Transient Asphyxia Research Articles

Paneth cell ontogeny in term and preterm ovine models.

Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

Umbilical arterial lactate levels after normal vaginal and elective cesarean delivery: The role of a longer active second stage most significant in high levels after vaginal delivery.

To evaluate umbilical arterial lactate concentrations after spontaneous vaginal delivery and after elective Cesarean delivery, and to study the simultaneous effects of maternal and obstetric variables in high lactate levels in vaginally delivered healthy term singletons. The birth register study included information about the umbilical artery lactate values and clinical perinatal data from 7723 women and their singleton newborns (7301 spontaneous vaginal and 422 elective cesareans) from Kuopio University Hospital, Finland. High lactate levels were evaluated more extensively among healthy term neonates (N = 6541), to evaluate high levels after normal vaginal labors. The mean lactate level was significantly lower after elective cesarean compared to vaginal delivery (2.42 [0.94] vs. 3.56 [1.62] mmol/l; p < 0.0001). Consequently, the 90th percentile limit values were 3.60 and 5.80 mmol/L. Among healthy term newborns born vaginally, higher lactate values (≥5.80 mmol/L) were independently associated with a longer duration of the active second stage of labor (ORs 1.91-10.97) and duration of ruptured fetal membranes (ORs 1.36-2.46), higher gestational age at birth (ORs 1.41-1.86), null parity (OR 2.17), maternal infection (OR 1.81) and short maternal stature (OR 1.45). We report 90th/95th limits for umbilical arterial lactate values in relation to the various durations of labors for term newborns who are delivered by the vaginal route. Even though the high umbilical lactate levels may indicate serious birth asphyxia, levels after vaginal birth reflect the physiological stress and subclinical transient asphyxia frequently seen in normal vaginal deliveries.

High-potential defense mechanisms of neocortex in a rat model of transient asphyxia induced cardiac arrest

Cardiac arrest (CA) is a common cause of disability and mortality and thus an important risk for human health. Circulatory failure has dramatic consequences for the brain as one of the most oxygen-consuming organs. Hippocampus, striatum and neocortex rate among the most vulnerable brain regions. The neocortex is less sensitive to hypoxia/reperfusion in comparison with the hippocampal CA1 region. That implicates the existence of efficient defense mechanisms in the neocortex against hypoxia/reperfusion injury, which we analyzed in a well-established CA rat model. We explored different immunohistochemical markers (NeuN, MAP2, GFAP, IBA1, NOX4, MnSOD, Bax, caspase 3, cfos, nNOS, eNOS, iNOS, TUNEL), amount of mitochondria, activities of respiratory chain complexes and amount/composition of cardiolipin.CA induced a moderate degeneration of cortical neurons. As possible defense mechanisms the study revealed: (i) increased activities of respiratory chain complexes of cortical mitochondria as response to increased energy demand after ACA-induced cell stress; (ii) increase of cardiolipin content as cellular stress response, which might contribute to the promotion of mitochondrial ATP synthesis; (iii) strengthening of the fast, effective and long-lasting mitochondrial MnSOD defense system; (iv) ACA-induced increase in expression of eNOS and nNOS in vasculature being able to reduce ischemic injury by vasodilation.

Using trend templates in a neonatal seizure algorithm improves detection of short seizures in a foetal ovine model

Seizures below one minute in duration are difficult to assess correctly using seizure detection algorithms. We aimed to improve neonatal detection algorithm performance for short seizures through the use of trend templates for seizure onset and end. Bipolar EEG were recorded within a transiently asphyxiated ovine model at 0.7 gestational age, a common experimental model for studying brain development in humans of 30–34 weeks of gestation. Transient asphyxia led to electrographic seizures within 6–8 h. A total of 3159 seizures, 2386 shorter than one minute, were annotated in 1976 h-long EEG recordings from 17 foetal lambs. To capture EEG characteristics, five features, sensitive to seizures, were calculated and used to derive trend information. Feature values and trend information were used as input for support vector machine classification and subsequently post-processed. Performance metrics, calculated after post-processing, were compared between analyses with and without employing trend information. Detector performance was assessed after five-fold cross-validation conducted ten times with random splits. The use of trend templates for seizure onset and end in a neonatal seizure detection algorithm significantly improves the correct detection of short seizures using two-channel EEG recordings from 54.3% (52.6–56.1) to 59.5% (58.5–59.9) at FDR 2.0 (median (range); p < 0.001, Wilcoxon signed rank test). Using trend templates might therefore aid in detection of short seizures by EEG monitoring at the NICU.

Neuroactive steroids induce changes in fetal sheep behavior during normoxic and asphyxic states

Allopregnanolone and related steroids are potent γ-aminobutyric acid receptor-A receptor agonistic allosteric modulators that suppress central nervous system (CNS) activity; in some species, these neurosteroids regulate normal CNS activity before birth. The aims of this study were to determine the effect of suppressing allopregnanolone production on behavioral responses to transient asphyxia in late gestation fetal sheep using the 5α-reductase (R)-2 inhibitor, finasteride. Specificity of the effects of finasteride was assessed by co-infusion of alfaxalone, a synthetic analog of allopregnanolone. Fetal catheters and electrodes for measurement of the electrocorticogram (ECoG) and nuchal electromyogram were implanted at 125 days of gestation, and an inflatable occluder was placed to allow umbilical cord occlusion (UCO). At of gestation, fetuses received carotid arterial infusion of vehicle (2-hydroxypropyl-β-cyclodextrin; 40% w/vol), finasteride (40 mg/kg/h), alfaxalone (5 mg/kg/h), or finasteride+alfaxalone. A further three groups of fetuses were subjected to 5 min UCO at 30 min after the start of each infusion regime. Finasteride treatment alone increased the incidence of arousal-like activity; this was reduced by co-infusion of alfaxalone. After UCO, finasteride treatment caused a prolongation of sub-low voltage (LV) ECoG activity and increase in aberrant ECoG spike activity when compared to vehicle-treated UCO fetuses. After UCO, alfaxalone treatment reduced the incidence of sub-LV, reduced the number of aberrant EEG spikes, and restored ECoG activity to the pattern observed after UCO in vehicle-treated fetuses. These results confirm that neurosteroids significantly modulate normal CNS activity in the late gestation fetus, modify, and limit the effects of asphyxia on the brain.

Ca2+ regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

Effects of CO2/HCO3− in Perilymph on the Endocochlear Potential in Guinea Pigs

The effect of CO(2)/HCO(3)(-) on the endocochlear potential (EP) was examined by using both ion-selective and conventional microelectrodes and the endolymphatic or perilymphatic perfusion technique. The main findings were as follows: (i) A decrease in the EP from approximately +75 to approximately +35 mV was produced by perilymphatic perfusion with CO(2)/HCO(3)(-)-free solution, which decrease was accompanied by an increase in the endolymphatic pH (DeltapH(e), approximately 0.4). (ii) Perilymphatic perfusion with a solution containing 20 mM NH(4)Cl produced a decrease in the EP (DeltaEP, approximately 20 mV) with an increase in the pH(e) (DeltapH(e), approximately 0.2), whereas switching the perfusion solution from the NH(4)Cl solution to a 5% CO(2)/25 mM HCO(3)(-) solution produced a gradual increase in the EP to the control level with the concomitant recovery of the pH(e). (iii) The perfusion with a solution of high or low HCO(3)(-) with a constant CO(2) level within 10 min produced no significant changes in the EP. (iv) Perfusion of the perilymph with 10 microg/ml nifedipine suppressed the transient asphyxia-induced decrease in EP slightly, but not significantly. (v) By contrast, the administration of 1 microg/ml nifedipine via the endolymph inhibited significantly the reduction in the EP induced by transient asphyxia or perilymphatic perfusion with CO(2)/HCO(3)(-)-free or 20 mM NH(4)Cl solution. These findings suggest that the effect of CO(2) removal from perilymphatic perfusion solution on the EP may be mediated by an increase in cytosolic Ca(2+) concentration induced by an elevation of cytosolic pH in endolymphatic surface cells.

The Effect of Mild Hypothermia on Insulin-like Growth Factors After Severe Asphyxia in the Preterm Fetal Sheep

Persistent reductions in insulin-like growth factor I (IGF-I) levels in the preterm infant are strongly associated with increased risk of retinopathy and other complications, and may result from exposure to severe hypoxia. The effects of a potential new treatment for hypoxic-ischemic encephalopathy, cerebral hypothermia, on the responses of the IGF axis to hypoxia are unknown. The aim of this study was to examine the effects of prenatal asphyxia and cerebral hypothermia on changes in IGF-I and -II, IGF-binding protein 1 (IGFBP-1), and insulin levels. Fetal sheep at 0.7 of gestation underwent either sham asphyxia and sham cooling (n = 7), asphyxia induced by 25 minutes of complete umbilical cord occlusion alone (n = 8), or asphyxia and head cooling (n = 8, extradural temperature 29.5 +/- 2.6C [vs 39.4 +/- 0.3C; P < .05]). Fetuses were studied for 3 days post-insult, during which time fetal blood samples were taken for endocrine measurements. There were no IGF axis changes during occlusion. Plasma IGF-I levels significantly decreased between 6 hours and 48 hours after asphyxia and IGF-II levels by 10 hours, in both asphyxia groups. IGFBP-1 rose from 4 hours, reaching a peak at 10 hours and returning to control values by 48 hours in the normothermia group, and by 24 hours in the hypothermia group. Insulin levels decreased between 2 hours and 10 hours after asphyxia in both asphyxia groups, and subsequently normalized. This study demonstrates for the first time that transient asphyxia in the preterm sheep fetus is associated with a significant decrease in IGF-II as well as IGF-I during recovery, and that these responses are not altered by mild systemic hypothermia.

Endolymphatic Perfusion with EGTA-Acetoxymethyl Ester Inhibits Asphyxia- and Furosemide-Induced Decrease in Endocochlear Potential in Guinea Pigs

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.

The treatment of Sleep Apnea Syndrome: looking for new answers for old problems

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Responses of single-unit carotid body chemoreceptors in adult rats.

1. Our goal was to describe the in situ responses in rats of single-unit carotid body chemoreceptors to changes in arterial PO2 and PCO2. We identified single-unit carotid chemoreceptor activity in male, adult Sprague-Dawley rats by their rapid responses to i.v. NaCN (20 microg) and transient (10 s) asphyxia. 2. Single-unit chemoreceptor responses to isocapnic changes in oxygenation within the arterial oxygen pressure range 34-114 mmHg were described by the power function: f(dis) = 74010(Pa,O2)-2.5; (r2 = 0.6), where f(dis) is the discharge frequency (spikes s-1), P(a,O2) is the arterial oxygen partial pressure (mmHg) and r2 is the correlation coefficient. 3. The responses to iso-oxic changes in CO2, assumed to be linear, had a slope of 0.089 spikes s-1 (mmHg Pa,CO2)-1 (r2 = 0.7). 4. We conclude that carotid body chemoreceptors in adult rats have responses to changes in Pa,O2 and Pa,CO2 similar to those of other species.

Hydroxyl radical formation in the perilymph of asphyxic guinea pig

To elucidate the role of hydroxyl radical ( OH) species in the generation mechanism of the cochlear pathology induced by transient asphyxia and subsequent re-ventilation, the concentrations of 2,3-hydroxybenzoic acid (DHBA) and 2,5-DHBA, major products arising from the attack of OH upon salicylate, were measured in the perilymph of the guinea pig by the high performance liquid chromatography–electrochemical/UV method. The mean value of 2,3-DHBA concentration in the perilymph significantly increased from the pre-asphyxic level (6.4 μM) to 7.6 μM and 8.8 μM during asphyxia of 3 min duration and at 5 min after the onset of re-ventilation, respectively. The 2,5-DHBA concentration was 7.9 μM before asphyxia, and also significantly increased to 11.5 μM and 16.2 μM during and after asphyxia, respectively. These results strongly indicated that OH was generated in the perilymph of the asphyxic and re-ventilated guinea pig cochlea, and the significance of this increased OH in generating anoxia and re-perfusion injury is discussed with respect to iron and oxygen-derived free radicals.

Boltzmann analysis of CM waveforms using virtual instrument software

We describe a modification to our technique for the rapid analysis of low-frequency cochlear microphonic (CM) waveforms in the basal turn of the guinea pig cochlea (Patuzzi and Moleirinho, 1998). The transfer curve relating instantaneous sound pressure in the ear canal to instantaneous receptor current through the outer hair cells (OHCs) is determined from the distorted microphonic waveform generated in the extracellular fluid near the hair cells, assuming a first-order Boltzmann activation curve. Previously, the analysis was done in real time using custom-built electronic circuitry. Here, the same task is performed numerically using virtual instrument software (National Instruments LabVIEW 4.1) running on a personal computer. The assumed theoretical function describing the CM waveform is V cm= V off+ V sat/{1+exp[( E o+ Z. P o.sin(2π f+φ tot))/ kT]}, where the six parameters are (i) a DC offset voltage ( V off); (ii) the frequency of the sinusoidal stimulus ( f); (iii) the phase of the sinusoidal stimulus (φ tot); (iv) the maximal amplitude of the distorted microphonic signal ( V sat); (v) the sensitivity of the transduction process ( Z); and (vi) the operating point on the sigmoidal transfer curve ( E o). The software obtains the least-squares fit to the CM waveforms by continuously deriving the six parameters at a speed of about one determination per second. The independent fitting of the frequency and phase allows the data to be analysed off-line from data previously recorded to tape (i.e. the frequency and phase of the microphonic response need not be known accurately beforehand). We present here an outline of the software we have used, and give an example of the changes which can be monitored using the technique (transient asphyxia). The method’s advantages and limitations have been discussed in our previous paper. The virtual instrument described here is available from the authors on request.

Interaction of methylprednisolone and transient asphyxia on the inner ear of the adrenalectomized rat

Methylprednisolone has been shown clinically to have beneficial effects on certain types of hearing loss. In the current study, compound action potential (CAP) thresholds, endocochlear potentials (EPs), and potassium concentration (C K +) values in the endolymph were determined under conditions of transient asphyxia (45 seconds) and methylprednisolone treatment (24 hours) in bilateral adrenalectomized rats. Treatment with methylprednisolone significantly reduced the effect of transient asphyxia on CAP thresholds as compared with nontreated animals. Methylprednisolone did not alter the dramatic short-term reduction in the EPs produced by anoxia. Potassium concentrations in treated adrenalectomized rats were significantly lower before transient asphyxia than in nontreated adrenalectomized rats. In the nontreated rats, transient asphyxia induced a reduction in C K + levels that was not seen in the methylprednisolone-treated animals. The data support the clinical application of methylprednisolone for certain forms of hearing loss and for potassium imbalance in the endolymph. (Otolaryngol Head Neck Surg 1998;118:338-43.)

Interaction of methylprednisolone and transient asphyxia on the inner ear of the adrenalectomized rat.

Methylprednisolone has been shown clinically to have beneficial effects on certain types of hearing loss. In the current study, compound action potential (CAP) thresholds, endocochlear potentials (EPs), and potassium concentration (CK+) values in the endolymph were determined under conditions of transient asphyxia (45 seconds) and methylprednisolone treatment (24 hours) in bilateral adrenalectomized rats. Treatment with methylprednisolone significantly reduced the effect of transient asphyxia on CAP thresholds as compared with nontreated animals. Methylprednisolone did not alter the dramatic short-term reduction in the EPs produced by anoxia. Potassium concentrations in treated adrenalectomized rats were significantly lower before transient asphyxia than in nontreated adrenalectomized rats. In the nontreated rats, transient asphyxia induced a reduction in CK+ levels that was not seen in the methylprednisolone-treated animals. The data support the clinical application of methylprednisolone for certain forms of hearing loss and for potassium imbalance in the endolymph.

Percutaneous dilatational tracheostomy in a medical ICU.

To evaluate the safety of percutaneous dilatational tracheostomy. A prospective clinical study. The intensive care unit of a university medical clinic. 137 critically ill patients admitted between May 1993 and September 1996. Percutaneous dilatational tracheostomy at the bedside. The median duration of translaryngeal intubation prior to tracheostomy was 8 days. Tracheostomy was carried out within 12.8 min (range 7-30 min). Acute complications were documented in 11.0% of the patients. There was one case of severe bleeding with transient asphyxia. Four patients had tracheal mucosal laceration treated conservatively. The postoperative in-hospital complication rate was 5.1%, the sole problem being stomal bleeding. Only two cases of stomal infection were documented. There was no procedure-related mortality. In the hands of the experienced, percutaneous dilatational tracheostomy is a safe and quick bedside procedure. It is also less expensive and incurs minimal stress for the patient compared with the surgical method. The technique can be easily mastered by non-surgical physicians and we feel that it is the method of choice for elective tracheostomy in the majority of intensive care patients.

Regulation of fetal breathing

Traditionally, the idea of transient asphyxia plus some degree of cold stimulation has been used to explain the establishment of continuous breathing at birth. This idea was nurtured by observations made in the acute fetal preparation at a time when fetal breathing was considered absent. Experimental observations made in the past two decades have challenged this traditional view. First, complete peripheral chemodenervation, essential to the hypoxic stimulus theory, did not affect fetal breathing or the establishment of continuous breathing at birth. Second, occlusion of the umbilical cord in utero, as long as some oxygenation is provided to the fetus in order to avoid fetal hypoxaemia, establishes continuous breathing in utero, in the absence of all sensorial input thought to be important for the establishment of continuous breathing. These observations led us to hypothesize the presence of a placental factor responsible for the inhibition of breathing in utero. This placental factor appears to be a peptide with a molecular mass between 3.5 and 10 kDa. This review will also explore some new observations regarding the generation of central respiratory activity in the fetus, and suggests that the rhythm generator is a neuronal network in which the unit is a pacemaker-like cell uniquely responsive to CO2.

Electrophysiological responses of the cochlea to transient asphyxia are influenced by arachidonate metabolites

The influence of a transient asphyxia on cochlear potentials, i.e. endolymphatic potential (EP), summating potential (SP) and cochlear microphonics (CM) was investigated in guinea pigs when pretreating the animals with various substances interacting with the arachidonic acid (AA) cascade at the level of thromboxane. The controls showed the well-known decline of the EP, CM and the SP increase. When infusing a thromboxane synthetase inhibitor (dazoxiben) or two different thromboxane receptor blockers (daltroban or sulotraban) before the 3-minutes' period of asphyxia was started, the electrophysiological responses of the inner ear (cochlea) could significantly be influenced. The results indicate that a shift of the thromboxane (TXA 2)/prostacyclin (PGI 2)-balance in favour of the last improve the metabolic conditions for a survival of the cochlea when it is challenged.

Functional changes in cultured neurons following transient asphyxia

Functional responses of cultured neurons from rat cortex and hippocampus to transient hypoxia/asphyxia was studied by the measurement of [3H]2-deoxy-D-glucose (2DG) specific uptake. Hypoxic insult was induced by incubating the cells in 5% CO2/95% N2 or in 1 mM NaCN-containing culture medium for various periods of time. Cell morphology, lactate dehydrogenase efflux and protein levels were also examined. Twenty-four hours following hypoxia (6 to 8 h in an anaerobic atmosphere or 90 min in the presence of NaCN) 2DG specific transport was significantly enhanced, without morphological alteration. Cell injury was evident three days post-hypoxia, with a significantly reduced rate of cellular metabolism. The data suggest indirectly that postanoxic release of excitatory amino acids may be responsible for 'delayed neuronal death'.

The effect of vasopressin upon the cochlear potentials in the guinea pig.

The change in the EP, CM, SP and AP during perilymphatic perfusion of vasopressin (antidiuretic hormone) was examined in the guinea pig. The EP was recorded with a microelectrode through the spiral ligament of the second turn. The CM, SP and AP were measured with the differential electrodes in the basal turn. The perfusion of vasopressin at concentration of more than 10(-5)M produced a reversible decrease in the EP. The extent of the EP decline was dependent upon the concentration of vasopressin. Abolition of the effect of vasopressin upon the EP by the resumption of respiration after transient asphyxia was observed. During the perfusion of vasopressin, the CM and AP decreased, while the negative component of the SP increased. The mechanism causing the effect of vasopressin upon the cochlear potentials is discussed.