bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fetal brain sparing in a mouse model of chronic maternal hypoxia
Authors: Cahill Lindsay S , Zhou Yu‐Qing , Hoggarth Johnathan , Seed Mike , Macgowan Christopher K , Sled John G 
Published: November 2023

Pregnancy complication may cause acute or chronic hypoxic stress to the fetus. The fetal response to acute hypoxia has been well characterized1 and is associated with brain sparing, a mechanism by which more oxygenated blood is directed to the brain at the expense of organs such as the liver and the lungs2–4. In contrast, the fetal response to chronic hypoxia is not well understood. In the present study, Doppler ultrasound was used to characterize the fetal hemodynamic responses to chronic maternal hypoxia during late gestation in pregnant mice.Pregnant CD‐1 mice were randomly assigned to either a chronic hypoxia (n=13) (11% O2 from gestational day E14.5 to E17.5) or control (n=13) group. Doppler ultrasound measurements were acquired at E17.5 (full term for CD‐1 dams is E18.5) of the middle cerebral artery, ductus venosus, main pulmonary artery, ductus arteriosus, and the umbilical artery. An acute hypoxic condition (8% O2 for 3 minutes) was used to challenge the fetus and assess its capacity to compensate for additional hypoxia stress. Fetal organ growth was assessed using ex vivo magnetic resonance imaging (MRI).The fetuses exposed to chronic maternal hypoxia weighed 15% less than controls, indicating that the hypoxia‐exposed fetuses failed to reach their growth potential. Fetal cerebral blood flow was elevated by 31%, ductus venosus blood flow was 69% higher and pulmonary blood flow was decreased by 74% in the fetuses exposed to chronic hypoxia compared to controls. This demonstrates that the brain sparing response persists during chronic fetal hypoxia and is mediated by “streaming”, where highly oxygenated blood preferentially flows through the ductus venosus towards the cerebral circulation, bypassing the liver and the lungs. Consistent with these changes in blood flow, the brain volume is preserved while the lung and liver volumes decreased by 27 and 22% respectively in fetuses exposed to chronic hypoxia compared to controls.With an acute hypoxic challenge, the cerebral and ductus venosus blood flow increased in control fetuses. The elevated flow was achieved by an increased fetal heart rate and vasodilation. In contrast, the blood flow in the fetuses exposed to maternal hypoxia decreased when challenged. The decrease was accompanied by a drop in fetal heart rate, suggesting the fetus can no longer compensate for the decreased oxygen transport across the placenta. This highlights that fetuses exposed to chronic maternal hypoxia are at increased risk of fetal demise caused by an acute episode of hypoxia; for example, those that commonly occur during labour and delivery.Support or Funding InformationFunded by Canadian Institutes of Health Research Grant MOP130403.
Prenatal food restriction causes adaptive changes in the metabolic regulation of reproductive physiology
Authors: Rhinehart Erin M 
Published: November 2023

Reproduction is the most energetically expensive undertaking that a female can perform, making energy the most important factor regulating reproduction. Reproductive physiology is sensitive to fluctuations in energy availability, and entrainment of female reproductive efforts with energy availability is especially critical because of the large amounts of energy required for gestation and lactation. To optimize reproductive fitness in an environment with reduced food availability, physiological mechanisms are needed to connect oxidizable fuel availability with reproductive physiology. When prenatal food availability is restricted, indicators of reduced maternal energy availability reach the fetus. These signals purportedly prepare the offspring for a food insecure postnatal environment. According to this idea, the energy availability in the intrauterine environment organizes the developmentally plastic mechanisms responsible for energy homeostasis, which would necessarily impact the regulation of reproduction. Therefore, we hypothesized that gestationally nutrient restricted, low birth weight (LBW) offspring would exhibit changes in energy balance and reproductive physiology that would improve survival and reproductive success in a food insecure postnatal environment. To test this hypothesis, we used a well‐characterized model of gestational nutrient restriction where pregnant rats were fed ad libitum or 50% of their normal caloric intake from embryonic day 10 to 21 and all offspring were cross‐fostered to non‐restricted dams for lactation. Consistent with previous studies, restricted female offspring were born LBW (5.6 ± 0.08 vs. 6.5 ± 0.06, p<0.01). LBW offspring also had decreased hypothalamic gonadotropin releasing hormone (GnRH) protein expression at postnatal day (PND) 1 and exhibited rapid catch‐up growth during lactation. LBW female offspring had earlier vaginal opening (33.5 ± 0.2 vs. 38.2 ± 0.2 PND, p<0.01), demonstrating early pubertal onset. AT PND 60–90, LBW female offspring had elongated estrous cycles (6.1 ± 0.4 vs. 4.7 ± 0.3, p<0.05) and significant alterations in ovarian histology. For example, ovaries from young adult LBW females were larger (3.6 ± 0.7 vs. 6.5 ± 0.6 mm2, p<0.05) due to large antral, cyst‐like follicles. By 8 months of age, 80% of LBW offspring were in persistent estrous and had stopped cycling, compared to 0% of control offspring. These data indicate that prenatal energy availability has a significant impact on the regulation of reproductive physiology. Early pubertal onset, in particular, may be an adaptive mechanism to ensure reproductive success in a food‐insecure postnatal environment, but early puberty has significant health ramifications. Therefore, a more thorough understanding of the mechanisms responsible for gestational programming of the metabolic regulation of reproduction should have a significant impact on treatment of female reproductive problems such as polycystic ovary syndrome.Support or Funding InformationNSF CAREER IOS‐1350448
Firing properties of pre‐inspiratory neurons are altered in female rats exposed to chronic intermittent hypoxia
Authors: Souza George Miguel P R , Amorim Mateus R , Moraes Davi JA , Machado Benedito H 
Published: November 2023

Female rats submitted to chronic intermittent hypoxia (CIH) presented a reduced time of inspiration, sympathetic overactivity and hypertension. The increase in sympathetic nerve discharge in CIH‐female rats is associated to an increase in the inspiratory modulation of sympathetic activity. In this context, we hypothesized that inspiratory neurons at the brainstem have change their activity in CIH‐female rats, which in turn, modulates the presympathetic neurons producing an increase in sympathetic activity during inspiration. To test this hypothesis, juvenile female Wistar rats (P20–21) underwent intermittent hypoxia for 10 days. One day after CIH, we performed the extracellular single unit recordings of respiratory neurons at the ventral surface of the medulla using the ventral approach of the working heart‐brainstem preparation. All the experimental protocols were approved by the institutional ethical committee (#091/2013). Pre‐inspiratory neurons (Pre‐I/I) of CIH‐female rats (n=16) have reduced their firing frequency (58.3 ± 9 vs 107.6 ± 12Hz, p=0.0027) compared to Pre‐I/I neurons of control (n=15). Synaptic blockade demonstrated that Pre‐I/I neurons (n=3) have auto‐depolarization property. Ramp‐I, Early‐I and Late‐I neurons presented no changes in their firing properties after CIH. Regarding the expiratory neurons, post‐inspiratory neurons (Post‐I) have increased the firing time relative to duration of expiration (92.2 ± 2 vs 81.3 ± 3 % of total duration of expiration, p=0.0127) in CIH‐female rats (n=15) compared to control (n=17). We conclude that CIH produces a reduction in the activity of pacemaker Pre‐I/I neurons that could explain the reduced time of inspiration and the increase in inspiratory modulation of sympathetic activity in CIH‐female rats. These findings contribute to a better understanding of the respiratory network activity associated with sympathetic overactivity and hypertension in female rats exposed to CIH.Support or Funding InformationFapesp, CAPES and CNPq
Ventral Medullary Inspiratory Neurons Recordings in Rats Submitted to Sino‐aortic Denervation
Authors: Amorim Mateus R. , Souza George Miguel P. R. , Bonagamba Leni G. H. , Machado Benedito H. 
Published: November 2023

There is experimental evidence showing that pre‐sympathetic neurons in the ventrolateral medulla have their firing frequency modulated by the respiratory network. After baroreceptors afferents removal [sino‐aortic denervation (SAD)], awake rats present normal levels of sympathetic activity and mean arterial pressure. However, the mechanisms involved in the respiratory modulation of the sympathetic activity after SAD remain unclear. In this study we hypothesized that rats present changes in the firing frequency of the inspiratory neurons after SAD. To test this hypothesis, male juvenile Wistar rats underwent to SAD or Sham surgery and on the 3rd day after the surgery they were surgically prepared for the in situ preparation. We recorded the activities of phrenic and thoracic sympathetic nerves as well as the single unit extracellular activities from the inspiratory neurons in the ventral medulla (CEUA #093/2013). The duration of inspiration significantly increased in SAD rats (P > 0.05). During inspiration, the firing frequency of Ramp‐I [Sham (n = 11) and SAD (n = 7, 52 ± 11 vs 56 ± 14 Hz)], Pre‐I / I [Sham (n = 13) and SAD (n = 11, 82 ± 15 vs 99 ± 20 Hz] and Late‐I neurons [Sham (n = 8) and SAD (n = 6, 8 ± 3 vs 9 ± 2 Hz] were not different between groups (P > 0.05). The data showed that the increase in time of inspiration observed in SAD rats was not associated with changes in the baseline firing frequency of the inspiratory neurons. However, these findings do not rule out the possibility that post‐inspiratory neurons are driving the activity of the pre‐sympathetic neurons after SAD. Further experiments are required to characterize the electrophysiological firing properties of the post‐inspiratory neurons in SAD rats.Support or Funding InformationFAPESP, CAPES and CNPq
Effects of lipopolysaccharide injection on in vivo breathing pattern in developing rats
Authors: Johnson Rhaya L. , Murray Samuel , Wilson Christopher G. 
Published: November 2023

Breathing pattern irregularities, which are common problems in pre‐term infants, are often exacerbated by infection. These irregularities can manifest as apneas, tachypnea, and periodic breathing. We have previously shown that lipopolysaccharide (LPS) injected into the airway of neonatal rats (postnatal days 10 to 12) causes up‐regulation of early pro‐inflammatory cytokines and blunts the response to an acute hypoxia challenge. Furthermore, we are using vagal nerve stimulation (VNS), an FDA approved treatment for epilepsy and depression, in an effort to reduce neuroinflammation in the brainstem regions for respiratory control. We have assessed cytokine expression in response to LPS injection using immunohistochemistry and mRNA quantification in nTS, XII, PBC.Our preliminary data suggests that LPS injection causes a blunted hypoxic ventilatory response to 10% oxygen (10 min, balance N2). Sham animals had a 46% increase in breath rate from normoxia to hypoxia, whereas LPS animals only had a 26% increase in breath rate from normoxia to hypoxia (n = 3 for each). LPS injection also significantly increases the expression of IL‐6 (p < 0.001) and is hypothesized to increase TNFa and IL‐1b in the respiratory control regions of the brainstem. VNS slows the baseline breath rate (13% lower than sham) over the two hour recovery period. The hypoxic ventilatory response begins to recover (31% change from normoxia to hypoxia) though the increase in breath rate with hypoxia is not as large as with sham animals. VNS does reduce cytokines IL‐6 and TNFa (p<0.5) in the nTS and XII.
EVIDENCE FOR ACE‐2 UBIQUITINATION AND PROTEASOMAL PROCESSING IN LUNG EPITHELIUM
Authors: Abdul‐Hafez Amal , Markey Jonathan , Uhal Bruce 
Published: November 2023

Background and AimThe angiotensin converting enzyme 2 (ACE‐2) was shown to play a protective role in the fibrogenesis and inflammation of many organs including liver and lung. Previous studies from our lab showed that ACE‐2 is downregulated in fibrotic conditions of the adult and neonatal human lung. The exact mechanisms of ACE‐2 downregulation and protein degradation in lung cells still remain unknown. This study aims to investigate whether ACE‐2 is regulated by ubiquitination/proteasome degradation pathways in the lung.MethodsHuman lung epithelial cell line (A549) and mouse lung epithelial cell line (MLE12) were cultured and cell lysates were used for immunoprecipitation using ACE‐2 antibodies. The immunoprecipitate was used to detect ubiquitination. Several proteasome inhibitors (Lactacystin, clasto‐Lactacystin β‐lactone, MG132, HMB‐Val‐Ser‐Leu‐VE, and Epoxomicin) were used on MLE12 cell culture to detect the effects on ACE‐2 protein bands by western blotting and to immunoprecipitate ACE‐2 and detect the effect on the ubiquitination of ACE‐2. Several dosing of the proteasome inhibitor MG132 on MLE12 cells and of clasto‐Lactacystin β‐lactone on A549 cells were used to detect changes in ACE‐2 bands by western blotting.ResultsImmunoprecipitation of ACE‐2 revealed ubiquitination of 3 major bands (150, 45, 40 kDa) detected by anti‐ubiquitin antibodies in MLE12 cells, while ubiquitination of ACE2 was markedly noted in 50 kDa band of A549 cells. On MLE12 cells, all proteasome inhibitors resulted in various degrees of increased total ubiquitinated protein levels. Lactacystin and clasto‐Lactacystin β‐lactone showed increased ACE‐2 isoform band at 70 kDa and were associated with the most increase in total ubiquitinated protein levels, more noted in clasto‐Lactacystin β‐lactone treatment than Lactacystin. Additionally, increasing doses of MG132 treatment on MLE12 cells resulted in a dose dependent increase of the 70 kDa ACE‐2 band with a corresponding decrease in 40 kDa band. Similarly, treatment of A549 cells with increasing doses of clasto‐Lactacystin β‐lactone resulted in a dose dependent decrease in the 50 kDa band.ConclusionThese data present the first evidence suggesting ubiquitination and proteasome dependent degradation of ACE‐2 in lung cells in a species‐dependent pattern. Future work will be directed to detect the roles of this ubiquitination/proteasome degradation in fibrotic lung diseases.Support or Funding InformationPHS HL‐45136; Department of Pediatrics, Michigan State University
Novel inflammatory mechanisms in the regulation of airway hyperresponsiveness: regulation of airway smooth muscle Kv7 channel expression by REST.
Authors: Brueggemann Lyubov I. , Zhang Chonyu , Kakad Priyanka P. , Cribbs Leanne L. , Byron Kenneth L. 
Published: November 2023

Inflammation contributes to airway hyperresponsiveness (AHR) in asthma, but the cellular mechanisms by which this occurs are not well understood. In previous research, inflammatory mediators were found to upregulate repressor element 1‐silencing transcription factor (REST) in neurons, and consequently, REST mediated transcriptional suppression of neuronal KCNQ genes, which was associated with decreased M‐current density and increased neuronal excitability. Functional binding sites for REST have been identified in both KCNQ4 and KCNQ5 genes which encode Kv7.4 and Kv7.5 potassium channels expressed in airway smooth muscle cells (ASMCs). We found that Kv7 currents and KCNQ gene expression were both decreased in ovalbumin‐(OA‐) sensitized guinea pigs (a model for allergic asthma) relative to saline‐treated control guinea pigs. Although REST has not been reported to be expressed in ASMCs, we found that it is expressed and that REST protein levels are significantly higher in the nuclei of ASMCs from sensitized guinea pigs relative to saline control guinea pigs. Furthermore, serum from the OA‐sensitized animals, but not from control animals, increased nuclear REST immunostaining in ASMCs of control animals. In previous research, serum from sensitized guinea pigs or from asthmatic humans was shown to induce features of AHR in ASMCs, and features of AHR could also be induced by treating human ASMCs with purified inflammatory mediators, including IL‐4 and IL‐13. We exposed strips of human trachealis tissue to IL‐13 overnight and observed a significant increase in nuclear REST expression in ASMCs, supporting a specific role for IL‐13 and providing evidence that this pathway is active in human ASMCs. These findings provide new evidence for an inflammatory mechanism that might account for airway smooth muscle hyper‐excitability in asthma.Support or Funding InformationThe project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.
Resveratrol Protects Against Hyperoxia‐Induced Airway Hyperreactivity in Neonatal Rats
Authors: SOPI RAMADAN B , Reçica Shkëlzen , Lajçi Azem , Kryeziu Islam , Thaçi Qëndrim , Dreshaj Ismail A , Basholli Mimoza 
Published: November 2023

Neonatal hyperoxia is associated with airway hyperreactivity and lung injury. We tested the hypothesis that supplementation of resveratrol will overcome the increased contractile responses of tracheal smooth muscle exposed to hyperoxia. Tracheal cylinders were obtained from Wistar rat pups (P12) exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study contractile responses of airway smooth muscle (ASM) evoked by methacholine (MCh, 10‐8 – 10‐4 M) in absence or presence of resveratrol (100 μM). MCh induced dose‐dependent contractile responses of ASM. The contractile responses towards MCh were significantly higher in preparations obtained from animals exposed to hyperoxia as compared to those obtained from control animals exposed to room air. The enhanced contractile responses in hyperoxic tissues were significantly attenuated in presence of resveratrol. The results of this study revealed that resveratrol diminishes the airway hyperreactivity induced by hyperoxia and we speculate that the use of resveratrol might be an effective therapeutic approach to prevent the adverse effects of hyperoxia.Support or Funding InformationSupported by MEST
Hypercapnic and hypoxic ventilatory response in streptozotocin‐model of Alzheimer's disease during wakefulness
Authors: Vicente Mariane C , Bícego Kênia C , Carrettiero Daniel C , Gargaglioni Luciane H 
Published: November 2023

Neurodegenerative diseases such as Alzheimer's may impair breathing function; however, how these breathing deficits occur is not completely understood. Therefore, it is important to evaluate how Alzheimer's disease (AD) can alter breathing activity. In light of this, we used an animal model for sporadic AD induced by intracerebroventricular (icv) injection of streptozotocin (STZ, 4 mg/kg) in adult male Wistar rats. After 30 days of STZ treatment, memory impairment (using Y‐maze and Barnes maze tests) was analyzed to validate STZ model. Then, ventilation (VE) was measured by whole‐body plethysmograph (barometric technique) during wakefulness and sleep under normocapnia, hypercapnia and hypoxia. STZ treatment caused learning and memory impairment and increased hypercapnia ventilatory response during wakefulness (VE STZ= 2460.9 ± 83.7 vs VE Vehicle= 1758.5 ± 234.3 mL.Kg−1.min−1) due to an effect on both tidal volume and breathing frequency. No difference was observed in the respiratory variables between STZ and vehicle groups during room air conditions (VE STZ = 775.5 ± 56.1 vs VE Vehicle= 651.3 ± 56.1 mL.Kg−1.min−1) and hypoxia (VE STZ = 1073,2 ± 53,7 vs VE Vehicle =1085.9 ± 127.2 mL.Kg−1.min−1) in wakefulness. Our data suggest that sporadic AD animals present an increased CO2 but no O2 chemosensitivity.Support or Funding InformationFAPESP and CNPq
Optogenetic Activation of Leptin‐Receptor Expressing Neurons in the Region of the Dorsomedial Hypothalamus Increases Respiratory Motor Output in Mice
Authors: Chang Zheng , Kowal Anthony S. , Sekerkova Gabriella , McKenna Kevin E. , Martina Marco , McCrimmon Donald R 
Published: November 2023

Administration of the anorexigenic hormone leptin to rodents increases energy expenditure with a parallel increase in respiratory motor output in order to maintain arterial blood gas and acid‐base balance. This occurs at least in part through activation of CNS neurons that express the long form of the leptin receptor (LepRb). LepRb expressing neurons are concentrated in several clusters, most notably in the hypothalamus and medulla. Previous studies have linked respiratory motor stimulation to activation of LepRb neurons in the medulla. In this study we sought to determine whether hypothalamic neurons also contribute to the respiratory stimulation. Specifically, LepRb neurons are concentrated in the dorsomedial nucleus of the hypothalamus (DMH) and activation of DMH neurons is known to stimulate breathing. However, whether LepRb expressing neurons contribute to the stimulation is not known. We have initiated studies to determine whether DMH LepRb neurons contribute to the respiratory stimulation using mice that express channelrhodopsin‐2 (ChR2) in LepRb neurons. Mice were anesthetized and an optical fiber (200 um diameter) conducting 473 nm wavelength light was used to activate the ChR2‐LepRb neurons. Light intensity was 4 mW, below the threshold for nonspecific activation or damage to tissue in the vicinity of the fiber. For stimulation, 3 min (or shorter) trains of light pulses at 5, 10, 20 and 50 Hz (≤10 ms pulse width) were used. In parallel control experiments, areas dorsal or lateral to the DMH were stimulated. Activating the DMH elicited a progressive increase in respiratory motor output, consisting primarily of an increase in the peak amplitude of integrated phrenic nerve activity with little change in the number of phrenic bursts per minute. The phrenic amplitude continued to increase after the end of the stimulation, averaging 189 +/− 23% of control (n = 3) at 30 min and 212 +/−17% (n = 2) at 60 min after the end of stimulation. Our preliminary conclusion is that the DMH contributes significantly to the leptin‐mediated stimulation of breathing.Support or Funding InformationWe are grateful for the support from NIH grant HL122921.