protection assay, relative quantitative RT-PCR and Western blot. Human hearts during early gestation (8- to 20-wk gestation), neonatal (1- to 4-d-old) and adult (18- to 48-year-old) stages were used. The results show that T-type Ca channel alpha(1H) subunit mRNA decreased and that L-type Ca channel alpha(1C) subunit mRNA increased with development. While the levels of sarcoplasmic reticulum ATPase (SERCA2a) mRNA did not significantly change with development, its protein levels increased with development. In conclusion, SERCA2a, L-type and T-type Ca channel transcripts were detected as early as 8-wk gestation. Defining the profile of Ca handling proteins during development is important to the understanding of excitation-contraction (EC)-coupling of the developing human heart.

Canonical Heat Shock Element in the αB-crystallinGene Shows Tissue-specific and Developmentally Controlled Interactions with Heat Shock Factor

Oligomerization of the heat shock factor (HSF) and its interaction with the heat shock element (HSE) are the hallmark of active transcriptional response to tangible physical or chemical stress. It is unknown if these interactions are subject to control and modulation by developmental cues and thus have tissue or stage specificity. By using promoter sequences containing a canonical HSE from the alphaB-crystallin gene, we demonstrate a tissue-specific transition from monomeric (in fetal and early neonatal stages that lack oligomeric HSF.HSE complexes) to oligomeric HSF-HSE interactions by postnatal day 10-21 in the ocular lens. Developmental control of these interactions is further demonstrated by induction of oligomeric HSF.HSE complexes in neonatal extracts by in vitro manipulations, interestingly, only in the lens and not in the brain, heart, or liver extracts. The exclusive presence of oligomeric HSF.HSE complexes in the postnatal/adult lens corresponds to known highly increased number of alphaB-crystallin transcripts in this tissue.

Galanin-like immunoreactive endocrine cells in bovine pancreas.

Pancreata of fetal, neonatal and adult cattle were studied immunohistochemically for galanin. The results revealed galanin-like immunoreactivity both in the endocrine cells and in the neural elements. The galanin-like immunoreactive endocrine cells (Gal-LIEC) were confined to the large islets, and were not observed in the islets of Langerhans and exocrine pancreas. They were first detected at the third prenatal month. Their developmental profile showed an increase from fetal to early neonatal stage with a subsequent decrease towards adulthood. The considerable number of Gal-LIEC from late prepartum to early postpartum stage may imply functional significance of galanin during the perinatal development of cattle. Coexistence of galanin and insulin was also observed which may suggest autocrine interaction between the 2 hormones.

Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene

To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-).delta(-/-) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPbeta(-/-).delta(-/-) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPbeta(-/-).delta(-/-) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-).delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrated that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta.

Developmental changes of cyclin dependent kinase 5 subcellular localization in the rat cerebellum.

Cyclin dependent kinase 5 (Cdk5) phosphorylates tau protein, a microtubule-associated protein, at pathological sites in vitro as well as in Alzheimer's disease brain. The enzyme is therefore regarded as an important candidate responsible for the progression of Alzheimer's disease. We and others have suggested that the enzyme has physiological roles in brain development and maturation. In this study, we investigated the exact distribution and developmental changes of the enzyme in cerebellum by immunohistochemistry and non-radioactive in situ hybridization. Immunohistochemistry demonstrated that Cdk5 was consistently expressed in the cerebellum at all developing stages. However, the subcellular localization of Cdk5 dramatically changed during maturation of the cerebellum. In the early neonatal stage, Cdk5 was strongly expressed in the cell bodies of neurones. With neuronal maturation Cdk5 immunoreactivity changed its subcellular localization from the cell body to axon. In terminally differentiated neurons, the immunoreactivity was only detected in the axon. These results suggest that subcellular localization of Cdk5 is strictly regulated and may play an important role in neuronal maturation.

The ontogeny of gene expression of progestin receptors in the female rat brain

The postnatal development of the progestin receptor (PR) system in the rat brain is a region-specific and stage-related process. In an attempt to analyze the molecular mechanism by which the dramatic change of gene expression of the PR occurs we have examined the level of PR mRNAs in the hypothalamus-preoptic area (HPOA) and cerebral cortex in development from fetal to postnatal stages of female rats. We used polymerase chain reaction to clone, from uterine cDNA, the cDNA corresponding to the steroid-binding domain of the PR forms ‘A’ and ‘B’ mRNA as well as the region around the translation-initiation site (ATG1) of the putative PR form ‘B’ mRNA. A quantitative reverse transcription-polymerase chain reaction assay was used to measure the level of mRNAs for PR forms ‘A’ and ‘B’ (total PR mRNAs) and PR form ‘B’. There was a regional difference in the intracerebral distribution between the total and form ‘B’ mRNAs, indicating possible distinct mechanisms responsible for regulating the expression of the PR mRNAs. The PR mRNAs in the brain, already detectable 2 days before birth, increased at early neonatal stages. The total PR mRNAs in the cortex developed in a manner essentially similar to the PR protein at the early stages, but, surprisingly, unlike the receptor, the message remained high at the later stages from day 18 to 8 weeks of life. On the other hand, the ontogeny of the cortical mRNA for form ‘B’, which predominantly existed in the region, resembled that of the cortical PR protein. In the HPOA the postnatal development of the form ‘B’ mRNAs was also roughly similar to the PR. These results suggest region-specific and stage-related gene expression of the PR isoform system in the developing brain: gene expression of form ‘B’ seems to be predominantly, first, “turned on” around birth, followed by form ‘A’ mRNA expression around days 8–12. Moreover, lowered levels of the cortical PR mRNAs in the propylthiouracil-induced hypothyroid rat, together with suppressed PR level, indicate a possible regulatory role of thyroid hormone on gene expression of the cortical receptor.

Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo.

A myocyte-specific enhancer-binding factor (MEF-2) DNA binding site was identified in the rat alpha-myosin heavy chain (MHC) gene adjacent to the E-box binding site for alpha-MHC binding factor-2 (BF-2). Mutation of the MEF-2 site, within the context of the full-length promoter, reduced activity by 85 and 80% in neonatal cardiomyocytes and the adult heart, respectively. Mutation of the BF-2 site reduced activity approximately 70% in both models. A MEF-2/BF-2 double mutant gave significantly less activity than the BF-2 mutant but not the MEF-2 mutant, suggesting the possibility that BF-2 and MEF-2 interact. Mutations in MEF-2, which decreased functional activity, also abolished MEF-2 DNA binding activity. MEF-2 DNA binding activity was present in the developing heart, reached a peak in the late fetal and early neonatal stages, and then declined to low levels in the adult heart. The adult levels were sufficient to support alpha-MHC gene expression. MEF-2 activity was increased 2-3-fold in the adult heart subjected to a pressure or volume overload. Two working models are proposed as possible explanations of the antithetic relationship between MEF-2 levels and alpha-MHC gene expression.

The mechanism of formation of alveolar pores (Kohn's pores) in neonatal rabbits

The mechanism of the formation of alveolar pores (Kohn's pores) in neonatal male rabbits was studied by scanning electron microscopy and scanning electron microscopic morphometric analysis. The number of alveolar pores per alveolus was increased after birth. About 80 percent of alveolar pores had Type II alveolar epithelial cells on their edges when the rabbits were 1-day old. This percentage decreased thereafter. Circle-like intercellular junctions of Type I cells were frequently observed in or around the alveolar pores and Type II cells frequently attached to these junctions in the early neonatal stage. The alveolar pores with Type II cells which were mainly faced this side of alveolar lumen, and Type II cells which were separated from the pores tended to be found at the points where three or more Type I alveolar epithelial cells gathered in the early neonatal stage. However, alveolar pores with Type II cells which were mainly faced other side of the alveolar lumen, and alveolar pores without Type II cells tended to be found between two Type I cells. At the rims of the pore, crescent-shaped Type II cells located between circle-like intercellular junctions were frequently observed in the early neonatal stage. In conclusion. 1) One of the function of Type II cells is to form the alveolar pores. 2) Alveolar pores are formed by Type II cells which have penetrated into the alveolar wall and faced both sides of the alveolar lumina. Type II cells combine the Type I alveolar epithelial cells of both sides of the alveolar wall and make pores with circle-like intercellular junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of connexin43 in the developing rat heart.

Connexin43 is the predominant gap junction protein expressed in the heart. To determine the relation between cardiac maturation and gap junction gene expression, the developmental profiles of connexin43 mRNA and protein were examined in the rat heart. Connexin43 mRNA levels accumulate progressively (eightfold) during embryonic and early neonatal stages, accompanied by a parallel, but temporally delayed, accumulation of connexin43 protein (15-fold). As the heart matures further, both mRNA and protein levels subsequently decline, to about 50% and 30% of their maximum levels, respectively. These observations suggest that increases in intercellular coupling that characterize cardiac development do not depend solely on modulation of connexin43 gene expression, but rather are likely to involve organization of gap junction channels into the intercalated disc.

Development of the epididymal adipose tissue in monosodium glutamate-induced obese mice.

Obesity was induced in neonatal mice by subcutaneous injections of monosodium glutamate (MSG) at an early neonatal stage. The process of adipocyte formation was studied comparatively in the developing epididymal adipose tissue of the MSG-treated mice and in normal mice during the period from the 6th to the 100th postnatal day. Tritiated thymidine autoradiographic studies showed that cell proliferation activity was the highest on the 6th postnatal day both in the MSG-treated and the control mice. In normal mice, however, cell proliferation took place less frequently after 6 days and had almost ceased after 49 days. In the obese mice, as evidenced by relatively high labeling indices, cell proliferation continued to occur even after 49 days. Ultimately there was no difference in the number of adipocytes counted by Hirsch's method in the MSG-treated and the control mice at the 100th postnatal day. The storage of fat droplets became more noticeable in obese mice than in normal mice after 35 days. The mean size of fat droplets of the obese mice was twice as large as that in normal mice on the 49th postnatal day. These results indicate that the MSG-induced obesity is of the hypertrophic type.

Are alpha-adrenoceptors involved in positive inotropic effects of phenylephrine in chick ventricles?

Effects of phenylephrine on contraction and Ca-action potentials were investigated to clarify whether the alpha-adrenergic mechanism may play a role in chick ventricles. Phenylephrine increased the contractile force of the ventricles isolated from both embryonic and hatched chicks, while methoxamine did not affect their contractility. Developmental changes in the sensitivity to phenylephrine, i.e., increase with age from late embryonic to early neonatal stages, were quite similar to those to a beta-agonist, isoproterenol. The positive inotropism of phenylephrine was antagonized by phentolamine and sotalol, but not antagonized by prazosin or yohimbine. Isobutylmethylxanthine augmented the effect of phenylephrine. Maximum upstroke velocity of Ca-action potentials recorded in partially depolarized ventricles were enhanced by phenylephrine, and the enhancement was eliminated by sotalol but not by phentolamine. The results suggested that the beta-adrenergic action of phenylephrine may be involved in part of its positive inotropic effect, which is mediated by increased Ca-influx through sarcolemma. Another mechanism may also participate in the effects of phenylephrine, but may not necessarily be classified as an "alpha-adrenergic effect".

Are α-Adrenoceptors Involved in Positive Inotropic Effects of Phenylephrine in Chick Ventricles?

Effects of phenylephrine on contraction and Ca-action potentials were investigated to clarify whether the α-adrenergic mechanism may play a role in chick ventricles. Phenylephrine increased the contractile force of the ventricles isolated from both embryonic and hatched chicks, while methoxamine did not affect their contractility. Developmental changes in the sensitivity to phenylephrine, i.e., increase with age from late embryonic to early neonatal stages, were quite similar to those to a β-agonist, isoproterenol. The positive inotropism of phenylephrine was antagonized by phentolamine and Sotalol, but not antagonized by prazosin or yohimbine. Isobutylmethylxanthine augmented the effect of phenylephrine. Maximum upstroke velocity of Ca-action potentials recorded in partially depolarized ventricles were enhanced by phenylephrine, and the enhancement was eliminated by Sotalol but not by phentolamine. The results suggested that the β-adrenergic action of phenylephrine may be involved in part of its positive inotropic effect, which is mediated by increased Ca-influx through sarcolemma. Another mechanism may also participate in the effects of phenylephrine, but may not necessarily be classified as an “α-adrenergic effect”.

Enzymes of serine metabolism in normal, developing and neoplastic rat tissues

1. 1.|The cellular pattern of serine metabolism was conceptualized into four main areas of metabolic sequences: the biosynthesis of serine from intermediates of the glycolytic pathway (the so-called “phosphorylated pathway”); and alternative pathways of serine utilization initiated by serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. 2. 2.|The known regulatory and adaptive properties of the enzymes involved in these pathways were reviewed in detail and key enzymes associated with each pathway (phosphoserine aminotransferase, serine dehydratase, serine aminotransferase, and serine hydroxymethyltransferase, respectively) were selected for further investigation. 3. 3.|Tissue distribution studies in the rat revealed that whereas serine dehydratase and serine aminotransferase activities were largely confined to the liver, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were more broadly distributed. In particular in tissues with a high rate of cell turnover, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were coordinately increased. An increase in serine hydroxymethyltransferase activity coincided temporally with the incorporation of [3- 14C]serine and thymidine into DNA in normal human lymphocytes during proliferation after mitogenic stimulation by phytohemagglutinin. The evidence suggested a primarily gluconeogenic role for serine dehydratase and serine aminotransferase. Serine hydroxymethyl-transferase has a role in providing glycine and one-carbon folate co-factors as precursors for nucleotide biosynthesis and in some situations serves to metabolically couple the pathway of serine biosynthesis to utilization for de novo purine and pyrimidine synthesis. 4. 4.|Multiple enzymic forms were distinguished for serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. For serine dehydratase the two cytosolic multiple forms had no apparent functional significance; the multiple forms were catalytically unmodified by conditions promoting phosphorylation-dephosphorylation in vitro. The mitochondrial form of serine aminotransferase showed adaptive responses in gluconcogenic situations, and the hypothesis was proposed that the mitochondrial isoenzyme of serine hydroxymethyltransferase is associated together with serine aminotransferase in a pathway for gluconeogenesis from protein-derived amino acids such as glycine and hydroxyproline. 5. 5.|The adaptive behaviour of the enzymes during the neonatal development of rat liver revealed that serine aminotransferase reached a peak in the mid-suckling period at a time when gluconcogenesis is known to be increased. Use of phosphoenolypyruvate carboxykinase inhibitors (mercaptopicolinate or quinolinate) supported a pathway via serine aminotransferase for gluconeogenesis from serine and hydroxyproline at this developmental stage. The concept of the involvement in a carbon salvage pathway to deal with increased body collagen turnover at this time was advanced. The developmental adaptation of serine aminotransferase at birth was shown to involve glucagon, acting via cyclic AMP, and to be dependent on transcriptional gene regulation. 6. 6.|Serine dehydratase showed a biphasic developmental pattern, similar to other enzymes involved in amino acid catabolism. The peaks of activity at the early neonatal and weaning developmental stages were shown to involve the joint action of glucagon, acting via cyclic AMP, and corticosteroid hormones. The inductions were dependent, at least initially, on transcriptional gene regulation but the precise mechanistic role of the two classes of hormone has yet to be defined. At both developmental peaks the distribution of serine dehydratase multiple forms was identical, and differential developmental regulation of the forms was not involved in determining the overall pattern of serine dehydratase development. 7. 7.|Phosphoserine aminotransferase and cytosolic serine hydroxymethyl-transferase showed similar developmental patterns with a peak of activity in the perinatal period. This coincides with an active period of hepatocyte proliferation and of nucleotide biosynthesis. A further rise of serine hydroxymethyltransferase coinciding with a second postnatal surge of proliferative hepatocellular growth was independent of de novo serine biosynthesis and reflected increased provision of serine from dietary sources. 8. 8.|A survey of the key enzymes of serine metabolism in transplantable rat neoplasms revealed that, in general, serine dehydratase and serine aminotransferase were deleted from the cellular repertoire of metabolic capacities. In contrast, phosphoserine aminotransferase and serine hydroxymethyltransferase were selectively retained to varying degrees in neoplastic tissues. 9. 9.|The pattern of serine metabolism displayed in normal, developing and neoplastic tissues revealed an integrated, genetically-programmed, response of enzymes of serine biosynthesis and of alternative enzymes of serine utilization. A major role for serine metabolism in cellular proliferation was emphasized by the coordination of serine synthesis from carbohydrate precursors with the biosynthesis of purine and pyrimidine nucleotides through a metabolic coupling via serine hydroxymethyltransferase.

ACTH Receptor Levels in Lamb Adrenals at Late Gestation and Early Neonatal Stages

ACTH Receptor Levels in Lamb Adrenals at Late Gestation and Early Neonatal Stages

Fine structural observations of ruthenium red binding in developing and adult rat lung.

AbstractThe ruthenium red staining technique of Luft ('71b) was utilized in an electron microscopic investigation of developing and adult rat lung. Electron‐dense deposits of ruthenium red‐positive material were observed on all exposed surfaces of the tissue block, regardless of the stage of development. In the more central areas of the block, sites of ruthenium red binding changed with age. In early prenatal lung (days 16 to 20) dense accumulations of ruthenium red‐positive material were found in association with the basement membranes of endodermal epithelial cells. Ruthenium red binding was also observed between adjacent epithelial cells; however, their luminal surfaces were negative. The main intracellular site of ruthenium red binding in intact cells was the lamellar body of the developing type II pulmonary epithelial cells. By day 21 of development, accumulations of granular product were observed in association with most lamellar bodies, as well as on epithelial cell luminal surfaces. Ruthenium red binding in postnatal tissue decreases with increasing age. By the second postnatal week, the predominant site of binding is the luminal surface of the type I and type II pulmonary epithelial cells. When compared to fetal and early neonatal stages, adult rat lung has a still more limited distribution of ruthenium red‐positive material. Changes in the distribution of ruthenium red‐positive material correlate with numerous morphologic and biochemical events in rat lung development.

Structural and biochemical maturation of the cerebral pallium in rabbit fetuses: morphogenesis and lipids.

Abstract—Morphological parameters of maturation of the cerebral pallium in rabbit fetuses ranging between 20 days of gestation (d.g.) and the early neonatal stage are expressed semi‐quantitatively and correlated with progressive changes in the brain lipids, glycolipids and nucleic acids. Dual expression of the chemical values, using as referents both the dry weight of the tissue and the DNA unit, reveal the crucial stage in brain development when rapid cell proliferation is replaced by rapid cell growth; this stage in rabbit fetuses occurred between 28 and 30 d.g. Between 20 d.g. and the early neonatal phase the RNA decreased moderately when expressed per unit of DNA. Even at the time of term birth the cortical nerve cells of the rabbit showed signs of immaturity including a relatively small nuclear volume.On the dry weight basis, the lipids of the cerebral pallium exhibited little change in composition during fetal development; however, cerebrosides rose substantially between 30 d.g. and the early neonatal phase. When expressed per unit of DNA, all lipids and glycolipids continued to increase progressively in a pattern characteristic of growth throughout the prenatal period studied. This increase was also apparent when the lipid constituents were expressed per total pallium at the progressive gestational stages.The molar ratios of phospholipids:cholesterol:cerebrosides in the pallium of rabbits of different ages were as follows: in adult rabbits‐2.9: 2.6: 1.0, in the newborn‐2.9: 1.3: 0.2 and in the 30 d.g. fetuses‐3.0: 1.2:0.44. These values reflect the fact that during maturation the content of phospholipids changes little, whereas that of cholesterol and especially erebrosides increases markedly.