Anoxic Challenge Research Articles

Synaptic and Network Contributions to Anoxic Depolarization in Mouse Hippocampal Slices

Ischemic stroke remains the third leading cause of death and leading cause of adult disability worldwide. A key event in the pathophysiology of stroke is the anoxic depolarization (AD) of neurons in the ischemic core. Previous studies have established that both the latency to AD and the time spent in AD prior to re-oxygenation are predictors of neuronal death. The present studies used hippocampal slices from male and female mice to investigate the electrophysiological events that affect latency to AD after oxygen deprivation. The results confirm that the epoch between AD and re-oxygenation largely determines the magnitude of synaptic recovery after anoxic challenge. Using a selective antagonist of adenosine A1 receptors, we also confirmed that adenosine released during anoxia (ANOX) suppresses synaptic glutamate release; however, this action has no effect on AD latency or the potential for post-anoxic recovery of synaptic transmission. In contrast, antagonism of AMPA- and NMDA-type glutamate receptors significantly prolongs the latency to AD and alters the speed and synchrony of associated depolarizing waves. Experiments using slices with fields Cornu ammonis 3 (CA3) and Cornu ammonis 1 (CA1) disconnected showed that AD latency is longer in CA1 than in CA3; however, the early AD in CA3 is propagated to CA1 in intact slices. Finally, AD latency in CA1 was found to be longer in slices from female mice than in those from age-matched male mice. The results have implications for stroke prevention and for understanding brain adaptations in hypoxia-tolerant animals.

Pharmacogenetic Modulation of the Noradrenergic System Reveals Protective and Vulnerable Roles in the Neonate Autoresuscitation Reflex

A key feature of neonatal respiration is the autoresuscitation reflex, which restarts breathing and cardiac function after an infant has fallen into respiratory arrest and bradycardia due to extreme hypoxic conditions. Abnormalities in this reflex have been hypothesized to play a role in life threatening pathologies such as Sudden Infant Death Syndrome (SIDS), where infants are often found face down, resulting in asphyxiation and death. Disruptions to the development and maturation of the brainstem autonomic neural circuitry, including the noradrenergic (NA) system, are thought to be an underlying factor in SIDS. Despite the number of studies implicating the NA system in cardiorespiratory control and in SIDS pathology, the role of the NA system in the autoresuscitation reflex remains poorly understood. We hypothesize that the NA system plays a critical role in modulating the neonatal autoresuscitation reflex and that increased NA tone may offer a protective effect.To test this hypothesis, we pharmacogenetically excited and inhibited the whole NA system in P7‐P8 mice by expressing neuro‐excitatory (hM3D) and inhibitory (hM4D) clozapine‐N‐oxide (CNO) activated DREADD receptors throughout all NA producing cells utilizing conditional DREADD and DBH_FlpO mouse lines developed in the Ray lab. Respiratory parameters were measured using a modified facemask pneumotachograph while recording heart rate (HR) through an electrocardiogram (ECG). To induce the autoresuscitation reflex, pups were given a brief exposure to mild hypercapnic anoxia (3%CO2, 97%N2) to induce respiratory arrest and bradycardia. Upon apnea, anoxia was reversed by flushing the pneumotachograph with room air to facilitate the autoresuscitation reflex and recovery. Pups were exposed to repeated bouts of hypercapnic anoxia, followed by recovery periods, until succumbing. We recorded HR and respiratory waveforms continuously throughout the assay. We obtained the number of episodes before death, exposure time, time until first gasping (gasp latency), and frequency of gasping (Gasp ƒB). HR and eupnea recovery were also analyzed. Differences between experimental mice and littermate controls were assessed by a repeated measurement two‐way ANOVA. A p‐value< 0.05 was considered statistically significant. Bonferroni correction was used where multiple comparisons were necessary.Contrary to our hypothesis, preliminary data suggests that acute excitation of the NA system during the anoxic challenge results in a decreased number of successful autoresuscitation events, a higher gasp latency and frequency, and longer delay time to recovery heart rate and eupnea during the last episode. Conversely, when the NA system was inhibited, pups showed a tendency to survive more episodes than controls with a longer exposure time before they succumbed. These results suggest that the NA system might act as an acute modulator of the autoresuscitation reflex, an important neonatal reflex implicated in SIDS pathology.Support or Funding InformationMcNair Medical Institute, March of Dimes Award #1‐FY18‐403, Basil O'Connor Starter Scholar Research Award, R01HL130249, CJ Foundation for SIDS Research AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intrauterine growth restriction impairs right ventricular response to hypoxia in adult male rats.

Intrauterine growth restriction (IUGR) predisposes to cardiovascular diseases in adulthood. The mechanisms of this phenomenon remain cryptic. We hypothesized that heart mitochondria in IUGR-born adult rats are more sensitive to acute hypoxia which translates into dysfunctional cardiac response to hypoxic stress. Adult IUGR-born male rats (the offspring of dams fed with calories-restricted diet during pregnancy) were exposed to acute hypoxic stress with echocardiographic assessment of cardiac function. In parallel, mitochondrial respiration in organelles isolated from left ventricle (LV) and right ventricle (RV) was tested in normoxic and anoxic conditions. The extent of post-anoxic inhibition of mitochondrial respiration and cardiac function was compared with controls, non-IUGR rats. Compared with controls, in the IUGR rats hypoxia significantly reduced only RV contractility, evidenced by decreased fractional shortening, functional area of contraction, and tricuspid annular plane systolic excursion. In isolated mitochondria, anoxic challenge inhibited respiratory chain in both groups of rats. However, compared with controls, the extent of anoxic mitochondrial depression was significantly greater in IUGR-born rats, but only in the organelles isolated from RV. In adult IUGR-born rats, mitochondria from RV are hypersensitive to oxygen deprivation and this translates into maladaptive RV cardiac response to acute hypoxia.

Caffeine improves survival in autoresuscitation‐impaired serotonin deficient rats

Our previous investigation demonstrated that an existing underlying vulnerability in rat neonates (i.e. brainstem 5‐HT deficiency) in combination with a stressor (i.e. pre‐ and early post‐natal nicotine exposure) exacerbates autoresuscitation failure from an anoxic event at a critical developmental period (P10 rats). During this critical period, we hypothesize that caffeine, a respiratory stimulant, will improve successful autoresuscitation rates in the 5‐HT deficient, nicotine exposed P10 neonates and reduce mortality. Preliminary data demonstrate that an intraperitoneal injection of 10mg kg−1 caffeine prior to anoxic challenge improves autoresuscitation rates (survival rate approximately 40%) in the 5‐HT deficient, nicotine exposed P10 rats in comparison to the rats who received IP saline vehicle injections (survival rate of 25%). Prior investigation suggests that eupnea and heart rate recovery following an anoxic event (which elicits an apnea accompanied by a bradycardia) is significantly delayed in 5‐HT deficient rats treated with nicotine, making them more susceptible to failure of autoresuscitation (p<0.05). Presence of caffeine is likely facilitating successful autoresuscitation by shortening apnea duration and promoting an early onset of gasping post‐apnea for a faster eupnea and heart rate recovery.Support or Funding InformationResearch is supported by the NIH Program Project Grant HD036379 (NICHD, PI, Dymecki, S. M.; Project PI, Nattie, E. E.). The authors would also like to acknowledge the generous support of the Tenney Fund and the Ryan Fellowship (awarded to S. Lee).

Evidence of hypoxic tolerance in weak upper airway muscle from young mdx mice

Duchenne muscular dystrophy (DMD) is a genetic disease characterised by deficiency in the protein dystrophin. The respiratory system is weakened and patients suffer from sleep disordered breathing and hypoventilation culminating in periods of hypoxaemia. We examined the effects of an acute (6h) hypoxic stress on sternohyoid muscle function (representative pharyngeal dilator). 8 week old male, wild-type (WT; C57BL/10ScSnJ; n=18) and mdx (C57BL/10ScSn-Dmdmdx/J; n=16) mice were exposed to sustained hypoxia (FIO2=0.10) or normoxia. Muscle functional properties were examined ex vivo. Additional WT (n=5) and mdx (n=5) sternohyoid muscle was exposed to an anoxic challenge. Sternohyoid dysfunction was observed in mdx mice with significant reductions in force and power. Following exposure to the acute in vivo hypoxic stress, WT sternohyoid muscle showed evidence of functional impairment (reduced force, work and power). Conversely, mdx sternohyoid showed an apparent tolerance to the acute hypoxic stress. This tolerance was not maintained for mdx following a severe hypoxic stress. A dysfunctional upper airway muscle phenotype is present at 8 weeks of age in the mdx mouse, which may have implications for the control of airway patency in DMD. Hypoxic tolerance in mdx respiratory muscle is suggestive of adaptation to chronic hypoxia, which could be present due to respiratory morbidity. We speculate a role for hypoxia in mdx respiratory muscle morbidity.

Glutamine and glutamate limit the shortening of action potential duration in anoxia-challenged rabbit hearts.

In clinical conditions, amino acid supplementation is applied to improve contractile function, minimize ischemia/reperfusion injury, and facilitate postoperative recovery. It has been shown that glutamine enhances myocardial ATP/APD (action potential duration) and glutathione/oxidized glutathione ratios, and can increase hexosamine biosynthesis pathway flux, which is believed to play a role in cardioprotection. Here, we studied the effect of glutamine and glutamate on electrical activity in Langendorff-perfused rabbit hearts. The hearts were supplied by Tyrode's media with or without 2.5 mmol/L glutamine and 150 μmol/L glutamate, and exposed to two 6-min anoxias with 20-min recovery in between. Change in APD was detected using a monophasic action potential probe. A nonlinear mixed-effects regression technique was used to evaluate the effect of amino acids on APD over the experiment. Typically, the dynamic of APD change encompasses three phases: short transient increase (more prominent in the first episode), slow decrease, and fast increase (starting with the beginning of recovery). The effect of both anoxic challenge and glutamine/glutamate was cumulative, being more pronounced in the second anoxia. The amino acids' protective effect became largest by the end of anoxia – 20.0% (18.9, 95% CI: [2.6 ms, 35.1 ms]), during the first anoxia and 36.6% (27.1, 95% CI: [7.7 ms, 46.6 ms]), during the second. Following the second anoxia, APD difference between control and supplemented hearts progressively increased, attaining 10.8% (13.6, 95% CI: [4.1 ms, 23.1 ms]) at the experiments' end. Our data reveal APD stabilizing and suggest an antiarrhythmic capacity of amino acid supplementation in anoxic/ischemic conditions.

Acute manipulation of serotonergic neurons alters the mouse neonatal response to homeostatic stressors.

Many sudden infant death syndrome (SIDS) cases are associated medullary serotonergic (5-HT) neuron abnormalities. We hypothesize that these abnormalities impair infants from adequately responding to stressors, such as hypoxia and hypercapnia (asphyxia). In response to asphyxia, young mammals initiate the “autoresuscitation” reflex – a survival promoting mechanism aimed at restoring homeostasis. Deficiency in this reflex is a proposed mechanism for SIDS mortality. To test the acute role of 5-HT neurons in autoresuscitation, we applied a chemogenetic neuronal inhibition strategy to inducibly suppress the activity of 5-HT neurons in unanesthetized mouse pups. Specifically, we expressed the synthetic inhibitory G-protein coupled receptor, Di, selectively in 5-HT neurons using mice transgenic for the driver Pet1::Flpe and the conditional Di-expressing knock-in allele RC::FDi. Selective 5-HT neuron inhibition follows upon intraperitoneal CNO injection. Using head-out plethysmography, we measured cardiorespiratory responses to repeated episodes of anoxia-induced (97% N2/3% CO2) apnea following acute inhibition of 5-HT neuron activity in P8 mouse pups. After injection with CNO, Di-expressing pups, but not controls, exhibited altered homeostatic parameters prior to an anoxic challenge. Additionally failure to autoresuscitate after anoxic challenge was more pronounced in CNO/Di-triggered pups than in controls, and was associated with a longer time to restore homeostasis. Results suggest that acute suppression of 5-HT neuron firing in mouse pups leads to altered baseline cardiorespiratory homeostasis and abnormal cardiorespiratory recovery from anoxia.

Perinatal hyperoxic exposure reconfigures the central respiratory network contributing to intolerance to anoxia in newborn rat pups

Perinatal exposure to hyperoxia (30-60% O2) alters the respiratory control system via modulation of peripheral arterial chemoreceptor development and function. Furthermore, hyperoxic exposure during the first two postnatal weeks of life can alternatively modulate the different phases of the hypoxic ventilatory response. Given the effects of perinatal hyperoxia, the aims of our study were 1) to determine the effect on survival time in response to lethal anoxic stimuli in rat pups and 2) to characterize the output of the isolated central respiratory network in response to acute hypoxic stimuli. We hypothesized that perinatal hyperoxic exposure would modify the neonatal rat ventilatory response to anoxia by affecting a central component of the respiratory network in addition to the maturation of the carotid body chemoreceptors. We found that animals continuously exposed to 60% oxygen up to age 5 days after parturition (P5) have reduced breathing frequency at baseline and within the first 10 min of a fatal anoxic challenge. Hyperoxic rat pups also have a shortened time to last gasp in response to anoxia that is not associated with lung injury or inflammation. This study is the first to demonstrate that these in vivo findings correlate with reduced phrenic burst frequency from the isolated brainstem ex vivo. Thus hyperoxic exposure reduced the phrenic burst frequency at baseline and in response to ex vivo anoxia. Importantly, our data suggest that perinatal hyperoxia alters ventilation and the response to anoxia at P5 in part by altering the frequency of phrenic bursts generated by the central respiratory network.

Reprint of: Effects of growth hormone transgene expression and triploidy on acute stress indicators in Atlantic salmon (Salmo salar L.)

Abstract Transgenic Atlantic salmon ( Salmo salar ) expressing an opAFP-csGH transgene exhibit 3–6-fold growth rate acceleration in the first years of life. Transgenics intended for production likely will be triploids for purposes of reproductive confinement. Growth hormone (GH) transgene expression and triploidy may affect physiological traits with bearing on fitness, animal welfare, and aquaculture production. The goal of our study was to determine the responses of juvenile GH-transgenic and triploid Atlantic salmon to stress. Groups of one-year old conventionally bred (termed wild-type), GH-transgenic, and triploid Atlantic salmon were subjected to no stress (control), one-week of fasting, or low dissolved oxygen (1.5–2.0 ppm) in triplicated tanks. Blood samples were taken from anesthetized fish, and nine markers of primary and secondary stress response were quantified. In addition, these stress-response markers were monitored over a time-course of 0, 1, 3, 6, and 24 h after handling and air exposure stress. For fish subject to no stress, parameters measured did not differ among genotypes, except that blood pH was higher and p O 2 and potassium levels lower in wild-type than in triploid or transgenic salmon. Immediately after one week of fasting, transgenic fish exhibited higher levels of sodium and chloride than other genotypes, suggesting osmoregulatory difficulty. Immediately after anoxic challenge, transgenic fish exhibited higher hematocrit, p CO 2 , glucose and sodium levels than other genotypes. In the time-course study, levels of stress-response indicators tended to peak at higher levels in GH-transgenic than in triploid than in wild-type salmon, and to not return to baseline levels through 24 h. Results of the experiments collectively demonstrated that wild-type fish maintained homeostasis more effectively than transgenic or triploid fish, exhibiting smaller changes in all measured stress-response parameters. Poor stress response may affect aquaculture performance of transgenic or triploid Atlantic salmon and hence the aquaculture practices needed for their production and maintenance of welfare, and also may reduce their fitness in the wild.

Effects of growth hormone transgene expression and triploidy on acute stress indicators in Atlantic salmon (Salmo salar L.)

Transgenic Atlantic salmon (Salmo salar) expressing an opAFP-csGH transgene exhibit 3–6-fold growth rate acceleration in the first years of life. Transgenics intended for production likely will be triploids for purposes of reproductive confinement. Growth hormone (GH) transgene expression and triploidy may affect physiological traits with bearing on fitness, animal welfare, and aquaculture production. The goal of our study was to determine the responses of juvenile GH-transgenic and triploid Atlantic salmon to stress. Groups of one-year old conventionally bred (termed wild-type), GH-transgenic, and triploid Atlantic salmon were subjected to no stress (control), one-week of fasting, or low dissolved oxygen (1.5–2.0ppm) in triplicated tanks. Blood samples were taken from anesthetized fish, and nine markers of primary and secondary stress response were quantified. In addition, these stress-response markers were monitored over a time-course of 0, 1, 3, 6, and 24h after handling and air exposure stress. For fish subject to no stress, parameters measured did not differ among genotypes, except that blood pH was higher and pO2 and potassium levels lower in wild-type than in triploid or transgenic salmon. Immediately after one week of fasting, transgenic fish exhibited higher levels of sodium and chloride than other genotypes, suggesting osmoregulatory difficulty. Immediately after anoxic challenge, transgenic fish exhibited higher hematocrit, pCO2, glucose and sodium levels than other genotypes. In the time-course study, levels of stress-response indicators tended to peak at higher levels in GH-transgenic than in triploid than in wild-type salmon, and to not return to baseline levels through 24h. Results of the experiments collectively demonstrated that wild-type fish maintained homeostasis more effectively than transgenic or triploid fish, exhibiting smaller changes in all measured stress-response parameters. Poor stress response may affect aquaculture performance of transgenic or triploid Atlantic salmon and hence the aquaculture practices needed for their production and maintenance of welfare, and also may reduce their fitness in the wild.

Measurement of the oxidation state of mitochondrial cytochrome c from the neocortex of the mammalian brain

Diffuse optical remission spectra from the mammalian neocortex at visible wavelengths contain spectral features originating from the mitochondria. A new algorithm is presented, based on analytically relating the first differential of the attenuation spectrum to the first differential of the chromophore spectra, that can separate and calculate the oxidation state of cytochrome c as well as the absolute concentration and saturation of hemoglobin. The algorithm is validated in phantoms and then tested on the neocortex of the rat during an anoxic challenge. Implementation of the algorithm will provide detailed information of mitochondrial oxygenation and mitochondrial function in physiological studies of the mammalian brain.

The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures

The epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are commonly found in periodically hypoxic environments. The ecophysiological time available for these animals to safely exploit these niches during different seasonal temperatures was examined. The time to loss of righting reflex (T (LRR)) was examined in response to an open ended anoxic challenge at three seasonal temperatures (23, 25 and 27°C). Ventilation rates were measured in an open ended anoxic challenge at 23°C and during 1.5h of anoxia followed by 2h of re-oxygenation at 23 and 25°C. The mean T (LRR) of epaulette and grey carpet sharks was inversely proportional to temperature. The T (LRR) was similar between species at 23°C; however, grey carpet sharks had significantly reduced T (LRR) at higher temperatures. During the standardised anoxic challenge, epaulette sharks entered into ventilatory depression significantly earlier at 25°C. During re-oxygenation, epaulette sharks exposed to anoxia at 23°C had no significant increase in ventilation rates. However, after anoxic challenge and re-oxygenation at 25°C, epaulette sharks showed a significant increase in ventilation rates during re-oxygenation. Grey carpet sharks displayed no evidence of ventilatory depression during anoxia. However, during re-oxygenation, grey carpet sharks had significantly elevated ventilation rates above pre-experimental levels and control animals. These data demonstrate that the anoxia tolerance times of both species were temperature dependent, with a significant reduction in the T (LRR) occurring at higher temperatures. Epaulette sharks had a significantly greater T (LRR) at higher temperatures than grey carpet sharks, which did not enter into a ventilatory depression.

Inspiratory neural network complexity of unusual gasping patterns in urethane‐anesthetized neonatal rats

Gasping is an autoresuscitative mechanism in response to anoxia, and unusual gasp patterns (i.e., two or three closely spaced successive gasps termed ‘doublet’ and ‘triplet’ gasps) have been observed in infants with sudden infant death syndrome (SIDS) and non‐SIDS conditions during agonal respiration. Previously, we identified doublet and triplet gasps in spontaneously breathing urethane‐anesthetized P0‐P12 neonatal rats in response to an anoxic challenge, and characterized the timing features associated with these bursts. Here, we evaluated the complexity of the central inspiratory neural network underlying these unusual gasping patterns using approximate entropy (ApEn) analysis. For these analyses, the neonatal rats were divided into two groups based on the marked difference in the number of doublet gasps. In P0‐P6 rats (n=12), ApEn decreased from baseline (BL) when compared to singlet (from 0.318±0.008 to 0.279±0.003; P<0.01), but not doublet or triplet gasps. In contrast, in P7‐P12 rats (n=7), ApEn decreased from BL when compared to singlet (from 0.355±0.009 to 0.242±0.004; P<0.01), doublet (P<0.01), and triplet gasps (P<0.01). These data suggest that while the central inspiratory neural network underlying singlet gasps may be unaffected by developmental age, the network underlying unusual gasping patterns appears to be differentially influenced by maturation.Supported by HL63175, NS049310

Hematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re‐oxygenation

We compared the hematological responses of wild and captive populations of two closely related sharks to a standardized anoxic challenge and during a 12 hr recovery period in normoxia: the epaulette shark (Hemiscyllium ocellatum, Bonnaterre, 1788) and the grey carpet shark (Chiloscyllium punctatum, Müller and Henle, 1838). Compared to normoxic controls, a significant increase in hematocrit (captive 22.3%; wild 35.9%) coupled with a decline in mean corpuscular hemoglobin concentration occurred in epaulette sharks indicating erythrocyte swelling in response to anoxia. However, the grey carpet shark had a significantly increased hematocrit (captive 27.2%; wild 29.3%), erythrocyte count (captive 37.6%; wild 46.3%) and hemoglobin concentration (captive 31.9%; wild 31.5%), suggesting a release of erythrocytes into the circulation and/or hemoconcentration in response to anoxia. Plasma glucose concentrations were maintained in both wild and captive epaulette sharks and in wild grey carpet sharks during anoxia but increased significantly after 2 hr of re-oxygenation (epaulette: captive 55.8%; wild 50.1%; grey carpet shark: wild 70.3%) and remained elevated for 12 hr. Captive grey carpet sharks had an immediate increase in plasma glucose concentrations after anoxia (96.4%), which was sustained for 12 hr of re-oxygenation. Lactate concentrations significantly increased in captive and wild animals of both species after anoxia, reaching a peak at 2 hr of re-oxygenation. Both species showed significant, yet divergent, hematological changes in response to anoxia and re-oxygenation, which may not only prolong their survival and assist in recovery but also reflect their respective ecophysiological adaptations to the extreme environments that they inhabit.

Interleukin-1β depresses hypoxic gasping and autoresuscitation in neonatal DBA/1lacJ mice

The pro-inflammatory cytokine interleukin-1beta (IL-1beta) has been proposed to act as an important mediator between infection and apnea in neonates. In this study, respiration and the ability to survive anoxic challenge were investigated between 70 and 95 min after intraperitoneal injection of IL-1beta (10 microg/kg) or NaCl in 9-day-old DBA/1lacJ mice. Using flow plethysmography, we show that mice given IL-1beta exhibited a decreased tidal volume (V(T)) and minute ventilation (V(E)) during normoxia compared to control animals. Hyperoxic challenge revealed functioning peripheral chemoreceptors in all animals, suggesting a central mechanism underlying the ventilatory effects of IL-1beta. In response to anoxia (100% N2), all animals irrespective of treatment displayed a biphasic ventilatory pattern. Mice given IL-1beta exhibited fewer gasps and were unable to sustain gasping efforts for as long as control animals. Additionally, they were less able to autoresuscitate and survive following severe hypoxic apnea. These findings indicate that infection may adversely affect central respiratory control in newborn mice via interleukin-1beta.

IL-1 beta depresses respiration and anoxic survival via a prostaglandin-dependent pathway in neonatal rats.

IL-1 beta has been proposed to be an important mediator linking infection, apnea, and sudden infant death syndrome. We hypothesized that IL-1 beta acts in this capacity by depressing brainstem respiratory neurons via a prostaglandin-dependent pathway. For studying the effects of IL-1 beta on respiration as well as the mechanism underlying its actions, 7-d-old rats received an initial injection (i.p.) of NaCl or a cyclooxygenase inhibitor (indomethacin, 10 mg/kg) followed by a second injection (i.p.) at 30 min of NaCl, recombinant rat IL-1 beta (10 microg/kg), or lipopolysaccharide (LPS; 100 microg/kg). Respiration during normoxia and in response to anoxia (100% N2) was examined at 60 min after the second injection using flow and barometric plethysmography. Animals given IL-1 beta breathed more slowly and died more often after anoxia. LPS also reduced the rats' ability to autoresuscitate and survive an anoxic challenge. Indomethacin prevented the depressive effects during normoxia and the adverse effects on survival. For investigating drug-induced changes in central respiratory activity, IL- 1 beta (1.0 or 1.25 ng/mL) and prostaglandin E2 (5 or 20 microg/L) was applied to the brainstem-spinal cord preparation of 0- to 4-d-old rats. Whereas IL-1 beta exerted no effect on respiration measured at the C4 ventral root during a 60-min period, prostaglandin E2 reversibly inhibited respiratory activity. These findings suggest that IL-1 beta does not inhibit respiratory neurons directly but may depress breathing and hypoxic defense via a prostaglandin-mediated mechanism.

Rapid T2* mapping using interleaved echo planar imaging.

Magnetic resonance imaging methods that are sensitive to T2* are widely used in the study of blood oxygenation changes, most notably in functional studies of the brain. In these studies the signal intensity change in T2*-weighted imaging is related to the coupling of cerebral blood flow and metabolism. Rapid measurement of T2* itself would offer a valuable method to quantify blood oxygenation changes indirectly and monitor their time course. An interleaved echoplanar imaging (EPI) sequence is presented here that allows maps of T2* to be generated in a few seconds. The sequence benefits from reduced geometric distortion and an improved point spread function compared with single-shot EPI. A comparison among a set of T2*-weighted interleaved EPI images, single-shot EPI, and conventional gradient-echo and spin-echo methods is made using a compartmentalized doped water phantom. The interleaved sequence yields accurate T2* values when compared with reference measurements made using the slower gradient-echo technique. Data acquired from the rat brain at 2.35 T prior to and during an anoxic challenge show, with high temporal resolution, the reduction in T2* associated with increased levels of deoxyhemoglobin.

Rapid T2* mapping using interleaved echo planar imaging

Magnetic resonance imaging methods that are sensitive to T2* are widely used in the study of blood oxygenation changes, most notably in functional studies of the brain. In these studies the signal intensity change in T2*-weighted imaging is related to the coupling of cerebral blood flow and metabolism. Rapid measurement of T2* itself would offer a valuable method to quantify blood oxygenation changes indirectly and monitor their time course. An interleaved echoplanar imaging (EPI) sequence is presented here that allows maps of T2* to be generated in a few seconds. The sequence benefits from reduced geometric distortion and an improved point spread function compared with single-shot EPI. A comparison among a set of T2*-weighted interleaved EPI images, single-shot EPI, and conventional gradient-echo and spin-echo methods is made using a compartmentalized doped water phantom. The interleaved sequence yields accurate T2* values when compared with reference measurements made using the slower gradient-echo technique. Data acquired from the rat brain at 2.35 T prior to and during an anoxic challenge show, with high temporal resolution, the reduction in T2* associated with increased levels of deoxyhemoglobin. Magn Reson Med 41:368–374, 1999. © 1999 Wiley-Liss, Inc.

Mechanisms of calcium and sodium fluxes in anoxic myelinated central nervous system axons

Electron probe X-ray microanalysis was used to measure water content and concentrations of elements (i.e. Na, K, Cl and Ca) in selected morphological compartments of rat optic nerve myelinated axons. Transaxolemmal movements of Na + and Ca 2+ were modified experimentally and corresponding effects on axon element and water compositions were determined under control conditions and following in vitro anoxic challenge. Also characterized were effects of modified ion transport on axon responses to postanoxia reoxygenation. Blockade of Na + entry by tetrodotoxin (1 μM) or zero Na +/Li +-substituted perfusion reduced anoxic increases in axonal Na and Ca concentrations. Incubation with zero-Ca 2+/EGTA perfusate prevented axoplasmic and mitochondrial Ca accumulation during anoxia but did not affect Na increases or K losses in these compartments. Inhibition of Na +–Ca 2+ exchange with bepridil (30 μM) selectively prevented increases in intra-axonal Ca, whereas neither nifedipine (5 μM) nor nimodipine (5 μM) influenced the effects of anoxia on axonal Na, K or Ca. X-ray microanalysis also showed that prevention of Na and Ca influx during anoxia obtunded severe elemental deregulation normally associated with reoxygenation. Results of the present study suggest that during anoxia, Na + enters axons mainly through voltage-gated Na + channels and that subsequent increases in axoplasmic Na + are functionally coupled to extra-axonal Ca 2+ import. Na + i-dependent, Ca 2+ o entry is consistent with reverse operation of the axolemmal Na +–Ca 2+ exchanger and we suggest this route represents a primary mechanism of Ca 2+ influx. Our findings also implicate a minor route of Ca 2+ entry directly through Na + channels.

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.