Infant Rat Brain Research Articles

Hypothermia enhances protective effect of MK-801 against hypoxic/ischemic brain damage in infant rats

Accumulating evidence suggests that the N-methyl- d-aspartate (NMDA) subtype of glutamate receptor may play an important role in hypoxic/ischemic (H/I) brain damage. Accordingly, it has been shown that the NMDA antagonist, MK-801, partially protects the infant rat brain against H/I damage. Here we show that reducing the body temperature of the infant rat also confers partial protetion against H/I brain damage and that mild hypothermia plus MK-801 treatment provides total protection against such damage. Relevance of these findings to the prevention of perinatal brain damage in humans is discussed.

Pathogenesis of brain abscess formation in an infant rat model of Citrobacter diversus bacteremia and meningitis.

The pathogenesis of Citrobacter diversus meningitis and brain abscess was studied in infant rats. Two-day-old rats were inoculated intraperitoneally and intranasally with C. diversus. C. diversus strain 4277, lacking the 32,000-molecular-weight outer membrane protein that appears to be a marker for strains causing meningitis in human neonates, was more likely to produce bacteremia, meningitis, and death in rats than was strain 4036, which possesses this outer membrane protein. Strain 4036 was, however, more likely than strain 4277 to produce ventriculitis and brain abscess. In the infant rat, central nervous system involvement by C. diversus begins with bacteremia and leptomeningitis, followed by ventriculitis and direct extension of infection into periventricular brain parenchyma. Large numbers of bacteria persist inside inflammatory cells, an observation suggesting resistance to intraphagocytic killing. Bacterial strain differences, possibly related to the presence of a 32,000-molecular-weight outer membrane protein, may account for histopathologic differences in the brains of infant rats with C. diversus meningitis.

Phenylalanine uptake in neonatal and infant rat brain.

At the same severe elevations in blood phenylalanine (Phe) levels maintained for 4 h, much higher cerebral Phe concentrations were found in 4-day-old than in 16- or 70-day-old rats. In order to compare this developmental change with 14C-Phe influx mediated by the L transport system, the rapid intracarotid injection method was adapted for use in neonatal rats. The brain uptake index (BUI) thus determined for the first time through the suckling period was significantly higher on the 4th day of age than on the 7th or 24th day, while no significant change occurred during subsequent life. This early period of change in influx across the blood-brain barrier overlapped with the age period of decrease of the hyperphenylalaninemia-associated accumulation of Phe in the brain. The results indicate that by the time when intermittent feeding begins, the brain has developed a considerable ability (a) to protect itself against physiological (e.g. postprandial) fluctuations in circulating Phe levels, and (b) to restrict the cerebral accumulation of Phe from pathologically elevated blood concentrations such as those in phenylketonuria.

Transplants of Normal Fetal Cerebral Cortical Tissue into Congenitally Malformed Brains of Infant Rats

Transplants of Normal Fetal Cerebral Cortical Tissue into Congenitally Malformed Brains of Infant Rats

Brain serotonin synthesis and Na +, K +-ATPase activity are increased postnatally after prenatal administration of l-tryptophan

The effect of prenatal l-tryptophan supplementation on the serotonin (5-HT) synthesis and the activity of Na +, K +-ATPase in the cerebral cortex was studied during postnatal development, from birth up to day 30. A parallel and significant elevation of the serotonin content and the activity of tryptophan-5-hydroxylase was observed in the brain of infant rats born to mothers treated with l-tryptophan, as related to non-treated controls. The activity of Na +, K +-ATPase was also significantly elevated at the different ages studied throughout the developmental period, as related to controls. These results suggest an important role of l-tryptophan in the early regulation of the serotonin-synthesizing machinery, which lasts postnatally. Elevation of ATPase activity seems to be associated to the elevation in the activity of the 5-HT system.

Brain serotonin synthesis and Na+,K+-ATPase activity are increased postnatally after prenatal administration of L-tryptophan.

The effect of prenatal L-tryptophan supplementation on the serotonin (5-HT) synthesis and the activity of Na+,K+-ATPase in the cerebral cortex was studied during postnatal development, from birth up to day 30. A parallel and significant elevation of the serotonin content and the activity of tryptophan-5-hydroxylase was observed in the brain of infant rats born to mothers treated with L-tryptophan, as related to non-treated controls. The activity of Na+,K+-ATPase was also significantly elevated at the different ages studied throughout the developmental period, as related to controls. These results suggest an important role of L-tryptophan in the early regulation of the serotonin-synthesizing machinery, which lasts postnatally. Elevation of ATPase activity seems to be associated to the elevation in the activity of the 5-HT system.

Antibodies to the capsular polysaccharide of Neisseria meningitidis group B or E. coli K1 bind to the brains of infant rats in vitro but not in vivo

The binding of monoclonal and polyclonal IgG antibodies specific to the capsular polysaccharide of Neisseria meningitidis group B and E. coli K1 was tested to the cross-reacting polysialosyl structures previously shown to be present in the brain of infant rats ( Lancet 1983; ii: 355–357). Strong immunofluorescence was obtained after in vitro incubation of the brains of 1 to 13 days old rats with the antibodies whereas the brains of adult rats remained negative. The number of antibody-binding structures decreased as a function of age, being highest at the age of 1 to 5 days. However, when the same antibodies were injected intraperitoneally into the infant rat, or into the mother rat 2 days before parturition, no binding of antibodies to the infant rat brain tissue was observed.

Phosphoprotein B-50 in nerve growth cones from fetal rat brain

The presynaptic, nervous tissue-specific phosphoprotein B-50 is present in infant and adult rat brain. In the present study we demonstrate that B-50 is a major phosphoprotein in nerve growth cones obtained from fetal rat brain. As this protein is an endogenous substrate for protein kinase C, an enzyme linked to cell growth and proliferation, a role for B-50 in nerve growth cone function is suggested.

Characterization of infant rat cerebral cortical membrane proteins phosphorylated in vivo: identification of the ACTH-sensitive phosphoprotein B-50.

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.

A method for stereotaxic implantation in neonatal rats

Presented are methods of implanting chronically indwelling electrodes or cannulas into brain of infant rats. Anesthesized pups are restrained in a flexible alginate mold that is placed in the stereotaxic instrument. Details of electrode implant are given. We routinely use these methods to accurately place electrodes into subcortical structures of 3- to 15-day-old pups with less than 2% loss of the implant.

Effects of branched-chain L-amino acids, L-phenylalanine, and L-methionine on the transport of L-glutamine in rat brain cortex in vitro. Influence of cations.

Uptake of L-glutamine (2 mM) by rat brain cortex slices against a concentration gradient is markedly inhibited (40%) by branched-chain L-amino acids (1 mM), L-phenylalanine (1 mM), or L-methionine (1 mM); that of L-asparagine (2 mM) is much less affected by these amino acids. Other amino acids investigated have little or no effect on cerebral L-glutamine uptake. The suppressions of L-glutamine uptake by the inhibitory amino acids are apparently blocked by high [K+], which itself has little or no effect on glutamine uptake. This abolition of suppression is partly explained by high [K+] retention of endogenous glutamine; in the absence of Ca2+ such retention disappears. The inhibitory amino acids (1 mM) also enhance the release of endogenous glutamine, exogenous glutamine with which slices have been loaded, or glutamine synthesized in the slices from exogenous glutamate. The enhanced release of endogenous glutamine is diminished by high [K+]. The suppression of glutamine uptake by the branched-chain amino acids is independent of the concentration of glutamine at low concentrations (0.25--0.5 mM), indicating non-competition, but is reduced with high concentration of glutamine. The inhibition by L-phenylalanine is noncompetitive. L-Glutamine (2mM) exerts no inhibition of the cerebral uptakes of the branched-chain L-amino acids or L-phenylalanine (0.25--2 mM). The inhibitory amino acids are as active in suppressing L-glutamine uptake with immature rat brain slices as with adult, although the uptake, against a gradient, of L-glutamine in the infant rat brain is about one-half that in the adult. They are also just as inhibitory on the concentrative uptake of L-glutamine by a crude synaptosomal preparation derived from rat brain cortex. Such a nerve ending preparation takes up L-glutamine (0.25 mM), against a gradient, at about ninefold the rate at which it is taken up by cortex slices (for equal amounts of protein), and the uptake process is markedly suppressed by high [K+] in contrast to the effects of high [K+] with slices. The possible physiological and pathological consequences of the suppression of glutamine uptake are discussed.

Experimental phenylketonuria: replacement of carboxyl terminal tyrosine by phenylalanine in infant rat brain tubulin.

In the brains of newborn rats, about half of the tubulin molecules are modified posttranslationally by the addition of an aromatic amino acid at the carboxyl terminus of the alpha chain. Of the added residues, 96 percent are tyrosine and 4 percent are phenylalanine. After induction of hyperphenylalaninemia, the proportion of tubulin molecules containing carboxyl terminal phenylalanine increases up to eightfold and the pool of tyrosine-containing molecules decreases by an equivalent amount.

Regulatory Aspects of Glucose and Ketone Body Metabolism in Infant Rat Brain

Summary: The present study was designed to investigate some of the possible factors responsible for the low rate of the oxidative use of glucose observed in neonatal rat brain. An evaluation of the role of pyruvate dehydrogenase (PDH, EC 1.2.4.1) showed that at all stages of brain development, the activities of both molecular forms of this enzyme are considerably less than those reported for the enzymes localized above in the glycolytic pathway. The proportion of both molecular forms is not altered during brain maturation. The activities of the active form and of the total enzyme in adult brain are 0.56 and 1.1 μmole/min/g brain, respectively. It is concluded that PDH represents the rate-limiting step in the oxidation of glucose by neonatal brain due to a delay in the synthesis of the enzyme protein. A “cross-over” plot of glycolytic intermediates in neonatal brain showed that only fructose 1,6-bisphosphate concentrations are significantly lower than in adult brain, indicating a block at the level of phosphofructokinase (PFK, EC 2.7.1.11). A marked increase in citrate concentrations in neonatal brain (values ranging from 0.44–0.82 μmole/g brain) with regard to adult levels (0.28 μmol/g brain) is suggested to be responsible for the inhibition of the enzyme. Glucose 6-phosphate concentrations in neonatal brain are not different from adult values. Despite increasing ketonemia neither consistent intracerebral accumulation of glucose nor a decrease in the brain/blood ratio of glucose is observed during brain maturation. These data argue against limitations of glucose use at the level of hexokinase or membrane transport in the neonatal brain. Speculation: Ketone bodies are major substrates of the neonatal brain in that they fulfill to a large extent both the energy requirements of the brain and provide carbon for the needs of biosynthetic processes. The dual function of ketone bodies cannot be substituted for alternatively by glucose due to a limitation in the oxidative breakdown of glucose observed in young rat brain. Thus, glucose is saved for preferential use in the pentosephosphate pathway, providing hydrogen equivalents for the synthesis of myelin lipids, a process which is very active in neonatal brain.

Brain Carbohydrate Metabolism during Experimental Haemophilus influenzae meningitis

Five-day-old infant rats which acquire Haemophilus influenzae b bacteremia and meningitis after intranasal inoculation have a transient depression in weight gain (2 days), but then continue to grow at the same rate as strain U--11 inoculated controls. Brain lactate, glucose, and glycogen concentrations increase during the first 5 days of disease in infected animals. The increase in brain glycogen can be accounted for by an influx of glycogen containing polymorphonuclear leukocytes. The increased concentrations of glucose and lactate were found not to be due to a change in brain weight to dry weight ratio or the volume of entrapped blood. The mean cerebrospinal fluid (CSF) glucose concentration was higher in animals with meningitis (2.7 mM) in comparison to U-11 inoculated controls (1.8 mM). This increase in brain and CSF glucose concentration appeared secondary to an increased brain uptake of hexoses as manifested by an increased [3H]mannitol uptake. Brain lactate accumulation was not explicable from the data available. There was no evidence of cerebral cortical cellular damage because in vitro oxygen uptake and lactate production were equivalent in control and meningitic animals. The ability of the infant rat brain to maintain cerebral adenosine triphosphate (ATP) content in menigitis and the failure of CSF glucose concentration to decrease might be a reflection of the importance of alternative oxidative substrate (e.g., beta-hydroxybutyrate) to the cerebral metabolism of the developing rat brain.

The effects of hyperthermia on polyribosomes and amino acid levels in infant rat brain

Brain polyribosomes prepared from infant rats exposed to elevated ambient temperatures (39.5°C for 45 min) are highly disaggregated. Disaggregation is evident 15 min after the animals have entered the hot environment and is nearly maximal after 25 min. Substantial recovery of polyribosomes is seen 20 min after the hyperthermic rats are transferred from 39.5 to 33°C ambient temperature, indicating that heat-induced disaggregation is quickly reversible. During hyperthermia the levels of certain amino acids in the brain, particularly essential amino acids, rise markedly. The increases in amino acid levels are reversed within 1 h after the rats are transferred from the hot environment to 33°C ambient temperature. The levels of amino acids in blood plasma also rise during hyperthermia; however, the increases are relatively smaller and less selective than in the brain but are also reversed by returning the animals to 33°C ambient temperature. Injection of phenylalanine into infant rats causes hyperphenylalaninemia, elevated brain phenylalanine levels, and brain polyribosome disaggregation ( Aoki & Siegel, 1970 ). The present observations show that elevated brain amino acid levels and polyribosome disaggregation are also linked in hyperthermic infant rats, but it is not yet clear if the two phenomena are causally related. Although the mechanisms of the increases of blood and brain amino acid levels remain to be elucidated, they may have a significant bearing on the functional status of the protein synthetic machinery of the brain, on the levels of neurotransmitter substances in the brain which are derived from amino acid precursors, and on the intermediary metabolism of the brain.

Effects of ammonium ions on spontaneous action potentials and on contents of sodium, potassium, ammonium and chloride ions in brain in vitro.

Abstract—The effects of ammonium ions on the frequency of spontaneous action potentials in guinea‐pig cerebellar slices, recorded with an extracellular microelectrode, and on the contents of sodium, potassium and chloride ions in incubated guinea‐pig cerebellar, and rat brain cortex, slices have been investigated. The frequencies of the spontaneous action potentials are partially suppressed by concentrations of NH4Cl less than 2 mm and completely abolished by concentrations exceeding 2 mm. The amplitudes of the spike discharges are unaffected. A lag period of at least 15 s precedes the inhibition. The suppressing action of NH on the spike frequency is reversible, as shown by complete recovery on removal of NH after short time intervals. Deficiency of Cl− in the superfusion medium causes conversion of inhibition by NH to excitation. Reduction of [K+], or of [Na+], causes increase of inhibition by NH in a normal [Cl1], and reduction of excitation in a low [Cl1], medium. The inhibitory effects of NH on spike frequency are unaffected by picrotoxin or strychnine. NH4Cl, even at 1 or 2 mm, causes a significant increase of aerobic glycolysis. It is suggested that the lag period preceding the suppression of the frequency of spike discharges by NH is partly due to a metabolic change induced by NH, perhaps a transient lowering of pH in the responsible neurons, causing changed permeability to Cl− and possibly to K+ and Na+, NH promotes, in guinea‐pig cerebellar slices, an inward flow of Na+ and an outward flow of K+, the latter being greater than that due to exchange of K+ for NH. NH4Cl at 1 or 2 mm causes an outward flow of K+ and an inward flow of Cl− in rat brain cortex slices. The movement of Cl− is biphasic, the first phase, seen with low [NH], consisting of an increase of tissue content of Cl− with little or no fluid uptake and a second phase, seen with high (> 5 mm) concentrations of NH, in which the uptake of Cl− is directly proportional to the fluid uptake. It is suggested that the first phase is largely neuronal in location whilst the second is largely glial. In infant rat brain cortex slices, there seems to be predominantly an equal exchange of NH for K+. There is little evidence of energy assisted concentrative uptake of NH by brain slices and this is thought to be due largely to the rapid diffusion of undissociated NH3 across cell membranes. It is suggested that some NH (amounting to about 2 mequiv/1) may be bound in the brain. It is concluded that changes in ionic permeabilities, particularly that of Cl−, partly due to a metabolic action, may be responsible for some of the acute cerebral effects of NH administration.

Circadian variations in plasma growth hormone and prolactin in the infant rat: Comparison with the adult pattern

Radioimmunoassayable (RIA) plasma growth hormone (GH) and prolactin (PRL) levels were determined at 3 hr intervals during a controlled 24-hr light-dark cycle in 10-day-old male and female rats; parallel measurements were made of brain monoamines (MA's), dopamine (DA), norepinephrine (NE) and serotonin (5-HT) concentration. Plasma GH and PRL and brain MA levels found in infant rats were compared to the same determinations made during the 24-hr cycle in 50-day-old male rats. GH levels were rather uniform and did not show circadian periodicity in the plasma of infant rats, while PRL levels showed a diurnal surge in the late afternoon hr (1800). In adult rats, GH levels exhibited wide fluctuations during the 24-hr cycle and no circadian periodicity, while PRL levels showed one diurnal (1500–1800) and one nocturnal (2400) surge. A pulsatile GH secretion was found in adult rats sampled at 15 min intervals over a period of 2 hr, which seemed to be lacking in infant rats. In the brain of infant rats, DA and NE levels exhibited circadian patterns which resembled the ones present in the brain of adult rats, whereas no circadian variations were present in 5-HT levels.

Metabolism of amino acids and ammonia in rat brain cortex slices in vitro: a possible role of ammonia in brain function.

Abstract— (1) The sum of the values of total (tissue + medium) amino acid‐N of glutamate, glutamine, γ‐aminobutyrate, and aspartate (referred to as the glutamate system) and of ammonia‐N of incubated rat brain cortex slices is approximately constant under a variety of metabolic conditions (presence or absence of glucose or of oxygen or in the presence of metabolic inhibitors such as aminooxyacetate, malonate, methionine sulfoximine, fluoroacetate, ouabain, 2:4 dinitrophenol, or Amytal). Fluctuations in the value of one constituent are compensated by fluctuations in the values of other constituents. The same applies to infant rat brain cortex slices and to rat brain synaptosome preparations. It is suggested that the constancy of the glutamate‐ammonia system implies a coupling of neurons and glia in such a manner that glutamate released from the neurons during excitation is taken up by the glia and there converted to glutamine. The glutamine is returned to the neurons where it is hydrolysed to glutamate and ammonia. The glia, on this view, exercise an important buffering effect on the extracellular content of the excitatory amino acid, glutamate, and possibly on that of other functionally active amino acids emanating from the neurons. (2) The magnitude of the glutamate‐ammonia system in the infant rat brain cortex is about 43% of that in the adult. It is suggested that, with maturity, the development of the glutamate‐ammonia system is linked with the development of the citric acid cycle of operations. (3) The ammonia in the system is tightly linked to the activity of the ATP‐controlled glutamine synthetase. (4) Proteolytic ammonia and amino acids are formed, during the incubation, to values that seem to be independent of a wide variety of metabolic conditions. The total value is approximately 10 μmol/g in the first h of incubation. (5) As the ammonium ion is necessary for the return of glutamate to the neuron in the form of glutamine, it is inferred that the ion plays a functional role in the nervous system by helping to maintain the steady state of glutamate in the neuron.

The occurrence of two types of deoxyribonucleic acid polymerase activity in the main classes of nuclei obtained from the brains of infant and adult rats

1. The influence of exogenous or activated DNA template on the DNA polymerase activity in the different types of intact nuclei from rat brain tissue was determined. The different amounts or physical state of the DNA template did not produce significant differences in the relative distribution of the DNA polymerase activity between the separate groups of nuclei. 2. The DNA polymerase activities, fractionated by sucrose gradient centrifugation into enzyme A and enzyme B, were found to be present in the extracts of all types of rat brain nuclei. The distribution of these two activities in the ;particulate' and ;soluble' fractions of the separate groups of nuclei from 10-day-old and adult rats was studied. The findings are related to the DNA-synthetic activity in vivo of the intact nuclei and the possible biological functions of the DNA polymerase activities are discussed.

The activity of pyruvate dehydrogenase in rat brain during postnatal development

Hawkins et al. [ 1 ] were the first to demonstrate, by arterio-venous blood difference measurements, that the brains of suckling rats show a net uptake of glucose, acetoacetate and D-/3-hydroxybutyrate and a net output of lactate. In fed adult rats net uptake by the brain of glucose only occurs and there is a negligible net output of lactate [ 1,2] . Furthermore, the amount of glucose utilized by infant rat brain (expressed as I.tmol/min per g fresh wt.) is considerably less than the amount metabolized by adult rat brain [3] . Changes in the rates of utilization of glucose and ketone-bodies by the brain during development are likely to be a consequence of changes in activities of certain key enzymes. There have been several investigations of the three main enzymes involved-in the conversion of D-P-hydroxybutyrate and acetoacetate to acetyl-CoA in rat brain from birth to adulthood [4-71. There appears to be no such report on the key enzyme forming acetyl-CoA from glucose, namely pyruvate dehydrogenase and it is the purpose of the present paper to present the result of such a study.