Acute Hypoxic Exposure Research Articles

Tandem Mass Tagging-Based Quantitative Proteomics Analysis Reveals Damage to the Liver and Brain of Hypophthalmichthys molitrix Exposed to Acute Hypoxia and Reoxygenation.

Aquaculture environments frequently experience hypoxia and subsequent reoxygenation conditions, which have significant effects on hypoxia-sensitive fish populations. In this study, hepatic biochemical activity indices in serum and the content of major neurotransmitters in the brain were altered markedly after acute hypoxia and reoxygenation exposure in silver carp (Hypophthalmichthys molitrix). Proteomics analysis of the liver showed that a number of immune-related and cytoskeletal organization-related proteins were downregulated, the ferroptosis pathway was activated, and several antioxidant molecules and detoxifying enzymes were upregulated. Proteomics analysis of the brain showed that somatostatin-1A (SST1A) was upregulated, dopamine-degrading enzyme catechol O methyltransferase (COMT) and ferritin, heavy subunit (FerH) were downregulated, and the levels of proteins involved in the nervous system were changed in different ways. In conclusion, these findings highlight that hypoxia–reoxygenation has potential adverse effects on growth, locomotion, immunity, and reproduction of silver carp, and represents a serious threat to liver and brain function, possibly via ferroptosis, oxidative stress, and cytoskeleton destruction in the liver, and abnormal expression of susceptibility genes for neurodegenerative disorders in the brain. Our present findings provide clues to the mechanisms of hypoxia and reoxygenation damage in the brain and liver of hypoxia-sensitive fish. They could also be used to develop methods to reduce hypoxia or reoxygenation injury to fish.

Aged Mouse Hippocampus Exhibits Signs of Chronic Hypoxia and an Impaired HIF-Controlled Response to Acute Hypoxic Exposures.

Altered hypoxia-inducible factor-alpha (HIF-α) activity may have significant consequences in the hippocampus, which mediates declarative memory, has limited vascularization, and is vulnerable to hypoxic insults. Previous studies have reported that neurovascular coupling is reduced in aged brains and that diseases which cause hypoxia increase with age, which may render the hippocampus susceptible to acute hypoxia. Most studies have investigated the actions of HIF-α in aging cortical structures, but few have focused on the role of HIF-α within aged hippocampus. This study tests the hypothesis that aging is associated with impaired hippocampal HIF-α activity. Dorsal hippocampal sections from mice aged 3, 9, 18, and 24 months were probed for the presence of HIF-α isoforms or their associated gene products using immunohistochemistry and fluorescent in situ hybridization (fISH). A subset of each age was exposed to acute hypoxia (8% oxygen) for 3 h to investigate changes in the responsiveness of HIF-α to hypoxia. Basal mean intensity of fluorescently labeled HIF-1α protein increases with age in the hippocampus, whereas HIF-2α intensity only increases in the 24-month group. Acute hypoxic elevation of HIF-1α is lost with aging and is reversed in the 24-month group. fISH reveals that glycolytic genes induced by HIF-1α (lactose dehydrogenase-a, phosphoglycerate kinase 1, and pyruvate dehydrogenase kinase 1) are lower in aged hippocampus than in 3-month hippocampus, and mRNA for monocarboxylate transporter 1, a lactose transporter, increases. These results indicate that lactate, used in neurotransmission, may be limited in aged hippocampus, concurrent with impaired HIF-α response to hypoxic events. Therefore, impaired HIF-α may contribute to age-associated cognitive decline during hypoxic events.

Cognitive Function is Unaffected during Acute Hypoxic Exposure but was Improved Following Exercise

Cognitive Function is Unaffected during Acute Hypoxic Exposure but was Improved Following Exercise

Acute hypoxia exposure following prenatal stress impairs hippocampus and novelty-seeking behavior in adolescent rats.

The present study aimed to investigate the effects of acute hypoxia exposure following prenatal stress on the novelty-seeking behavior and hippocampus of adolescent rats. The offspring were divided into prenatal stress (PS) and non-stress (NS) groups. Both groups were exposed to hypoxia on postnatal day 10 (P10) while control groups were undisturbed. Novel object recognition task was performed in each group. Next, brains were collected to examine hippocampus via immunohistochemical and biochemical studies on postnatal day 35 (P35). PS decreased novelty discrimination and synaptophysin (SYN) expressions in both CA1 and CA3 of the hypoxia group prominently (p < 0.05). Nestin-expressing cells were reduced while vascular endothelial growth factor (VEGF) expression was enhanced in the subgranular zone (SGZ) of PS-hypoxia group (p < 0.05). VEGF enhancement triggered angiogenesis in the CA1 and CA3 significantly (p < 0.05). PS also increased thiobarbituric acid reactive substances (TBARS) levels in the hypoxia group as a result of oxidative stress (p < 0.05). These findings demonstrated that PS exacerbates neurodevelopmental deficits in the hippocampus of acute hypoxia-induced offspring in adolescence.

Ketone Bodies Impact on Hypoxic CO2 Retention Protocol During Exercise.

Exogenous ketone esters have demonstrated the capacity to increase oxygen availability during acute hypoxic exposure leading to the potential application of their use to mitigate performance declines at high altitudes. Voluntary hypoventilation (VH) with exercise reliably reduces oxygen availability and increases carbon dioxide retention without alterations to ambient pressure or gas content. Utilizing a double-blind randomized crossover design, fifteen recreational male distance runners performed submaximal exercise (4 × 5 min; 70% VO2 Max) with VH. An exogenous ketone ester (KME; 573 mg⋅kg–1) or iso-caloric flavor matched placebo (PLA) was consumed prior to exercise. Metabolites, blood gases, expired air, heart rate, oxygen saturation, cognition, and perception metrics were collected throughout. KME rapidly elevated R-β-hydroxybutyrate and reduced blood glucose without altering lactate production. KME lowered pH, bicarbonate, and total carbon dioxide. VH with exercise significantly reduced blood (SpO2) and muscle (SmO2) oxygenation and increased cognitive mean reaction time and respiratory rate regardless of condition. KME administration significantly elevated respiratory exchange ratio (RER) at rest and throughout recovery from VH, compared to PLA. Blood carbon dioxide (PCO2) retention increased in the PLA condition while decreasing in the KME condition, leading to a significantly lower PCO2 value immediately post VH exercise (IPE; p = 0.031) and at recovery (p = 0.001), independent of respiratory rate. The KME’s ability to rapidly alter metabolism, acid/base balance, CO2 retention, and respiratory exchange rate independent of respiratory rate changes at rest, during, and/or following VH exercise protocol illustrates a rapid countermeasure to CO2 retention in concert with systemic metabolic changes.

Key role of Extracellular RNA in hypoxic stress induced myocardial injury.

Myocardial infarction (MI), atherosclerosis and other inflammatory and ischemic cardiovascular diseases (CVDs) have a very high mortality rate and limited therapeutic options. Although the diagnosis is based on markers such as cardiac Troponin-T (cTrop-T), the mechanism of cTrop-T upregulation and release is relatively obscure. In the present study, we have investigated the mechanism of cTrop-T release during acute hypoxia (AH) in a mice model by ELISA & immunohistochemistry. Our study showed that AH exposure significantly induces the expression and release of sterile inflammatory as well as MI markers in a time-dependent manner. We further demonstrated that activation of TLR3 (mediated by eRNA) by AH exposure in mice induced cTrop-T release and Poly I:C (TLR3 agonist) also induced cTrop-T release, but the pre-treatment of TLR3 immuno-neutralizing antibody or silencing of Tlr3 gene or RNaseA treatment two hrs before AH exposure, significantly abrogated AH-induced Caspase 3 activity as well as cTrop-T release. Our immunohistochemistry and Masson Trichrome (MT) staining studies further established the progression of myocardial injury by collagen accumulation, endothelial cell and leukocyte activation and adhesion in myocardial tissue which was abrogated significantly by pre-treatment of RNaseA 2 hrs before AH exposure. These data indicate that AH induced cTrop-T release is mediated via the eRNA-TLR3-Caspase 3 pathway.

Effects of Hypoxia on Selected Psychophysiological Stress Responses of Military Aircrew.

There is a lack of information on the psychophysiological response of pilots under hypoxic conditions. The study of the physiological, psychological, cardiorespiratory, neurological, behavioural, sensory, and cognitive symptoms that may appear during training in hypobaric chambers is essential to optimize the training processes of aircrew members. Thus, the present study is aimed at analyzing the psychophysiological responses of aircrew members in an incremental hypoxia training protocol. Psychophysiological responses of 44 aircrew members (34 males and 10 females) in an incremental hypoxia training protocol (3 minutes at 0 meters, 8 minutes at 5,000 meters, and maximum time at 7500 meters) were measured. Results suggested that the incremental hypoxia training protocol did not affect cortical arousal and handgrip strength; however, it increased the sympathetic tone, perceived stress, perceived effort, and heart rate and decreased forced expiratory volume and blood oxygen saturation. Thus, we concluded that acute hypoxic hypobaric exposure leads to decreased parasympathetic tone, blood oxygen saturation, and maximal spirometry values, without negatively affecting handgrip strength and cortical arousal. This information will lead to find specific training systems that meet the real needs of aircrew.

PATET ratio by Doppler echocardiography: noninvasive detection of pediatric pulmonary arterial hypertension.

Pulmonary artery acceleration time (PAT) and PAT: ejection time (PATET) ratio are echocardiographic measurements of pulmonary arterial hypertension (PAH). These non-invasive quantitative measurements are ideal to follow longitudinally through the clinical course of PAH, especially as it relates to need for and/or response to treatment. This review article focuses on the current literature of PATET measurement for infants and children as it relates to shortening of the PATET ratio in PAH. At the same time, further development of PATET as an outcome measure for PAH in pre-clinical models, particularly mice, such that the field can move forward to human clinical studies that are both safe and effective. Here we present what is known about PATET in infants and children and discuss what is known in pre-clinical models with particular emphasis on neonatal mouse models. In both animal models and human disease, PATET allows for longitudinal measurements in the same individual, leading to more precise determinations of disease/model progression and/or response to therapy.

Acute Exposure to Normobaric Hypoxia Impairs Balance Performance in Sub-elite but Not Elite Basketball Players.

Although high and simulated altitude training has become an increasingly popular training method, no study has investigated the influence of acute hypoxic exposure on balance in team-sport athletes. Therefore, the purpose of this study was to investigate whether acute exposure to normobaric hypoxia is detrimental to balance performance in highly-trained basketball players. Nine elite and nine sub-elite male basketball players participated in a randomized, single-blinded, cross-over study. Subjects performed repeated trials of a single-leg balance test (SLBT) in an altitude chamber in normoxia (NOR; approximately sea level) with FiO2 20.9% and PiO2 ranging from 146.7 to 150.4 mmHg and in normobaric hypoxia (HYP; ~3,800 m above sea level) with FiO2 13.0% and PiO2 ranging from 90.9 to 94.6 mmHg. The SLBT was performed three times: 15 min after entering the environmental chamber in NOR or HYP, then two times more interspersed by 3-min rest. Peripheral oxygen saturation (SpO2) and heart rate (HR) were recorded at four time points: after the initial 15-min rest inside the chamber and immediately after each SLBT. Across the cohort, the balance performance was 7.1% better during NOR than HYP (P < 0.01, = 0.58). However, the performance of the elite group was not impaired by HYP, whereas the sub-elite group performed worse in the HYP condition on both legs (DL: P = 0.02, d = 1.23; NDL: P = 0.01, d = 1.43). SpO2 was lower in HYP than NOR (P < 0.001, = 0.99) with a significant decline over time during HYP. HR was higher in HYP than NOR (P = 0.04, = 0.25) with a significant increase over time. Acute exposure to normobaric hypoxia detrimentally affected the balance performance in sub-elite but not elite basketball players.

Stroke-Related Mortality at Different Altitudes: A 17-Year Nationwide Population-Based Analysis From Ecuador.

Introduction: Worldwide, more than 5.7% of the population reside above 1,500 m of elevation. It has been hypothesized that acute short-term hypoxia exposure could increase the risk of developing a stroke. Studies assessing the effect of altitude on stroke have provided conflicting results, some analyses suggest that long-term chronic exposure could be associated with reduced mortality and lower stroke incidence rates.Methods: An ecological analysis of all stroke hospital admissions, mortality rates, and disability-adjusted life years in Ecuador was performed from 2001 to 2017. The cases and population at risk were categorized in low (<1,500 m), moderate (1,500–2,500 m), high (2,500–3,500 m), and very high altitude (3,500–5,500 m) according to the place of residence. The derived crude and direct standardized age-sex adjusted mortality and hospital admission rates were calculated.Results: A total of 38,201 deaths and 75,893 stroke-related hospital admissions were reported. High altitude populations (HAP) had lower stroke mortality in men [OR: 0.91 (0.88–0.95)] and women [OR: 0.83 (0.79–0.86)]. In addition, HAP had a significant lower risk of getting admitted to the hospital when compared with the low altitude group in men [OR: 0.55 (CI 95% 0.54–0.56)] and women [OR: 0.65 (CI 95% 0.64–0.66)].Conclusion: This is the first epidemiological study that aims to elucidate the association between stroke and altitude using four different elevation ranges. Our findings suggest that living at higher elevations offers a reduction or the risk of dying due to stroke as well as a reduction in the probability of being admitted to the hospital. Nevertheless, this protective factor has a stronger effect between 2,000 and 3,500 m.

MEDEX 2015: Prophylactic Effects of Positive Expiratory Pressure in Trekkers at Very High Altitude.

Purpose: Positive expiratory pressure (PEP) breathing has been shown to increase arterial oxygenation during acute hypoxic exposure but the underlying mechanisms and consequences on symptoms during prolonged high-altitude exposure remain to be elucidated.Methods: Twenty-four males (41 ± 16 years) were investigated, at sea level and at 5,085 m after 18 days of trekking from 570 m. Participants breathed through a face-mask with PEP = 0 cmH2O (PEP0, 0–45th min) and with PEP = 10 cmH2O (PEP10, 46–90th min). Arterial (SpO2), quadriceps and prefrontal (near infrared spectroscopy) oxygenation was measured continuously. Middle cerebral artery blood velocity (MCAv, transcranial Doppler), cardiac function (2D-echocardiography), extravascular lung water accumulation (UsLC, thoracic ultrasound lung comets) and acute mountain sickness (Lake Louise score, LLS) were assessed during PEP0 and PEP10.Results: At 5,085 m with PEP0, SpO2 was 78 ± 4%, UsLC was 8 ± 5 (a.u.) and the LLS was 2.3 ± 1.7 (all P < 0.05 versus sea level). At 5,085 m, PEP10 increased significantly SpO2 (+9 ± 5%), quadriceps (+2 ± 2%) and prefrontal cortex (+2 ± 2%) oxygenation (P < 0.05), and decreased significantly MCAv (−16 ± 14 cm.s–1) and cardiac output (−0.7 ± 1.2 L.min–1) together with a reduced stroke volume (−9 ± 15 mL, all P < 0.05) and no systemic hypotension. PEP10 decreased slightly the number of UsLC (−1.4 ± 2.7, P = 0.04) while the incidence of acute mountain sickness (LLS ≥ 3) fell from 42% with PEP0 to 25% after PEP10 (P = 0.043).Conclusion: PEP10 breathing improved arterial and tissue oxygenation and symptoms of acute mountain sickness after trekking to very high altitude, despite reduced cerebral perfusion and cardiac output. Further studies are required to establish whether PEP-breathing prophylactic mechanisms also occur in participants with more severe acute mountain sickness.

Spleen Contraction During Sudden Eupneic Hypoxia Elevates Hemoglobin Concentration.

The spleen contracts progressively during moderate normobaric hypoxia exposure of 20 min, which elevates hemoglobin concentration (Hb). However, acute hypoxia exposure could be shorter and more severe when oxygen systems fail during, e.g., high-altitude sky diving, aircraft cabin pressure drop, balloon flights, extreme altitude climbing, and in some maladies. We aimed to evaluate the speed and magnitude of spleen contraction during short exposure to extreme eupneic hypoxia and its subsequent recovery on oxygen. Eight female and seven male volunteers were exposed to normobaric hypoxia (10% oxygen) for 10 min during sitting rest, followed by 10 min on 100% oxygen. Heart rate (HR), arterial oxygen saturation (SpO2), and mean arterial blood pressure (MAP) were measured continuously. The spleen was measured via ultrasonic imaging every minute for volume calculations, and venous blood samples were drawn before and after exposure for hemoglobin concentration (Hb). Mean (SD) spleen volume was 279 (115) mL before exposure, 219 (75) mL (21% reduction; P = 0.005) at 3 min of exposure, and 201 (93) mL after 10 min exposure to hypoxia (28% reduction; P < 0.001). Hb was 138.8 (7.6) g·L−1 before and 142.9 (8.1) g·L−1 after 10 min of exposure (2.9% increase; P < 0.001). SpO2 was 96.4 (1.7)% before exposure and 74.7 (8.4)% during the last minute of exposure (22.5% reduction; P < 0.001). HR increased from 80 (14) to 90 (17) bpm during exposure (12% increase, P < 0.05). MAP remained unchanged. After 10 min recovery on oxygen, values had been restored for spleen volume and Hb, while SpO2 was higher and HR lower compared with before hypoxia exposure. We concluded that acute normobaric hypoxia of only 10 min caused significant spleen volume contraction with Hb increase. This rapid spleen response, evident already after 3 min of exposure, could have a protective effect during sudden exposure to severe hypoxia.

Ampakine pretreatment enables a single hypoxic episode to produce phrenic motor facilitation with no added benefit of additional episodes.

Repeated short episodes of hypoxia produce a sustained increase in phrenic nerve output lasting well beyond acute intermittent hypoxia (AIH) exposure (i.e., phrenic long-term facilitation; pLTF). Pretreatment with ampakines, drugs which allosterically modulate AMPA receptors, enables a single brief episode of hypoxia to produce pLTF, lasting up to 90 min after hypoxia. Here, we tested the hypothesis that ampakine pretreatment would enhance the magnitude of pLTF evoked by repeated bouts of hypoxia. Phrenic nerve output was recorded in urethane-anesthetized, mechanically ventilated, and vagotomized adult male Sprague-Dawley rats. Initial experiments demonstrated that ampakine CX717 (15 mg/kg iv) caused an acute increase in phrenic nerve inspiratory burst amplitude reaching 70 ± 48% baseline (BL) after 2 min (P = 0.01). This increased bursting was not sustained (2 ± 32% BL at 60 min, P = 0.9). When CX717 was delivered 2 min before a single episode of isocapnic hypoxia (5 min, [Formula: see text] = 44 ± 9 mmHg), facilitation of phrenic nerve burst amplitude occurred (96 ± 62% BL at 60 min, P < 0.001). However, when CX717 was given 2 min before three, 5-min hypoxic episodes ([Formula: see text] = 45 ± 6 mmHg) pLTF was attenuated and did not reach statistical significance (24 ± 29% BL, P = 0.08). In the absence of CX717 pretreatment, pLTF was observed after three (74 ± 33% BL at 60 min, P < 0.001) but not one episode of hypoxia (1 ± 8% BL at 60 min, P = 0.9). We conclude that pLTF is not enhanced when ampakine pretreatment is followed by repeated bouts of hypoxia. Rather, the combination of ampakine and a single hypoxic episode appears to be ideal for producing sustained increase in phrenic motor output.NEW & NOTEWORTHY Pretreatment with ampakine CX717 created conditions that enabled an acute bout of moderate hypoxia to evoke phrenic motor facilitation, but this response was not observed when ampakine pretreatment was followed by intermittent hypoxia. Thus, in anesthetized and spinal intact rats, the combination of ampakine and one bout of hypoxia appears ideal for triggering respiratory neuroplasticity.

Hypoxic Ventilatory Response to Acute and Prolonged Hypoxic Exposures in Subjects Susceptible to High-altitude Pulmonary Edema

Hypoxic Ventilatory Response to Acute and Prolonged Hypoxic Exposures in Subjects Susceptible to High-altitude Pulmonary Edema

Physiological changes and transcriptional modulation of HIF-αs in Siberian sturgeon in response to hypoxia

To clarify the effects of acute hypoxia on physiological response and mRNA expression of Hypoxia-inducible factors (HIFs) in Siberian sturgeon, a chondrostean fish, severe hypoxia treatment was implemented by transfer of sturgeons from normoxia (DO =7 mg/mL) to hypoxia (DO = 2 mg/mL). We found that the levels of serum cortisol, GLU, SOD, LDH, CK and CREA increased significantly at the early time-points post hypoxia exposure. Whereas the activity of immune related enzyme — AKP showed a sudden drop, followed by a recovery. Further, we identified and characterized three hif-αs cDNA sequences, namely Abhif-1α, Abhif-2α, and Abhif-3α. The mRNA abundance of three hif-αs was all rich in brain, heart and liver. During acute hypoxia exposure, Abhif-1α and Abhif-3α mRNA expression levels increased significantly in the heart, gill and brain at early time-points, and gradually declined later on. Abhif-2α only showed a slight increase in the gill upon hypoxia stress. In conclusion, we observed that, acute hypoxia affected the muscle function and immune related enzyme activity of Acipenser baeri; in response to hypoxia stress, sturgeons enhanced antioxidant capacity, anaerobic metabolism and up regulated the transcriptional expression of Abhif-1α and Abhif-3α. Our results might be helpful for further understanding of the potential effect of hypoxia on chondrostean fish and their molecular anti-hypoxia mechanisms.

Time course of recovery from acute hypoxia exposure as measured by vigilance and event-related potentials

Exposure to reduced levels of breathable oxygen is known to cause a number of deleterious effects on human performance. Previous work has demonstrated that in healthy adults, hypoxia results in decrements on a wide range of sensory, cognitive, and motor tasks. However, very little is known about the time course of recovery of cognitive functions following a hypoxic exposure. While previous studies have shown that physiological responses like heart rate and oxygen saturation rebound almost immediately, one previous study has shown a delayed recovery for response time (RT) measures following hypoxia. In the current study, we assessed the time course of neurocognitive recovery following a hypoxic exposure in healthy adults using the psychomotor vigilance task (PVT), passively elicited event-related potentials (ERPs) that assess auditory processing, and physiological measures. We also compared whether speed of recovery differed when participants were provided with 21% or 100% oxygen immediately following hypoxic exposure. Participants underwent a baseline testing session and two separate recovery sessions where they were assessed during a hypoxic exposure and at regular intervals for up to four hours post-exposure. Results demonstrated that RT, as measured by the PVT, significantly slowed during hypoxia compared to baseline and continued to be impaired until 60 min post-exposure. We assessed the mismatch negativity (MMN) and P3a ERP components in response to an auditory oddball paradigm and found a significant reduction in the amplitude of the MMN during hypoxia compared to baseline and that attenuation in amplitude persisted for up to 120 min post-exposure. Together, these results indicate that both RT and auditory processing showed a delayed recovery following hypoxia. We found no strong evidence for differential recovery speed based on recovery gas administered (21% versus 100% oxygen). These results have implications for guidance regarding return-to-duty status for military aviators following a hypoxic exposure.

Acute hypoxic exposure: Effect on hemocyte functional parameters and antioxidant potential in gills of the pacific oyster, Crassostrea gigas

Bivalve mollusks are frequently subjected to fluctuations of dissolved oxygen concentration in the environment which can represent a significant threat to bivalve antioxidant status. In this work the effects of hypoxia on hemocyte reactive oxygen species (ROS) production and level of mitochondrial potential as well as the activity and expression level of catalase (CAT) and superoxide dismutase (SOD) in gills of Crassostrea gigas were investigated after 24 h and 72 h exposure. 24 h hypoxia promoted an increase of mitochondrial membrane potential in agranulocytes and induced ROS accumulation in granulocytes. 72 h exposure substantially decreased hemocyte mitochondrial potential and intracellular ROS level in all hemocyte types. No significant changes in the activity of CAT in gills were observed following both 24 h and 72 h exposure periods compared to control. SOD activity in gills decreased after 72 h exposure to hypoxia but did not change under 24 h hypoxia. Significant up-regulation of SOD gene and no changes in expression level of CAT were observed in all experimental groups. The results indicate an overall shift in antioxidant status in gills and hemocytes of the Pacific oyster that may act as compensatory mechanisms to maintain redox homeostasis after a short-term (24 h) exposure and represent the occurrence of oxidative stress conditions at the end of 72 h hypoxia.

Coping with climate change: Phenotypic plasticity in an imperilled freshwater fish in response to elevated water temperature

Abstract Climate change has emerged as an increasingly important threat to freshwater systems. To cope with rapidly changing thermal regimes, freshwater fishes must either relocate or adjust through genetic adaptation and/or phenotypic plasticity. Short‐term responses to elevated water temperature have been well studied in freshwater fishes; however, far less is understood about change induced by long‐term exposure. Furthermore, few studies have investigated the effects of temperature on already imperilled species, which may be more sensitive to environmental change. This study investigated the effects of rearing temperature on critical thermal maximum (CTmax), agitation temperature (Tag, temperature at which fish show behavioural signs of thermal stress) and gill size in pugnose shiner, Notropis anogenus, a threatened species in Canada. Juvenile pugnose shiner were reared for 4 months across five different ecologically relevant temperatures. CTmax and Tag were measured under normoxia and acute exposure to hypoxia to test for oxygen sensitivity of the upper thermal limits in this species. CTmax increased with elevated water temperature. Tag also increased with rearing temperature and occurred, on average, 4.3°C above acclimation temperatures. The CTmax and Tag were lower when fish were exposed acutely to hypoxia. Interestingly, gill size (e.g. total gill filament length) increased with rearing temperature, which may increase oxygen uptake capacity and support increased metabolic demands of warmer waters. Overall, pugnose shiner show plasticity in several traits in response to long‐term exposure to elevated water temperature that may facilitate persistence in warmer waters. However, acute hypoxia exposure reduced thermal tolerance, stressing the importance of evaluating interactive effects of multiple stressors. Identifying source populations of pugnose shiner with greater thermal tolerance or implementing captive breeding under higher temperature regimes may improve the success of re‐introduction efforts in the face of climate change, but the consequences to fitness of increased thermal tolerance should be examined.

High‐Altitude Exposure and Sustained Hypoxia Increase Expression of Inflammatory Genes and Enhance the Peripheral Blood Inflammatory Response to LPS

Transcriptional responses to hypoxia and inflammatory stimuli are linked via hypoxia inducible factor (HIF) and NF-kB crosstalk. Tissues experience hypoxia during infection or inflammatory states due to increased metabolic demand of immune cells, or impaired oxygen delivery by occluded blood supply or fluid buildup. However, while the evolutionarily conserved inflammatory responses to oxygen limitation are necessary for modulating the physiological responses to hypoxic stress, during chronic and/or systemic hypoxia, the crosstalk between these two pathways can become maladaptive. For example, hypoxia-induced inflammation may contribute to the development of Acute and Chronic Mountain Sickness. We hypothesized that acute sustained hypoxia exposure induces a systemic inflammatory response and changes sensitivity to subsequent inflammatory stimuli. To test this hypothesis, we used a high-altitude exposure model (1-3 days). We compared the RNA profiles in whole blood collected in the morning during fasting at sea level, and after 1 night and 3 nights at high altitude (3800m elevation) in 14 healthy sojourners (4 women, 10 men). RNA sequencing was coupled with Nanostring direct digital detection of 255 inflammatory pathway genes. 127 genes were significantly upregulated, and 140 genes were significantly downregulated with a fold change threshold > 1 and adjusted p-value < 0.05 on the first day at high altitude. Of all significant genes, 13 were also detected as significantly upregulated via Nanostring: HMGB1, TGFBR1, HMGN1, DDIT3, TLR8, MAPK1, TLR1, IFIT1, IFI44, MAPK8, HMGB2, TLR5, IFIT2 (adj. p < 0.05 for all). Among the significant gene ontology terms for all differentially expressed genes with adj p < 0.05 were: toll-like receptor signaling pathway (p<0.0001), MyD88-dependent toll-like receptor signaling pathway (p=0.012), interleukin-1-mediated signaling pathway (p=0.003), and I-kappaB kinase/NF-kappaB signaling (p<<0.0001). To test if upregulation of toll-like receptor signaling pathways increased sensitivity of peripheral blood mononuclear cells to inflammatory stimuli, we cultured whole blood and isolated monocytes in the presence of normoxia (18% O2, 5% CO2 at sea level) or hypoxia (1% O2, 5% CO2 at sea level) and with or without a lipopolysaccharide (LPS) stimulus for 24 hours. Both LPS (p<<0.0001) and hypoxia (p=0.01) independently increased TNF-a expression in whole blood and there was a significant interaction between hypoxia and LPS (p=0.002) in which hypoxia augmented TNF-a expression in response to LPS. TNF-a expression was higher in monocyte cultures exposed to LPS (p<<0.0001) and showed a nonsignificant trend to be higher in the presence of hypoxia, but there was no interaction. In conclusion, our results indicate that acute sustained hypoxia exposure may exacerbate the response to subsequent inflammatory stimuli. This may be particularly important in the context of pulmonary diseases if hypoxemia exacerbates the inflammatory response to viral or bacterial infection.

The Role of Cytoplasmic Polyadenylation Element Binding Protein 2 Alternative Splicing in Chronic Hypoxia

Cytosolic polyadenylation element binding (CPEB) proteins are established as mRNA binding proteins that, upon interacting with a 3 ‘UTR consensus sequence, regulate the translation of their mRNA targets. Moreover, the stress-induced CPEB family member, CPEB2, has been shown to interact with the oxygen-dependent subunit of the HIF1 (hypoxia-inducible factor 1) transcription factor, HIF1a, in order to repress its translation. However, our lab has previously demonstrated in cancer cells that alternatively spliced isoforms of CPEB2 regulate the translation of HIF1a in opposing fashions: the CPEB2A isoform represses its translation, while the CPEB2B isoform activates its translation. Although it is well established that during hypoxia, HIF1α levels are initially upregulated due to a decrease in proteasomal degradation, we hypothesize that during chronic hypoxia, the expression of HIF1α is maintained through a stress-induced translational mechanism, likely alongside a decrease in proteolytic activity. In this study we demonstrate that targeting the CPEB2B splice isoform via antisense oligonucleotides has an inhibitory effect on the translation of nascent HIF1α protein during chronic hypoxia, but not during acute hypoxia exposure, and that this this regulatory pathway is dependent on the initiation factor eIF3H. We further demonstrate that CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences, which may depend on surrounding sequence context that can be detected through RNA foot-printing assays. Finally, we characterize how CPEB2A and B expression impacts new blood vessel growth in a 3D vasculature cell model. These findings are important, since they provide evidence that this splicing event could potentially act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.