bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative stress and chronic obstructive pulmonary disease: The impact of oral antioxidants on skeletal muscle fatigue
Authors: Rossman Matthew J , Groot H. Jonathan , Amann Markus , Richardson Russell S
Published: June 2021

Oxidative stress may contribute to exercise intolerance in patients with COPD. This study sought to determine the effect of an acute oral antioxidant cocktail (AOC: vitamins C, E, and alpha‐lipoic acid) on skeletal muscle function in patients with COPD. Ten patients with COPD (FEV1/FVC < 0.7, FEV1 ≤ 80% predicted) performed knee extensor exercise to exhaustion as well as isotime trials following either the AOC or placebo. Changes in quadriceps maximal voluntary contractions (MVCs) and potentiated twitch forces (Qtw,pot) quantified quadriceps fatigue. Despite increasing plasma ascorbate levels (10.1±2.2 to 24.1±3.8 ug/ml, p<0.05) and attenuating the electron paramagnetic resonance (EPR) spectroscopy free radical signal (AUC: 11.6±3.7 to 4.8±2.2 AU, p<0.05), AOC consumption did not alter endurance time or the development of quadriceps fatigue. Revaluation of the EPR spectroscopy data revealed that the AOC was most efficacious in patients with high (n=5, AUC: 19.7±5.8 to 5.8±4.5 AU) compared to low (n=4, AUC: 1.6±0.5 to 3.4±1.1 AU) basal free radical levels. This baseline index of free radicals was inversely correlated with FEV1 (r=−0.54, p<0.05) and MVC force (r=−0.56, p<0.05). Thus, while a potentially beneficial role for the AOC in COPD can be inferred, an acute reduction in free radicals does not appear to influence exercise performance in COPD. Supported by the National Institutes of Health grant PO1 HL 09830.
Validity of the oxygen uptake efficiency slope to assess aerobic fitness following bed rest
Authors: Scott Jessica , Goetchius Elizabeth , Buxton Roxanne , Ploutz‐Snyder Lori
Published: June 2021

Achieving true peak oxygen uptake (VO2peak) is often difficult after the deconditioning that results from musculoskeletal unloading via bed rest or spaceflight. Consequently, submaximal measures predicting VO2peak are crucial for the effective assessment of cardiorespiratory functional reserve. One proposed index, oxygen uptake efficiency slope (OUES), is effort independent; however, its sensitivity to change in aerobic performance because of disuse and exercise training is unknown. Twenty‐five subjects performed a maximal exercise test before and after 14 days of 6° head‐down tilt bed rest. During bed rest, 9 subjects completed a high‐intensity exercise program (EX) while 16 were sedentary (CONT). In addition to VO2peak and ventilatory threshold (VT), OUES at 75% and 100% of total exercise time were determined. VO2peak (−13%), VT (−17%), OUES100 (−12%), and OUES75 (−10%) decreased in the CONT group; while VO2peak (+10%), VT (+8%), OUES100 (+6%), and OUES75 (+10%) increased in the EX group. The change in both OUES100 and OUES75 was significantly related to the change in VO2peak (OUES100: r = 0.85; OUES75: r = 0.75; P < 0.05). Bland‐Altman plots showed high agreement between OUES and VO2peak. These findings indicate that OUES is an objective submaximal index that can detect changes in cardiopulmonary reserve following deconditioning and exercise training.

Support: NASA Johnson Space Center Human Research Program
Adiponectin mediates cellular plasticity of osteoblasts towards adipocytes
Authors: Abbott Marcia J , O'Carroll Dylan , Nissenson Robert A
Published: June 2021

Adiponectin has been implicated in regulating skeletal homeostasis. Studies show that adiponectin is inversely related to bone mineral density (BMD) and positively related to fracture risk in adults. We sought to identify the role of adiponectin on differentiating osteoblasts in vitro. Mouse bone marrow stromal cells were treated with adiponectin (2.5 μg/ml) acutely for 24 hrs at various stages (day 7, 14, 21 & 28) or chronically (day 14–28) of osteoblastic differentiation. Osteoblast properties were observed in all experiments by day 21 of differentiation. Acute adiponectin decreased osteoblast markers, osterix (OSX) and runx2, mRNA at day 14 (35–29%, respectively) and day 21 (34–53%, respectively). At day 21 with acute adiponectin, osteocalcin (OC) and collagen type 1 (Col1) mRNA decreased (56–59%, respectively). Chronic adiponectin reduced colony formation, decreased mineralized nodules and decreased mRNA of OSX, runx2, alkaline phosphatase, Col1 and OC (53–82%). Adipocyte factors PPARγ and C/EBPα mRNA increased with acute and chronic adiponectin (39–64% & 39–64%, respectively). Chronic adiponectin increased lipid colonies and lipid droplet size as visualized by Oil Red O. Here we show that adiponectin mediates cellular plasticity by exerting negative osteoblastic effects in favor of adipogenesis in mature osteoblasts. These data may explain the negative association between adiponectin and BMD in humans.
Ethanol induces skeletal muscle autophagy and sarcopenia by an AMPK independent, PI3K dependent mechanism
Authors: Dasarathy Srinivasan , Thapaliya Samjhana , Runkana Ashok , Garber Ari , McMullen Megan , Nagy Laura E , Prasad Sathyamangala V Naga
Published: June 2021

Loss of skeletal muscle mass or sarcopenia is the most common but understudied complication of alcoholic liver disease. Skeletal muscle proteolysis due to ethanol has not been systematically evaluated.

A comprehensive approach was used to study the proteolytic pathways in muscle of patients with alcoholic cirrhosis and alcohol fed rodent along with in vitro studies in C2C12 myotubes exposed to 100mM ethanol for 24 hrs. Ubiquitin proteasome activity and autophagy were studied using immunoblots, real time PCR, proteasome activity, standard autophagy readouts including LC3 lipidation, p62 degradation and Beclin overexpression. C2C12 cells transfected with GFP‐LC3 and GFP‐mCherry‐LC3 reporter were also studied. Pulse chase experiments were performed with and without 3‐methyladenine. Immunoblots for components of the PI3K pathway‐Akt, mTOR, p70s6k and 4EBP activation, pAMPK as well as protein phosphatase 2A and PI3Kγ activity were quantified by thin layer chromatography. Ubiquitin proteasomal activity was unaltered and autophagy was increased in the myotubes and in vivo with ethanol. mTOR and its target protein phosphorylation was lower with ethanol exposure. Increased PP2A activity resulted in an AMPK independent mTOR dependent increase in autophagy.
Temperature regulation of fish‐specific paralogs of cardiac TnC
Authors: Genge Christine Elizabeth , Tibbits Glen F
Published: June 2021

Temperature‐dependent regulation of factors involved with cardiac contractility is crucial for ectothermic fish facing changes in temperature. The teleost‐specific whole genome duplication created multiple copies of genes allowing for sub‐functionalization of isoforms. The Ca2+‐binding troponin C (TnC), responsible for initiating myocyte contraction is highly conserved across vertebrates due to its crucial role in regulating contractility. TnC expressed in the hearts of many teleosts is the product of two distinct genes: cardiac TnC (cTnC) and the fish‐specific slow skeletal TnC (ssTnC). Tissue‐specific mRNA profiles in adult zebrafish revealed a distinct localization of gene expression, with ssTnC preferentially expressed in the atrium and cTnC is almost exclusively expressed in the ventricle. Chamber‐specific localization varies between teleost species, with trout preferentially expressing cTnC in both chambers of the heart. Isoform composition of the chambers is also modulated by changes in temperature. Possible sub‐functionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber‐specific contractile properties. Funded by NSERC.
Chronic hypoxia (10% O2) alters cardiovascular regulation and gene expression in Snapping turtle embryos (Chelydra serpentina)
Authors: Eme John , Rhen Turk , Tate Kevin B , Gruchalla Kathryn , Kohl Zachary F , Slay Christopher E , Crossley Dane A
Published: June 2021

Reptile embryos are basal amniotic vertebrate developmental models that tolerate severe chronic hypoxia. We measured heart rate (fH) and arterial blood pressure (Pm) for normoxic (N21) and chronically hypoxic‐incubated (H10; 10% O2) turtle embryos following pharmacological blockade, and we quantified heart tissue mRNA expression for genes using RT‐qPCR. Compared to N21 embryos (controls), H10 embryos were hypotensive and tachycardic, smaller, had relatively larger hearts, and showed reduced intrinsic fH. Unlike other reptile embryos, cholinergic (atropine) and ganglionic receptor blockade (hexamethonium) increased fH, and similar to bird/reptile embryos, adrenoreceptor blockade with propranolol (β) (reduced fH) and phentolamine (α) (reduced Pm) caused pronounced responses. H10 embryos’ heart tissue had lower mRNA levels for adrenergic receptors (Adrb1, Adra1A, Adra2C) and growth‐related products (Igf1, PdgfB). Cholinergic tone was reduced in H10 embryos, but cholinergic receptor mRNA levels (Chrm2) were unchanged, indicating reduced cardiac receptor gene expression is not responsible for reduced cholinergic tone. Hypoxia likely altered the normal balance between cholinergic receptor and adrenoreceptor function reflected in altered intrinsic fH and adrenoreceptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a reptile embryo. NSF CAREER IBN IOS‐0845741 DAC
3‐Dimensional Visual Receptive Fields of Dragonfly Neurons Directing Prey Interception
Authors: Wainman Lauren M , Shah Ankur , Olberg Robert M
Published: June 2021

Dragonflies are visual predators that intercept and capture flying insects, a behavior thought to be guided by a small group of interneurons. This small group comprises eight bilateral pairs of interneurons called target‐selective descending neurons (TSDNs). TSDNs are large, feature‐detecting interneurons that project to the thoracic ganglia from the brain of the dragonfly and receive information from the part of the dragonfly's compound eye where the ommatidial facets are the largest and the vision is most acute (Olberg 2000, 2007). The receptive fields for these interneurons are located in the forward and upward portion of visual space, the region of the eye that views the prey during approach. Most of what is known about the TSDNs is derived from recording responses to moving target projected on a flat screen in front of the animal. Using single‐unit and intracellular recording from the ventral nerve, we studied the responses of TSDNs to real objects, glass beads of various sizes moved robotically in 3 dimensions. Our results show that the directional preferences of at least 2 of the TSDNs include a component in the depth direction (ie approaching or receding objects).
Huddling and nest building do not completely ameliorate the cold stress of typical housing in mice
Authors: Maher Rebecca Lynn , Swoap Steven J
Published: June 2021

Huddling and nest building are two methods of behavioral thermoregulation commonly utilized by mice under cold stress. Lab mice are typically housed at an ambient temperature (Ta) of 20°C, approximately 10°C below their thermoneutral zone of 30°C. We tested the hypothesis that the thermoregulatory benefits of huddling and nest building would lower heart rate (HR), blood pressure (BP) and metabolic rate (MR) of mice housed at 20°C to levels typical of mice housed at 30°C. C57Bl/6J mice were implanted with EKG/BP/core body temperature (Tb) telemeters, housed in metabolic cages and, using a repeated‐measures crossover design, exposed to one of four conditions: 1) singly housed at 20°C, 2) group housed at 20°C, 3) singly housed with nesting material at 20°C, 4) singly housed at 30°C. Mice singly housed at 20°C had a 24 hour HR of 607 ± 20 bpm, mean BP of 98 ± 4 mm Hg, MR of 1.5 ± 0.1 ml O2/min and food consumption of 16.6 ± 5.0 kcal/day. The nesting only condition significantly changed only HR (Δ‐51 bpm). Group housing changed both HR and food consumption (Δ‐62 bpm and Δ‐2.6 kcal/day). Singly housed mice at 30°C had changes in all measured parameters (Δ‐239 bpm; Δ‐5 mm Hg; Δ‐0.6 ml O2/min; Δ‐5.8 kcal/day). No significant difference was found in Tb among the groups. These findings indicate that huddling and nest building lessen but do not completely ameliorate cold stress at 20°C.
Altitude acclimatization and acute mountain sickness during a graded ascent to 5896 m
Authors: Muza Stephen Raymond , Kenefick Robert W , Andrew Sean P , Beidleman Beth A , Fulco Charles S , Hamilton Scott W
Published: June 2021

Trekkers ascending Mt. Kilimanjaro (5896 m) with only 4 sleep nights above 2500 m experience a high incidence (~75%) of acute mountain sickness (AMS) and only ~60% success reaching the summit (Karinen, 2008). This study determined the effect of adding 2 sleep nights of altitude exposure at 4040 to 4681 m on arterial oxygen saturation (SaO2), AMS prevalence, and summit success ascending Mt. Kilimanjaro. Over 4 days participants ascended to 4642 m (4th night), descended to 4040 m for one night and ascended to 4681 m (6th night) prior to summiting on day 7. Resting and sleeping SaO2 by pulse oximetry, and prevalence of AMS using the Environmental Symptoms Questionnaire were assessed in 24 low‐altitude residents each morning and evening on the 4th and 6th days. From the 4th to the 6th day, evening resting & sleep SaO2 improved (p<0.001) from (81±5 and 78±5%) to (84±4 and 81±4%) while AMS prevalence fell from 13 to 4%. All participants successfully reached the summit on the afternoon of day 7. These results show that adding 2 days to the ascent increased SaO2, decreased AMS and greatly improved summit success. Funding: USAMRMC. Authors’ views; not official U.S. Army or DoD policy.
Two Days of Staging at Moderate Altitude Reduces Acute Mountain Sickness Upon Further Ascent to 4300 m in Unacclimatized Lowlanders
Authors: Beidleman Beth A , Fulco Charles S , Kenefick Robert W , Cymerman Allen , Staab Janet E , Muza Stephen R
Published: June 2021

Four or more days of staging at moderate altitude reduces acute mountain sickness (AMS) in unacclimatized lowlanders during subsequent ascent to high altitude. The impact of two days of staging at moderate altitude on reducing AMS is unknown. Two independent groups of unacclimatized men were staged at either 2500 m (n=12, 23±3 yr, 80±6 kg, mean±SD) or 3000 m (n=7, 23±2 yr, 79±6 kg) for 2 days prior to ascent to 4300 m and were compared to a control group that directly ascended to 4300 m (n=7, 21±3 yr, 76±4 kg). Following 22 h of exposure to 4300 m, AMS was measured using the Environmental Symptoms Questionnaire and AMS‐C score and arterial oxygen saturation (SaO2) was measured using pulse oximetry. AMS prevalence and severity at 4300 m were lower (P<0.05) in the 2500 m group (42% and 0.59±0.38) and 3000 m group (29% and 0.51±0.39) compared to the control group (73% and 1.21±0.48). AMS prevalence and severity at 4300 m was not different (P>;0.05) between the two staged groups. SaO2 was higher (P<0.05) in the 2500 m group (82±4%) and 3000 m group (84±4%) at 4300 m compared to the control group (79±3%) but was not different (P>;0.05) between the two staged groups. These results demonstrate that two days of staging at either 2500 m or 3000 m is effective for reducing AMS and increasing SaO2 during subsequent exposure to 4300 m. Funding: USAMRMC. Authors’ views; not official U.S. Army or DoD policy.
Two days of staging at moderate altitude results in similar time‐trial performance impairments at 4300m compared to direct ascent and stay for two days at 4300m
Authors: Andrew Sean P. , Beidleman Beth A. , Fulco Charles S. , Yates Brandon A. , Muza Stephen R.
Published: June 2021

Staging at moderate altitude or direct ascent and stay at high altitude (HA) are strategies used to reduce time‐trial (TT) performance impairments at HA. To determine whether 2 days (d) of staging at 2500m or 3000m prior to ascent is as effective as 2 d of continuous living at 4300m, we measured TT performance following 2 h of exposure to 4300m in young fit men. Two groups of unacclimatized men staged at either 2500m (n=12) or 3000m (n=7) for 2 d prior to ascent to 4300m were compared to a 3rd group that directly ascended and lived for 2 d at 4300m (n=7) prior to testing. Acute mountain sickness (AMS‐C) was assessed prior to performing an all out 5‐mile treadmill TT at sea level (SL) and HA. Heart rate (HR), arterial oxygen concentration (SaO2), and rating of perceived exertion (RPE) were recorded every 5 min during the TT and the mean value was calculated. From SL to ALT, the %increase in TT‐completion time was similar (P>;0.05) in the 2500 m (44±27%), 3000 m (39±27%) and direct ascent group (58±28%). There was no difference (P>;0.05) in the mean exercise HR, SaO2, or RPE between groups at SL or HA. AMS prevalence was lower (P<0.05) in the staged groups (both 0%) than the direct ascent group (29%). These results suggest that 2 d of staging at moderate altitude reduces AMS but results in similar TT performance impairments as direct ascent and stay for 2 d at 4300 m. Funding: USAMRMC. Authors’ views; not official US Army or DoD policy.
Influence of Inhaled Amiloride on Lung Fluid Regulation During Normobaric Hypoxia in Healthy Humans
Authors: Baker Sarah E , Wheatley Courtney M , Miller Andrew D , Kasak Alexander J , Carlson Alex R , Taylor Bryan J , Hulsebus Minelle L , Snyder Eric M , Johnson Bruce D
Published: June 2021

Background

Fluid flux in the lungs is influenced by factors altering accumulation or removal (e.g., stimulation of epithelium sodium channels, ENaC).

Purpose

To determine if ENaC are necessary in the normal fluid regulation with simulated altitude exposure.

Methods/Results

On separate occasions, 19 healthy subjects (27 ± 5 y) received either nebulized amiloride (ENaC antagonist) or placebo (nebulized saline) during 17 h normobaric hypoxia (FIO2 = 12.5%) in a randomized, double‐blind fashion. Lung diffusing capacity for carbon monoxide (DLCO), alveolar‐capillary membrane conductance (Dm) and pulmonary capillary blood volume (Vc) were measured via a rebreathe method before and after hypoxic exposure. DLCO was not different at baseline for the amiloride vs. placebo treatments (19 ± 5 vs. 21 ± 8 mL/min/mmHg). Hypoxic exposure increased Vc but did not change Dm in both treatments (Placebo: Vc = 43 ± 22 vs. 59±23 mL p=0.011, Dm = 35 ± 10 vs. 37 ± 12 mL/min/mmHg p=0.312; Amiloride: Vc = 38 ± 17 vs. 57 ± 22 mL p<0.01, Dm = 31 ± 8 vs. 34 ± 10 mL/min/mmHg p= 0.206).

Conclusion

Normobaric hypoxia did not differentially affect alveolar‐capillary conductance in amiloride and placebo conditions, suggesting that ENaC do not have a role in normal lung fluid regulation during acute hypoxic exposure. HL71478
Heart rate variability at rest during repeated 6‐hour water immersions
Authors: Marrs Kevin L. , Simmons Erin E. , Chon Ki H. , Shykoff Barbara E. , Florian John P
Published: June 2021

Short‐duration water immersion (WI) alters heart rate variability (HRV), but the responses to long‐duration and repeated (WI) in comfortably warm water are unknown. Ten healthy young men were exposed to a 6‐hr resting WI breathing air (1.35 ATA) on 5 consecutive days. Heart rate (HR), SDNN and RMSSD (statistical measures of NN intervals), ApEn (approximate entropy), LFPSD and HFPSD (low and high frequency components of power spectral density), LFnPSD and HFnPSD (normalized units from PSD), and cardiac sympathetic and parasympathetic nervous system (SNS and PNS) dynamics using principal dynamic mode (PDM) analysis were measured. Data were compared to pre‐dive baseline HRV data and post‐dive recovery data obtained from the subjects. Compared to baseline, LFPSD increased on dive days 4 (p < 0.0001) and 5 (p=0.0005); HFPSD increased on dive days 2 (p=0.0066) and 3(p=0.0004); SNS increased on dive day 2 (p=0.0086); SDNN increased during dives 2–5 (p < 0.0001); RMSSD decreased during dive days 2 and 3(p < 0.0001). Significant and consistent decreases in ApEn measures compared with baseline were observed across dive days 1–5 (p < 0.0001). The results suggest that 1) the complexity of HR responses (ApEn) is reduced during single and repeated WI, and 2) time‐and frequency‐domain measures of HRV are altered with repeated WI.

Support: ONR and NAVSEA DSBDP
The effect of hypergravity exposure for two weeks on propofol anesthesia in rats
Authors: Iwata Chihiro , Abe Chikara , Morita Hironobu
Published: June 2021

General anesthesia in space flight is a big challenge for now. To achieve this, understanding the influence of gravitational change on central nervous system (CNS) modulation is required, however it is still unclear. The vestibular system is known to be a highly plastic organ. Previous studies reported that the hypergravity exposure induced plastic alteration of the glutaminergic and GABAergic system via the vestibular system. We hypothesized that hypergravity exposure‐induced CNS modulation might change the effectiveness of anesthetic agent. To clarify this hypothesis, we examined the effect of the intravenous propofol anesthesia in rats reared in 1 G and 3 G environments for 14 days. We measured electroencephalogram (EEG), electrical stimulation‐induced pressor response, propofol metabolism, and GABAA β2/3 subunits in the brain. EEG data showed that the hypnotic effect of propofol (20mg/kg) in 3 G rats was prolonged (43.7 ± 2.9 min) than that in 1 G (26.0 ± 1.0 min) rats. This effect was completely abolished by vestibular lesion (29.7 ± 1.6 min) in 3 G rats. Since there was no difference in propofol metabolism among groups, hypergravity exposure might alter the sensitivity of the propofol via vestibular system.
The genetic response of human endothelial cells to exposure to varying oxygen concentration.
Authors: Bowman Phillip , Bynum James A , McFaul Steve J
Published: June 2021

Most cell culture is performed under atmospheric oxygen (20.9%), but this level is not present in vivo. Cells cultivated under more physiologic O2 levels have been reported to exhibit different responses, but O2 levels in the culture media have not been reported in such studies. We utilized a SensorDish Reader® to monitor real time O2 levels in the culture media of human umbilical vein endothelial cells (HUVEC) exposed to 170 μM (20.9%), 30 μM (3%), and 10 μM (1%) O2 concentrations for up to 24 hrs. Approximately 6 hrs were required for the media of unstirred cultures to achieve the final level of O2. By western blot analysis, hypoxia inducible factor 1 alpha (HIF‐1A) was not detectable at 170 μM O2 but was readily detectable at 30 and 10 μM. At the end of 24 h RNA was isolated and subjected to gene expression analysis with human whole genome microarrays. About 740 genes were statistically altered in their expression with a false discovery rate <5%. The gene most altered in its expression by reduced O2 was EGLN3, a key enzyme in regulating HIF‐1A levels. The growth of HUVEC over 6 days did not differ at 170 μM and 30 μM, but experienced a 20% increase in time to confluence at 10 μM O2 indicating that the level of oxygen was limiting for growth. Some discrepancies reported between behavior of freshly isolated cells versus cells cultured for only a few days in vitro might be explained by their growth in hyperoxic conditions.
The histone deacetylase inhibitor valproic acid (VPA) pleotropiclly modulates cytokine and hypoxia‐dependent dysregulation of genes that predispose the formation of intraabdominal adhesions in cultured human mesothelial cells (HMCs)
Authors: Gruver Charles , Heydrick Stanley , Stucchi Arthur F
Published: June 2021

Mesothelial injury initiates the secretion of a fibrinous exudate, the formation of a fibrin matrix and the formation of abdominal adhesions. Inflammation suppresses peritoneal fibrinolysis and mitigating this decrease reduces adhesions. We showed that VPA inhibits adhesions by a mechanism independent of peritoneal fibrinolysis. Our objective was to examine the mechanism(s) by which VPA counters the dysregulation of key adhesiogenic genes. Cultured HMCs were treated with either the cytokines TGFβ+IL1β+IFNγ (C), the hypoxia mimetic dimethyloxaloglycine (D) or both, +/− 2 mM VPA, and mRNA levels for vascular endothelial growth factor (VEGF; vascular extravasation), thrombomodulin (TM; inhibits thrombin), and tissue plasminogen activator (tPA); activates plasmin) were measured at 6 hrs via RT‐PCR. VPA 1) blunted the increase of VEGF mRNA by D (7.4±1.4 vs 4.0±0.5 fold over control) and D+C (23.9 ±9 vs 10.1±1.5 fold over control, both p<.01); 2) increased TM mRNA 2‐fold in all groups (p<.05 for all), inhibiting thrombin activation; and 3) induced tPA mRNA to increase in response to C (1.4±0.3 vs 4.3±1.2 fold over control) and D+C (1.1±0.4 to 2.7±10.6 fold over control, both p<.05). VPA may decrease fibrinogen levels in the peritoneum, its conversion to fibrin, and blunt the decrease in fibrinolytic activity. Funded by Cooper‐Tyler Endowment, Department of Surgery, Boston University Medical Center.
Influence of hypoxia on lipopolysaccharide (LPS)‐induced chemokine CX3CL1 production by human amniotic epithelial cells (HAEC) – correlation with CX3CR1 receptor expression
Authors: Szukiewicz Dariusz , Pyzlak Michal , Mittal Tarun Kumar , Stangret Aleksandra
Published: June 2021

Placental cytokine network includes fetal membranes. Precise autoregulation of chemokine CX3CL1 production in HAEC via CX3CL1/CX3CR1 pathway is involved in some mechanisms of both physiologic and pathologic response. Depending on the stage of pregnancy, hypoxic intrauterine environment may be beneficial or deteriorating for the utero‐placento‐fetal unit. We examined influence of low oxygen tension (5% O2) on LPS‐induced CX3CL1 production in relation to CX3CR1 expression and functional status. After normal full‐term deliveries HAEC were isolated from the amnion using trypsinization, and cultured under hypoxia (Group I; N=12) vs. normoxia (20% O2; Group II, controls; N=12). According to experimental design, LPS (1ìg/ml) or neutralizing anti‐CX3CR1 antibodies + LPS, were added at respective timepoints. Mean CX3CL1 concentration in the culture supernatant were determined by ELISA. Expression of immunostained CX3CR1 within HAEC cultures was analyzed by quantitative morphometry. Hypoxia transiently decreased (p < 0.05) CX3CL1 concentration. Blockade of CX3CR1 produced irreversible lowering in CX3CL1 production, especially in Group I (the mean 6.7‐fold decrease vs. 1.9‐fold in Group II). CX3CR1 expression was significantly augmented in Group I. The results indicate that compensatory chemokine CX3CL1 response to hypoxia may depend on CX3CR1 expression.
Ketamine (KET) decreases inflammatory and apoptotic gene expression in ovine fetal frontal cortex exposed to global acute hypoxic hypoxia (HH)
Authors: Chang Eileen I‐Ling , Rabaglino Maria Belen , Richards Elaine M. , Wood Charles E.
Published: June 2021

HH activates fetal neuroendocrine stress responses, perhaps by excessive glutamatergic signaling, which could stimulate inflammation and apoptosis in fetal brain. We have shown that HH stimulates inflammation and apoptosis genomic response with ovine gene array. We propose that treatment with KET, an N‐methyl‐D‐aspartate receptor antagonist, during HH will interrupt inflammatory and apoptotic signaling pathways in fetal frontal cortex. Fetal sheep (n=3–5/group) were chronically catheterized (125 d gestation). KET (3 mg/kg) was administered intravenously to the fetus 10 min prior to the induction of HH (30 min) stimulation or normoxic control (NC). Fetal frontal cortex was collected 24 hrs post‐HH, and cDNA was synthesized from mRNA using random primers. CASP8, MYD88, and NFKB mRNA expression were measured using real‐time PCR and normalized to β‐actin mRNA. At 24 hrs post‐HH, CASP8, MYD88, and NFKB mRNA expressions increased vs. NC, 2.01‐fold, 1.32‐fold, and 1.40‐fold, and KET treatment attenuated this increase to 0.98‐fold, 1.08‐fold, and 0.91‐fold, respectively (CASP8, P<0.05, one‐way ANOVA; MYD88, P<0.05, one‐way ANOVA; NFKB, P<0.05, t‐test 24 HH vs. 24 HH+KET). Fetal inflammatory (MYD88, NFKB) and apoptotic (CASP8) responses were activated with HH and reduced with KET. The results are consistent with our gene array analysis. Research supported by NIH grants HD033053, HD057561, and DK076541.
Validation of Saline Contrast to Measure and Quantify Intrapulmonary Arteriovenous Anastomoses
Authors: VanDerWeele Hannah , Bates Melissa , Pegelow David , Sobakin Aleksey , Kellihan Heidi , Eldridge Marlowe
Published: June 2021

Intrapulmonary arteriovenous anastomoses (IPAVs) are large diameter pathways with the potential to allow blood to bypass pulmonary capillaries. In humans, saline contrast echocardiography has been used to demonstrate their opening with exercise and hypoxia. Using a 0–5 scale, the intensity of right‐to‐left bubble passage has been shown to increase with exercise and hypoxia severity. The limitations of this method are that bubbles are of unknown size and stability and the 0–5 scale has never been quantitatively validated. We aimed to validate the bubble method using the gold standard microsphere method in piglets, hypothesizing that there would be agreement. We studied eight anesthetized piglets (7–10 kg) breathing 21% or 8% O2 for five minutes in random order. One million, 50 μm fluorescent (red or green) microspheres were injected into the right ventricle in each condition while a simultaneous saline contrast echocardiogram was obtained. The brains were removed, hydrolyzed with 5N KOH, filtered through an 8 μm filter, and the presence of microspheres was assessed. Bubble and microsphere passage was not observed at 21% O2. In 8% O2, microspheres were noted in the brains of 8/8 piglets. However, bubbles were noted in only 6/8 piglets. This suggests that microspheres are a more sensitive measure of IPAVs recruitment. The fact that bubbles traversed the lungs coincidentally with 50 μm microspheres suggests they are traveling through large diameter pathways. In our subsequent analysis, also presented here, we have aimed to quantify microsphere passage to determine if the 0–5 scale has quantitative meaning.
The effects of hypoventilation on the physiological and biochemical properties of hindlimb muscles
Authors: IMAGIATA HIDETAKA , YAMAGAMI TAKU , OKADA KEISUKE , KAWATA SHINNOSUKE
Published: June 2021

The purpose of this study was to determine physiological and biochemical properties of hindlimb muscles after bilateral phrenic nerve denervation (DNV).

Male Wistar rats (10 wk) underwent DNV (n=18) by cutting the phrenic nerve at the cervical level under the stereomicroscope or sham surgery (n=18, CON). Analyses were performed 4, 8 and 12 wks after surgery. The myosin heavy chain (MHC) isoform profile and in vitro isometric contractile properties of the soleus (SOL) and extensor digitram longus (EDL) were analyzed.

DNV induced changes in SpO2 characteristic of hypoventilation syndromes. After 12 wks, increases in MHC 1 and decreases in MHC 2a (both p<0.05) were observed in the DNV SOL. Increases in MHC 1 (7%), MHC 2a (7%) and MHC 2d (9%) and decreases in MHC 2b (23%) (all p<0.05) were measured in the DNV EDL. After 8 wks and 12 wks of DNV, the peak twitch force of EDL decreased (~30%) and after 12 wks the fatigue index increased (19%) (all p<0.05). However, DNV did not affect the contractile properties of SOL.

In conclusion chronically low SpO2 and decreased hindlimb contractile activity induced by DNV resulted in a unique fast‐to‐slow MHC shift in SOL and EDL. This was accompanied by enhanced fatigue resistance and impaired contractile function in the EDL but not the SOL.

This work was supported by Grants‐in‐Aid for Scientific Research from the Japanese Ministry of Science and Technology, no 24500628.
Transdiaphragmatic pressure measurements reveal age‐related diaphragm muscle dysfunction during non‐ventilatory behaviors
Authors: Greising Sarah M , Mantilla Carlos B , Sieck Dylan C , Sieck Gary C
Published: June 2021

The diaphragm muscle (DIAm) is responsible for breathing and must provide sufficient force to accomplish both ventilatory and non‐ventilatory behaviors. Age‐related changes to the DIAm, especially sarcopenia, may limit the ability to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance, and thus contribute to respiratory complications. Age‐related changes in load (as a result of chest wall stiffening or lung compliance) may impact DIAm function. Adult male mice (6 and 24 months old) underwent measurements of respiratory system compliance and transdiaphragmatic pressure (Pdi) during: eupnea, hypoxia (10% O2) –hypercapnia (5% CO2), spontaneously occurring deep breaths, airway occlusion, and bilateral phrenic nerve stimulation. Overall respiratory system compliance decreased with age (increased load), while Pdi required for ventilatory behaviors did not change, consistent with the reserve capacity of the DIAm. The Pdi generated during deep breaths and airway occlusion were reduced 30–45% with age. These results indicate that DIAm dysfunction during aging is associated with reduced force to perform expulsive, non‐ventilatory behaviors. Reduced reserve capacity in old age may underlie increased susceptibility to lung infections and augment respiratory compromise associated with other age‐related diseases. Supported by T32‐HL‐105355 and the Mayo Clinic.
Orderly Recruitment of Diaphragm Motor Units Across Ventilatory and Non‐Ventilatory Motor Behaviors
Authors: Seven Yasin Baris , Mantilla Carlos B , Sieck Gary C
Published: June 2021

Respiratory excitatory premotor drive from the medulla is widely distributed to phrenic motor neurons, and it is generally assumed that motor neuron recruitment order is determined by intrinsic, size‐related electrophysiological properties (Size Principle). During ventilatory behaviors, fatigue‐resistant diaphragm muscle (DIAm) motor units (smaller motor neurons) are recruited, but during more forceful non‐ventilatory behaviors such as airway occlusion, deep sighs and sneezing, additional more fatigable motor units are recruited. We hypothesized that a fixed recruitment order of DIAm motor units is maintained across different ventilatory and non‐ventilatory behaviors. Unitary motor unit discharge was recorded across eupnea, hypoxia‐hypercapnia (10% O2–5% CO2), airway occlusion and deep sighs. The recruitment order displayed by motor units during eupnea and hypoxia‐hypercapnia was conserved during more forceful behaviors (82% of pairs maintained their recruitment order). Reversals of motor unit recruitment order occurred predominantly when the difference in onset times were within 20 ms. Onset discharge rates of motor units recruited during eupnea and hypoxia‐hypercapnia were lower than those of units recruited only during airway occlusion or deep sighs. These results support the Size Principle as an organizing principle of DIAm motor unit recruitment across a range of motor behaviors.
Selective activation of respiratory thoracic efferents in the spinal cord of the in situ rat preparation
Authors: Guarnieri Lauren , Sandhu Milap , Fuller David , Baekey David M
Published: June 2021

Previous studies of respiratory plasticity have focused primarily on phrenic motoneurons (PM) in the cervical spinal cord, while little is known about the intercostal (IC) motor system. Recent reports suggest that IC muscle activity is only modestly impaired after unilateral cervical spinal cord injury (SCI), therefore, we hypothesized that respiratory thoracic motoneurons are capable of greater plasticity than PMs. To test this hypothesis, we simultaneously recorded activity from the left phrenic nerve (PNA) and thoracic ventral roots in the perfused in situ rat. Preparations were exposed to repeated bouts of hypercapnia (9% CO2/91% O2) alternated with baseline recovery periods (5%CO2/95%O2); and/or to periods of relative hypoxia (40% O2/5% CO2/55% N2) and recovery (95%O2/5%CO2). During both exposures, the relative increase in inspiratory‐related activity was significantly greater in the thoracic efferents compared to the phrenic nerve. Further, thoracic activity showed enhanced progressive augmentation compared to the phrenic nerve during each successive perturbation. We conclude that respiratory thoracic efferents have a greater capacity for dynamic plasticity in response to both hypercapnia and hypoxia. We suggest that compensation by the IC motor system may be a significant spontaneous mechanism for respiratory recovery after SCI and could be a key target for therapeutic interventions.
Ontogeny of low threshold myelinated vagal afferents in the rat is sexually dimorphic.
Authors: Qian Zhao , Li Bai‐Yan , Schild John
Published: June 2021

We have previously reported on the existence of a sex‐specific subpopulation of low threshold myelinated vagal afferents. These “Ah‐type” fibers are rarely found in male rats and exhibit an excitability profile strongly influenced by circulating sex hormones. Postnatally, these “Ah‐type” afferents are present in both sexes but by adulthood Ah‐type visceral afferents have nearly disappeared from the male population. This study aims to quantify the time course of the differential ontogenetic development of Ah‐type afferents using the patch clamp technique. Current clamp protocols were carried out on vagal ganglion neurons (VGN) acutely dissociated from 4, 6, 8, 11, 13, 15, 21, 23, 25, 28, 30, 32 day old rats of both sexes. A cluster analysis of select measures from the current clamp recordings revealed three statistically unique populations of VGN at each time point, low threshold myelinated A‐ and Ah‐type VGN and high threshold unmyelinated C‐type VGN. The relative expression of these three afferent subtypes in females remains relatively steady from 4 – 32 days. In stark contrast, the relative expression of Ah‐type afferents in males declines from levels comparable to those in female at birth to a near absence in adulthood. Our results quantify the time course of visceral afferent ontogeny and provide additional support for our hypotheses regarding sexual dimorphism in visceral afferent function. HL072012 HL081819
Caffeine alters sleep and breathing without an increase in brain serotonin in developing rat pups
Authors: Schneider Robert W , Phillips Louisa , Ryu Young Sang , Keyes Lauren M , Darnall Robert A
Published: June 2021

In rat pups a reduction in serotonin (5HT) input to respiratory networks impairs anoxic auto‐resuscitation and impairs arousal during hypoxia. In contrast, blockade of adenosine receptors enhances auto‐resuscitation and attenuates the VE decline during the biphasic hypoxic ventilatory response. Caffeine (Caf), an adenosine receptor antagonist, used to treat apnea of prematurity in human infants, has well known effects on sleep and vigilance in adults and in some reports increases the levels of brain 5HT. We therefore examined the pharmacodynamics and the physiological effects of single IP injections of Caf (base) at 5 to 40 mg/kg in P9–P11 pups. Plasma Caf levels reached 10.9 and 76.3 mg/l for 5 and 40 mg/kg, respectively, at 60 min, and were stable until at least 90 min. Caf increased the % time awake at 10 and 40 mg/kg and at 40 mg/kg decreased the % time in active sleep. Caf did not change baseline heart or respiratory rate but the effect of Caf on VT and VE was dependent on state and dose. At 40 mg/kg, VT was increased during wake and quiet sleep whereas VE was increased only during wake. No increases in brainstem 5HT were found at either 10 or 40 mg/kg. In rat pups, a low dose of Caf alters sleep and at higher doses stimulates respiration. The effects of Caf on auto‐resuscitation and ventilation during hypoxia do not appear to be related to an increase in brainstem 5HT levels. NICHD 5‐PO1 HD036379 15
“Full term” prenatal nicotinic exposure (fPNE) increases the pulmonary C‐fiber (PCF)‐mediated apneic response in rat pups
Authors: Zhuang Jianguo , Xu Fadi
Published: June 2021

Different from traditional PNE, fPNE consists of the periods before pregnancy and during pregnancy and lactation. Our recent study has shown that fPNE significantly depresses hypoxic ventilatory response (HVR) often followed by respiratory failure and death in postnatal rats (P12–14) (FASEB 2012, Xu and Zhuang), but the relevant mechanisms remain unexplored. PCFs are sensitized/stimulated by nicotine and inhibitory to ventilation and HVR. These results, along with an over‐expression of vagal C‐fibers in SIDS victims, allow us to hypothesize that fPNE would augment the PCF‐mediated apneic response via sensitizing PCFs and elevating PCF expression in the airways. We compared the ventilatory response to right atrial bolus injection of capsaicin (1 μg/kg) to stimulate PCFs in anesthetized fPNE and control pups, and then the PCF density in the airways was identified by using substance P immunoreactivity (SP‐IR). We also tested if fPNE was able to sensitize PCFs by extracellularly recording pulmonary C neurons (cell bodies of PCFs) in nodose ganglia of anesthetized and paralyzed control and fPNE pups. We found that fPNE: 1) significantly prolonged the apneic response to capsaicin by ~3‐fold with little change in baseline cardiorespiratory activities; 2) augmented pulmonary C neural firing response to capsaicin; and 3) markedly increased the SP‐IR fibers’ density in the airways. Our results suggest that fPNE augments the PCF‐mediated apneic response via sensitizing PCFs and elevating airway SP‐IR fibers’ density. Further studies are required for determining the role of PCFs in generating the depressed HVR and respiratory failure observed in fPNE pups. (Supported by HL 107462 and ALA RG‐191095‐N)
Deletion of the transient receptor potential vanilloid type 1 channel exacerbates renal inflammation induced by lipopolysaccharide in mice
Authors: Wang Youping , Wang Donna H
Published: June 2021

To determine the role of the transient receptor potential vanilloid type 1 (TRPV1) channel in the regulation of renal inflammation, lipopolysaccharide (LPS, 3 mg/kg) was intraperitoneally injected into wild type (WT) and TRPV1‐null mutant (TRPV1−/−) mice. Glomerular hypercellularity and red blood cell congestion were found in the kidneys of WT and TRPV1−/− mice 24 hours after LPS injection with greater magnitude in TRPV1−/− mice. Serum creatinine levels were higher in TRPV1−/− than in WT mice 24 hours after LPS injection. Neutrophil infiltration in the kidneys was greater in TRPV1−/− than in WT mice 6 or 24 hours after LPS injection. Serum cytokine levels (TNF‐α, IL‐1β, and IL‐6) and renal chemokine levels (KC and MIP‐2) were higher in TRPV1−/− than in WT mice 6 hours after LPS injection. Western blot analysis showed that renal VCAM‐1 but not ICAM‐1 expression was increased in WT and TRPV1−/− mice 6 hours after LPS injection with greater magnitude in TRPV1−/− mice. Renal NF‐κB activity was elevated further in TRPV1−/− compared to WT mice 6 hours after LPS injection. Thus, TRPV1 gene ablation exacerbates LPS‐induced renal injury, including aggravated renal neutrophil infiltration, chemokine and adhesion molecule levels, and glomerular hypercellularity accompanying with further increased serum creatinine and cytokine levels. Our findings indicate that TRPV1 is activated during LPS challenge and protects against LPS‐induced renal injury possibly via reducing renal inflammatory responses. (Supported in part by the National Natural Science Foundation of China: No. 81170243)
Mitochondria‐Targeted Ogg1 and Aco‐2 Prevent Oxidant‐Induced Mitochondrial DNA Damage
Authors: Kim Seok‐Jo , Cheresh Paul , Tularisam Sandhya , Schumacker Paul , Weitzman Sigmund A , Bohr Vilhelm , Kamp David W.
Published: June 2021

Objective

Mitochondria‐targeted hOgg1 (mt‐hOgg1; a DNA base excision repair enzyme) and aconitase‐2 (Aco‐2; Kreb's cycle enzyme) each reduce oxidant‐induced alveolar epithelial cell (AEC) apoptosis but it is unclear whether protection occurs by preventing mtDNA damage.

Methods

Quantitative PCR‐based measurement of mitochondrial DNA damage was assessed following transient transfection with wild‐type mt‐hOgg1, mt‐long α/β Ogg1 mutant (mt‐Lα/β Ogg1), Aco‐2 and siRNA for Aconitase‐2 (siAco‐2) in A549 cells with asbestos (5–25 μg/cm2) or H2O2 (100–250 μM) for 24 h. Apoptosis was assessed by cleaved caspase‐9/3 and DNA fragmentation.

Results

Asbestos and H2O2 induced mtDNA damage and apoptosis that was mitigated by overexpression of mt‐hOgg1, mt‐Lα/β Ogg1 or Aco2. Compared to scrambled siRNA, siAco‐2 enhanced mtDNA damage and apoptosis induced by asbestos or H2O2.

Conclusions

Our data demonstrate a novel role for mt‐hOgg1 and Aco‐2 in preventing oxidant‐induced AEC mtDNA damage and apoptosis. Given the importance of AEC apoptosis in pulmonary fibrosis, we reason that the mt‐hOgg1/Aco‐2 effects on AEC mtDNA is an innovative target for preventing pulmonary fibrosis. Funding: NIH‐RO1 ES020357‐01A1 (DK) and VA Merit (DK)
Effect of surfactants on polystyrene nanoparticle (PNP) interactions with primary rat alveolar epithelial cell monolayers (RAECM)
Authors: Kim Yong Ho , Sipos Arnold , Fazlollahi Farnoosh , Borok Zea , Kim Kwang‐Jin , Crandall Edward D
Published: June 2021

We investigated effects of apical 0.1% (w/v) Pluronic (P) or 1 mg/mL Infasurf (I) on flux of 20 nm carboxyl‐modified (negatively charged) PNP (20‐PNP) or 20 nm amidine‐modified (positively charged) PNP (20+ PNP) across RAECM, across which the bioelectric properties (transepithelial resistance (Rt) and equivalent short circuit current (Ieq)) were unchanged in the presence of these two surfactants. Results of flux measurements, along with hydrodynamic diameter (HD) measured by dynamic light scattering and zeta potential (ZP) measured in the presence and absence of P or I, are summarized below.

20+ PNP
20‐ PNP

Flux (pg/cm2/sec)
PNP alone
38±7
1.6±0.1

PNP with P
35±7
2.5±0.2

PNP with I
42±6
0.9±0.2

HD (nm)
PNP alone
543±28
156±7

PNP with P
374±13
80±3

PNP with I
316±9
278±9

ZP (mV)
PNP alone
37±10
−61±6

PNP with P
33±2
−29±2

PNP with I
−44±1
−82±4

These data indicate that there is remarkably little effect of P or I on PNP flux despite significant changes in measured HD and ZP. We conclude that PNP (and RAECM) exposure to either surfactant P or I surprisingly does not cause meaningful alterations in PNP translocation across RAECM, which may be explainable by the behavior of PNP in the extracellular microdomain adjacent to the apical alveolar epithelial cell membrane.

(Funded by NIH and Hastings & Whittier Fdns.)
Nanodiamond (ND) interactions with primary rat alveolar epithelial cell monolayers (RAECM)
Authors: Kim Yong Ho , Sipos Arnold , Fazlollahi Farnoosh , Borok Zea , Kim Kwang‐Jin , Crandall Edward D
Published: June 2021

We investigated interactions of positively and negatively charged 5 nm ND (zeta potentials of 50 and −60 mV, respectively; provided by Carbodeon, Vantaa, Finland) with RAECM. Transepithelial resistance (Rt) and equivalent short circuit current (Ieq) of RAECM in the presence and absence of ND in apical fluid for up to 24 h did not change. We estimated cellular uptake and transepithelial trafficking of ND across RAECM over 24h spectrofluorometrically with excitation/emission wavelengths at 275/555 nm. Cell uptake of positively charged ND was ~3 times greater than that (~9 pg/μg cell protein) of negatively charged ND, while transepithelial trafficking of positively and negatively charged ND was about the same at ~20 pg/cm2/s. Trafficking of ND is ~10‐fold faster than that of negatively or positively charged quantum dots (core diameter 5 nm) and negatively charged polystyrene nanoparticles (PNP, 20 nm), but ~2‐fold slower than that of positively charged PNP (20 nm). These data indicate that positively or negatively charged ND do not affect barrier function of alveolar epithelium and suggest that ND surface charge affects accumulation in but not translocation across alveolar epithelium. Mechanisms underlying ND entry into/egress from alveolar epithelial cells remain to be determined.

(Funded by NIH and Hastings & Whittier Fdns.)