Effect of Perinatal Asphyxia on Systemic and Intracerebral pH and Glycolysis Metabolism in the Rat

doi:10.1006/exnr.1997.6482

Experimental Neurology

Volume 145, Issue 2, June 1997, Pages 390-396

https://doi.org/10.1006/exnr.1997.6482 Get rights and content

Abstract

The effects of perinatal asphyxia on systemic and brain pH and glycolysis metabolism were studied in the rat. Perinatal asphyxia was induced by immersing pup-containing uterus horns, obtained by cesarean section from rats within the last day of gestation, in a water bath at 37°C for various periods of time (0–23 min). Subcutaneous levels of pyruvate (Pyr), lactate (Lact), glutamate (Glu), and aspartate (Asp) were monitored with microdialysis 40–80 min after delivery. In parallel experiments, the pups were sacrificed 40 min after delivery and the heart and brain were removed for measuring pH. Brain (striatum) Pyr, Lact, Glu, and Asp levels were also analyzed. A decrease in the rate of survival was first observed following asphyctic periods longer than 16 min, and no survival could be observed after 22 min of asphyxia. In control (cesarean-delivered) pups, heart and brain pH were 7.36 ± 0.01 (N = 8) and 7.30 ± 0.01 (N = 8), respectively. Significant decreases in pH were first observed following 5–6 and 10–11 min of asphyxia, in heart and brain, respectively. In both regions pH decreased along with the length of asphyxia, but a decrease below 7 was only observed in the brain, following asphyctic periods longer than 16 min. A significant increase in subcutaneous Lact levels was first observed following 2–3 min of asphyxia, with a maximum after 20–21 min of asphyxia. In the brain, the increase in Lact levels was delayed compared to that observed in subcutaneous tissue. Pyr and Asp levels increased in subcutaneous tissue following perinatal asphyxia and decreased in brain tissue following >15 min of asphyxia. Glu levels were increased subcutaneously by moderate (5–16 min) asphyctic periods, but, in the brain, were only transiently increased by 10–11 min of asphyxia. Thus, changes in systemic pH, glycolysis, and excitatory amino acid metabolism are observed following shorter asphyctic periods than are changes in the brain. In particular, increases in subcutaneous Lact levels precede: (i) a decrease in brain pH, (ii) an increase in brain Lact levels, (iii) a decrease in the rate of survival, and, probably, (iv) brain damage. It is suggested that monitoring Lact levels by subcutaneous microdialysis is a useful method for predicting the outcome produced by hypoxic–ischemic insults.

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After routine disinfection, the blood samples were collected through cardiac puncture. The rat pups were anesthetized with ether and were euthanized according to institutional protocols [19,20]. The blood samples of the rat pups were collected using the decapitating method.
Maternal diet during pregnancy can influence offspring's health by affecting development and metabolism. This study aimed to analyze the influence of maternal folic acid (FA) supplementation on the metabolism of rat pups using targeted metabolomics. Twenty female rats were randomly assigned to a FA supplementation (FAS group, n = 10) or control group (n = 10), which were fed AIN93G diet with 2 or 10 mg/kg FA, respectively. We then measured amino acids and their derivatives, biogenic amines, and fatty acids in the female rats and their pups by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC/MS-MS) and gas chromatography–mass spectrometry (GC/MS-MS). In maternal rats, the significant changes of three metabolites (proline, γ-aminobutyric acid and esterified octadecatetraenoic acid, P < 0.05) were observed in FAS group. For the rat pups, FAS pups had significantly lower homocysteine and higher FA levels than control pups. The lower levels of amino acids (leucine, isoleucine, serine, proline) were obtained in FAS pups. Furthermore, there were the decreased esterified fatty acids (arachidonic acid, eicosapentaenoic acid, and docosatetraenoic acid) and free fatty acids (oleic acid, linoleic acid, γ-linolenic acid, octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid and selacholeic acid) in FAS pups. Metabolic changes in the FAS pups were characterized by changes in fatty acids and amino acids. These results suggested that FA supplementation during pregnancy influenced amino acids and fatty acids metabolism in rat pups. This study provides new insights into the regulation of amino acids and fatty acids metabolism during early life.
Active forms of Akt and ERK are dominant in the cerebral cortex of newborn pigs that are unaffected by asphyxia
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ATP depletion will soon lead to the depression of neuronal activity soon followed by anoxic depolarization. Anoxic depolarization is characterized by the loss of transmembrane ionic gradients eliciting the accumulation of intracellular Na+, H+, and Ca2 + ions causing edema and worsening tissue acidosis [18,19], the unregulated release of excitatory amino acids along with the inhibition of astrocytic uptake can also contribute to excitotoxic damage via the N-methyl-d-aspartate (NMDA) receptor [20,21]. Upon reventilation/reoxygenation, the high energy phosphate levels and the transmembrane ionic gradients are gradually restored, but the initially still increased intracellular and intramitochondrial Ca2 + levels will activate a host of intracellular proteases and nucleases, as well as enzymatic and non-enzymatic synthesis of reactive oxygen species (ROS).
Perinatal asphyxia (PA) often results in hypoxic–ischemic encephalopathy (HIE) in term neonates. Introduction of therapeutic hypothermia improved HIE outcome, but further neuroprotective therapies are still warranted. The present study sought to determine the feasibility of the activation of the cytoprotective PI-3-K/Akt and the MAPK/ERK signaling pathways in the subacute phase of HIE development in a translational newborn pig PA/HIE model.
Phosphorylated and total levels of Akt and ERK were determined by Western blotting in brain samples obtained from untreated naive, time control, and PA/HIE animals at 24–48 h survival (n = 3–3–6,respectively). PA (20 min) was induced in anesthetized piglets by ventilation with a hypoxic/hypercapnic (6%O₂20%CO₂) gas mixture. Furthermore, we studied the effect of topically administered specific Akt1/2 and MAPK/ERK kinase inhibitors on Akt and ERK phosphorylation (n = 4–4) in the cerebral cortex under normoxic conditions.
PA resulted in significant neuronal injury shown by neuropathology assessment of haematoxylin/eosin stained sections. However, there were no significant differences among the groups in the high phosphorylation levels of both ERK and Akt in the cerebral cortex, hippocampus and subcortical structures. However, the Akt1/2 and MAPK/ERK kinase inhibitors significantly reduced cerebrocortical Akt and ERK phosphorylation within 30 min.
The major finding of the present study is that the PI-3-K/Akt and the MAPK/ERK signaling pathways appear to be constitutively active in the piglet brain, and this activation remains unaltered during HIE development. Thus, neuroprotective strategies aiming to activate these pathways to limit apoptotic neuronal death may offer limited efficacy in this translational model.
25 years of research on global asphyxia in the immature rat brain
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Whilst global brain acidosis was already evident after short durations of PA (for 5 min), only sustained insults exceeding 15–16 min decreased the cerebral pH below pH 7 and caused marked glycolysis (Chen et al., 1997a; Engidawork et al., 1997). The increases in brain lactate were preceded by peripheral markers of glycolysis and excitotoxicity; including increased subcutaneous lactate, pyruvate, glutamate and aspartate levels (Chen et al., 1997a; Dell’Anna et al., 1995; Engidawork et al., 1997). Increased peripheral and brain lactate levels were not accompanied by corresponding increased lactate metabolism (the oxidation of lactate, glucose and glycine was not changed) at 60 min after moderate PA (for 15 min) (Frizzo et al., 2010).
Hypoxic-ischemic encephalopathy remains a common cause of brain damage in neonates. Preterm infants have additional complications, as prematurity by itself increases the risk of encephalopathy. Currently, therapy for this subset of asphyxiated infants is limited to supportive care. There is an urgent need for therapies in preterm infants – and for representative animal models for preclinical drug development. In 1991, a novel rodent model of global asphyxia in the preterm infant was developed in Sweden. This method was based on the induction of asphyxia during the birth processes itself by submerging pups, still in the uterine horns, in a water bath followed by C-section. This insult occurs at a time-point when the rodent brain maturity resembles the brain of a 22–32 week old human fetus. This model has developed over the past 25 years as an established model of perinatal global asphyxia in the early preterm brain. Here we summarize the knowledge gained on the short- and long-term neuropathological and behavioral effects of asphyxia on the immature central nervous system.
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In the human fetus, an increased lactate and glucose level can be anticipated when hypoxia and stress are present and is likely to be a function of both anaerobic metabolism and catecholamine-mediated glycogenolysis/glycolysis.
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Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2–3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2–3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference memory in the Morris water maze task. Compared to control subjects, anoxic animals exhibited increased latencies and path lengths to reach the platform, as well as decreased searching specifically for the platform location. In contrast, no significant differences were observed for swimming speeds and frequency within the target quadrant. Together, these behavioral results indicate that the poorer performance by anoxic subjects is related to spatial memory deficits and not to sensory or motor deficits. Therefore, this model of neonatal anoxia in rats induces hippocampal changes that result in cell losses and impaired hippocampal function, and these changes are likely related to spatial memory deficits in adulthood.
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Intrapartum CTG has low specificity with many non-acidemic fetuses having CTG changes, and FBS with lactate analysis can be used to exclude metabolic acidemia in cases of non-reassuring CTG, as false negative tests are unlikely [2]. Since fetal lactate increases specifically during anaerobic metabolism, FBS also reliably detect fetuses at risk of developing hypoxemia [3–5]. Lactate concentration in fetal scalp blood correlates with lactate and pH in the umbilical artery, and is shown to be more sensitive than fetal scalp pH in predicting low Apgar score at 5 min and hypoxic ischemic encephalopathy [6].
To investigate if repeat (≥3) fetal scalp blood sampling (FBS) is associated with increased risk of caesarean delivery and worse neonatal outcome than occasional (1–2) FBS.
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During the study period there were 2134 FBSs performed on 1070 laboring women with a median of two samplings (range 1–8). There were no differences in Apgar score <7 at 5 min or metabolic acidemia in umbilical artery blood at birth between labors with 1–2 FBS and ≥3 FBS. Among women who underwent 1–2 FBS, 23% had a caesarean delivery as compared with 42% of those having ≥3 FBS. After adjustment for confounders, repeat FBS remained an independent risk factor for caesarean delivery (adj OR 2.05; 95%C.I 1.5–2.8).
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^☆: J. Gross, Ed.

¹: To whom correspondence should be addressed.

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Regular ArticleEffect of Perinatal Asphyxia on Systemic and Intracerebral pH and Glycolysis Metabolism in the Rat☆

Abstract

Brain Res.

Neuroscience

Brain Res.

Int. J. Biochem.

Pediatr. Neurol.

Neuroscience

Neurosci. Lett.

Neuroscience

Life Sci.

Pediatr. Neurol.

Asphyxia-induced lesion of the rat hippocampus (CA1, CA3) and the nigro-striatal dopamine system

Hypoxia and Ischemia, CNS

Perinatal asphyxia increases bFGF mRNA levels and DA cell body number in the mesencephalon of rats

NeuroReport

Excitatory amino acids contribute to the pathogenesis of perinatal hypoxic–ischemic brain injury

Brain Pathol.

Respiration of mitochondria of red and white skeletal muscle

Am. J. Physiol.

Short-term effects of perinatal asphyxia studied with Fos-immunocytochemistry andin vivo

Exp. Neurol.

Delayed neuronal death following perinatal asphyxia in rat

Exp. Brain Res.

ATP and brain function

J. Cereb. Blood Flow Metab.

In vivo release of endogenous amino acids from rat neostriatum: Further evidence for a role of glutamate and aspartate in corticostriatal neurotransmission

J. Neurochem.

Cerebral histological and electrocorticographic changes after asphyxia in fetal sheep

Pediatric Res.

Hydroxymalonate inhibits uptake by the rabbit hindlimb

J. Appl. Physiol.

Cerebral extracellular glucose and lactate concentrations during and after moderate hypoxia in glucose- and saline-infused rats

Anesthesiology

Regular Article
Effect of Perinatal Asphyxia on Systemic and Intracerebral pH and Glycolysis Metabolism in the Rat☆