bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid synthesis and distribution during the annual cycle of a hibernator
Authors: Otis Jessica P , Sahoo Daisy , Drover Victor A , Eric Yen C. L. , Carey Hannah V
Published: June 2021

Although the seasonal shift from carbohydrate to lipid metabolism in hibernators is well established, a comprehensive study of lipid regulation in one species is lacking. To address this we studied triglyceride (TG) and lipoprotein metabolism in spring (SPR), summer (SUM) and hibernating (HIB) 13‐lined ground squirrels (5–6 per group). Plasma TGs are carried in VLDL particles in all seasons. There are no differences among the groups in ApoB mRNA in liver and gut or ApoB protein in liver and plasma. Fasting decreases plasma TGs 2‐fold in SPR (p<0.05), but not in SUM. Plasma TGs are lower in torpid HIB (76.7±43.9 mg/dl) than interbout arousal (IBA) HIB (178.0±17.4 mg/dl), and both are less than SUM (278.2±44.4 mg/dl) (P<0.05). Plasma free fatty acids (FFAs) are highest in torpid HIB, equal in SUM and IBA HIB (P<0.001), and are not affected by fasting in SPR or SUM. Liver TGs are higher in torpid HIB than in SPR or IBA HIB (P<0.05), but liver FFAs are highest in torpid HIB (P<0.05). White adipose tissue (WAT) TGs do not vary among states, but WAT FFAs are lower in torpid HIB than SUM (P<0.05). mRNA levels of the TG synthesis enzymes MGAT2 and DGAT2 are greater in HIB than SUM (P<0.05). The results provide insight into how hibernating species regulate synthesis and distribution of lipids throughout the body during their annual cycle of feeding and fasting.
The proapoptotic factor BIM is necessary and sufficient for cAMP/PKA‐induced apoptosis in murine S49 lymphoma cells
Authors: Zambon Alex C , Ho Angela , Wilderman Andrea , Seibold Millie , Insel Paul A
Published: June 2021

The intracellular second messenger cyclic AMP (cAMP) acts via protein kinase A (PKA) to induce G1 phase cell‐cycle arrest by 24h and subsequent apoptosis at 48h in murine S49 lymphoma cells. The molecular mechanisms for cAMP/PKA growth arrest and killing are undefined. Using genome‐wide microarray analysis, we found that treatment of WT and Deathless (D‐) S49 cells with the PKA agonist 8‐(4‐chlorophenylthio)‐cAMP (CPT‐cAMP) differentially regulates several pro‐ and anti‐apoptotic transcripts at 24 h, thus identifying putative mediators of cAMP/PKA killing. The D‐ (cAMP‐deathless) S49 cell variant has wild‐type levels of PKA activity, undergoes cAMP‐promoted G1 growth arrest but is protected from cAMP/PKA‐induced apoptosis by an unknown mechanism. We hypothesized that the pro‐apoptotic BH3‐only family member Bim, which shows increased expression in WT, but not D‐cells 24h after CPT‐cAMP treatment, is a key mediator of PKA‐mediated killing in S49 cells. Conditional expression of the BimL isoform in both WT and D‐ S49 cells dose‐dependently increased apoptosis in both cell lines, indicating that its up‐regulation is sufficient to induce apoptosis of S49 cells. Furthermore, inhibition of cAMP‐mediated BimL induction via short hairpin RNAi knockdown studies abolished CPT‐cAMP‐mediated apoptosis of WT S49 cells, indicating that Bim up‐regulation by PKA is necessary for PKA‐mediated apoptosis. Taken together, these results indicate that the transcriptional up‐regulation of the pro‐apoptotic protein Bim is necessary and sufficient for cAMP/PKA‐mediated apoptosis.
Short Sleep Duration and Endothelial Progenitor Cell Function
Authors: Morgan Richard Garrett , Weil Brian Raymond , MacEneaney Owen James , Kushner Erich James , Greiner Jared James , Stauffer Brian , DeSouza Christopher A
Published: June 2021

Chronic short sleep duration is associated with increased cardiovascular (CV) morbidity. Mechanisms underlying this increased CV risk are not clear. Endothelial progenitor cells (EPCs) contribute to both reendothelialization and neovascularization. Numerical and functional impairment of EPCs has been linked to CV risk. Currently, there is no information regarding the influence of short sleep duration on EPC function. We tested the hypothesis that EPC function is impaired in adults who sleep less than 7 hrs/night (short sleep) compared with those who sleep 7 to 9 hrs/night (normal sleep). Cells with phenotypic EPC characteristics were isolated from 37 healthy, sedentary adults: 20 normal sleepers (13M/7F; age: 59±1 yr; sleep: 7.7±0.1 hrs/night) and 17 short sleepers (9M/8F; 56±2 yr; 6.0±0.2 hrs/night). There were no significant differences between groups in EPC number via FACS (0.001±0.0004 vs 0.001±0.0003 %), colony‐forming capacity (6.1±1.5 vs 5.4±1.7 CFUs), or migration to VEGF in a Boyden chamber (1410.1±151.2 vs 1334.3±111.1 AU). Furthermore, were no group differences in basal and staurosporine stimulated intracellular concentrations of active caspase‐3 (0.3±0.03 vs 0.5±0.1 ng/mL; and 2.9±0.4 vs 2.7±0.3 ng/mL) and cytochrome c (16.4±0.7 vs 14.5±1.4 ng/mL), markers of apoptotic susceptibility. Short sleep duration is not associated with EPC dysfunction in healthy adults.
Histaminergic modulation during nicotinamide‐stimulated differentiation of amniotic epithelial cells (AC) into insulin producing cells (IC)
Authors: Szukiewicz Dariusz , Pyzlak Michal
Published: June 2021

AC resemble stem cells and express histamine H1 and H2 receptors. We examined an effect of histamine on the generation of IC from nicotinamide‐stimulated AC and expression of H1 and H2 at different stages of this process. AC were isolated from human amnion (N=12) and cultured for 16 days using Okita's method with nicotinamide (10mM). Mean concentration of

C‐peptide (CCP) in the culture media was measured every 3 days as a marker of the secretion of insulin. At the same intervals the cultures were fixed and immunostained for H1 and H2. Quantitative morphometry was applied for evaluation of H1 and H2 expression. Influence of histamine (100microM) on CCP was studied in subgroups with/without H1 and H2 antagonists.

C‐peptide was detected on Day 6 and CCP gradually raised until Day 12, then stayed at similar levels, 4,1‐fold higher than initially. Expression of H2 was unchanged (compared to control AC) until Day 9, then significantly decreased. Differences in H1 expression were not observed. Histamine produced significant increase in CCP, more evident after 6 days of culture than after 15 days. H2 blockade reduced histamine‐related increase of CCP, whereas H1 antagonist did not affect CCP. Nicotinamide‐induced, pancreatic differentiation of AC in vitro may be augmented by histamine acting on H2 receptors. Variable expression of H2 may define a time‐point, indicating involvement of histamine at earlier stages.
Transplantation of mesenchymal stem cells fails to provide a synergistic effect on functional recovery of transected nerve with exercise‐treated rats
Authors: Yang Chen‐Chia , Hsieh Yueh‐Ling , Ou Hsiu‐Chung
Published: June 2021

This study examined whether transplantation of mesenchymal stem cells (MSC) in combination with exercise had synergistic effects leading to functional recovery that is greater than exercise alone. Adult male SD rats received a sciatic nerve transection were divided into four groups: denervated (control), denervated + exercise (control + Ex), denervated + MSC transplantation (MSC), and denervated + MSC transplantation + exercise (MSC + Ex). MSC was injected into the lesion site in MSC‐treated groups and culture medium was injected as control. Twelve hours after surgery, two exercise groups swam for 7 days. Functional assessments including sciatic function index (SFI), vertical activity (VA) of locomotion, angle of ankle (AA), and electrophysiological studies were performed for monitoring the recovery of motor function. No significant differences in SFI, VA and AA were found between MSC + Ex and control + Ex groups. Furthermore, there were no significant differences in amplitudes and onset latencies of compound muscle action potentials between two groups. Morphological study revealed prominent axonal degeneration in lesion nerves. Results showed no noticeably synergistic effect of MSC transplantation on functional recovery of transected nerve treated with swimming exercise. This study was supported by a grant from the National Science Council (95‐2314‐B‐241‐003) of Taiwan.
Impact of 12 wks Exercise on Renal Nitric Oxide and Antioxidant Status: A Strain Difference Comparison
Authors: Moningka Natasha , Cunningham Mark , Sterling Myrline , Baylis Chris
Published: June 2021

One mechanism by which the vasculature benefits from exercise (EX) is by increases in blood flow (BF). This causes increases in shear stress and induction of the endothelial nitric oxide synthase (eNOS) and antioxidant, extracellular superoxide dismutase (EC SOD). The kidney, however, undergoes reduced BF during EX and therefore may be at risk for falls in NO and antioxidant bioavailability. Genetic differences may also dictate this response. Therefore, we investigated kidney cortex (KC) responses to EX in the Sprague Dawley (SD) and Fisher 344 (F344) rat. Male SD and F344 rats (10–12 wks old) voluntarily wheel ran for 12 wks (24 hr access) or remained sedentary (SED). Running activity was similar between strains. Nitrate/nitrite (NOx) levels and eNOS and EC SOD abundance in the KC of F344 rats increased with EX (Table; *p≤0.05 vs. SED). In contrast, SD rats decreased their renal eNOS and EC SOD with no change in KC NOx. The oxidative stress marker p22phox also increased in the KC of EX F344 but decreased in the EX SD rat. Another oxidative stress marker, H2O2, measured in KC, increased only in EX F344 rats. These findings indicate that a strain difference exists in the response to 12 wks voluntary EX between the F344 and SD rat. F344 rats increased their KC eNOS and EC SOD and this was interestingly also associated with increased oxidative stress. Conversely, SD rats had the opposite response to EX in their KC: reduced eNOS and EC SOD and either decreased p22phox or no change in H2O2. Mechanisms responsible for these marked differences in renal responses to EX remain to be determined.

eNOS
NOx
EC SOD
p22phox
H2O2

n
Arbitrary Units
nmol/mg
Arbitrary Units
Arbitrary Units
nmol/mg

F344

SED
12
6±1.2
8±1.7
68±23
5±0.5
0.2±0.04

EX
12
18±3.2*
15±6.3*
122±27*
8±0.6*
0.6±0.14*

SD

SED
6
64±2.6
9±0.4
13±1.0
9±0.4
0.4±0.07

EX
6
56±2.2*
10±0.4
8±0.7*
7±0.6*
0.5±0.07
Aging‐Related Renal Damage Is Associated with Decreased Klotho Expression and Increased Superoxide Production
Authors: Zuo Zhong , Lei Han , Wang Xiuqing , Wang Yuhong , Sonntag William , Sun Zhongjie
Published: June 2021

Klotho is a recently‐discovered anti‐aging gene. The purpose of this study was to determine changes in klotho, endothelin (ET) receptors, NADPH oxidases, and superoxide production in kidneys of aged rats. Twenty aged rats (male, 36 months) were divided into an impaired group (n=9) and an intact aging group (n=11) according to a cognitive function test. A group of 12 month‐old rats (n=10) was used as a young group. Plasma creatinine was significantly greater in the impaired group, suggesting impaired renal function. Aged rats showed a decrease in the number of glomeruli, an increase in collagen staining (fibrosis) around glomeruli and tubules, and an increase in periodic acid Schiff (PAS) staining (matrix remodeling) in and around glomeruli, indicating renal structural damage. These pathological changes were markedly aggravated in the impaired than in intact animals. Notably, aged rats demonstrated a significant decrease in klotho protein expression in renal cortex and medulla. Protein expression of interleukin‐6, Nox2 and ETA receptors and superoxide production were increased whereas MnSOD and ETB receptor expression were decreased in kidneys of aged rats. Interestingly, these changes were more pronounced in the impaired than in the intact rats. Conclusions: Aging‐related renal damage was associated with decreased klotho, increased inflammation, and increased superoxide production. Renal damage parallels with cognitive function impairment. Further studies are required to evaluate the role of klotho, ET receptors, and Nox2 and their relationship in aging‐related renal damage.
Shear Stress Induced Nitric Oxide (NO) Production In Macula Densa Cells Is Mediated By The Primary Cilia
Authors: Liu Haifeng , Lu Deyin , Fu Yiling , Zhu Xiaolong , Juncos Luis A. , Liu Ruisheng
Published: June 2021

The macula densa (MD) senses NaCl/tubular flow and initiates tubuloglomerular feedback. We hypothesize that sheer stress along the luminal membrane of macula densa cells bends the primary cilia, which causes an increase in intracellular calcium and enhances nNOS activity at the MD. To test this hypothesis we investigated whether increasing shear stress from 0.5 to 5 dynes/cm2 (with a circular flow chamber) across MMDD1 cells, a macula densa‐like cell line, increased intracellular calcium (measured by Fura‐2) and NO levels (measured with DAF‐FM). Increasing sheer stress caused intracellular calcium to increase from 0.94 ± 0.07 to 1.38 ± 0.41(p < 0.01), and NO levels to increase from 174.8 ± 21.5 to 276.1 ± 24.3 units/min (p < 0.01). We next determined whether the primary cilia mediated this increase in intracellular calcium and NO. For this, we removed the primary cilia of MMDD1 cells by treating them with a siRNA against polaris, measured with immunostaining of cilia, and then repeated the above experiments. In contract to the previous experiments, increasing shear stress had no effect on either intracellular calcium (0.94 ± 0.07 vs. 0.98 ± 0.13) or NO levels (48.6 ± 17.5 vs. 62.7 ± 15.2 units/min). These data indicate that: 1) shear stress enhances NO generation in MMDD1 cells; 2) cilia can be removed with a siRNA against polaris; 3) shear stress‐induced NO generation is mediated by cilia via elevated intracellular calcium.
Laser speckle microscopy measurement of arteriolar blood flow in many individual nephrons simultaneously
Authors: Holstein‐Rathlou Niels‐Henrik , Sosnovtseva Olga , Laugesen Jakob , Cupples William , Marsh Donald J
Published: June 2021

Tubular pressure oscillations in cortical nephron pairs often synchronize. To determine the extent and duration of synchronization we used laser speckle microscopy on the renal surface of rats. The camera measures changes in surface roughness associated with blood flow changes in 442K pixels at 1/sec. A 15×15 pixel matrix captures one star vessel, and up to 100 star vessels are sampled simultaneously for 30 min or longer. Star vessel locations are identified on the renal surface by ordinary digital microscopy during intravenous dye injection. The digital camera image is overlaid on the time averaged speckle image in Photoshop; star vessel locations are registered on the speckle image. Time series are sampled from the speckle image at each star vessel location. Single star vessel flow changes in response to angiotensin II and acetyl choline mimic those in whole kidney blood flow. The time series were analyzed with wavelet transforms and show oscillations associated with both tubuloglomerular feedback and the myogenic mechanism. Synchronization is present among nephrons but persists for variable periods of time; the size of synchronization clusters also varies with time.
Role of connexin 40 in renal autoregulation
Authors: Sorensen Charlotte Mehlin , Braunstein Thomas Hartig , Holstein‐Rathlou Niels‐Henrik
Published: June 2021

The expression of connexins in renal arterioles is believed to have profound impact on regulation of arteriolar tonus and renal blood flow. Connexin 40 (Cx40), which is primarily expressed in the endothelial cells of the renal afferent arterioles, has been shown to play a significant role in the regulation of renin secretion. Also, connexins are believed to be involved in the transmission of the calcium signal initiating the TGF response and connexins may therefore affect renal autoregulation.

We wished to examine the renal autoregulatory response by measuring changes in afferent arteriolar diameter in kidneys from Cx40 knockout (KO) mice in response to a step increase in perfusion pressure. We used the isolated perfused juxtamedullary nephron preparation to visualize afferent arterioles. As previously reported Cx40 KO mice had a higher arterial blood pressure than age‐matched litter‐mate controls. Also, the plasma renin concentration was significantly higher in the Cx40 KO mice compared to wild‐type mice. The change in afferent diameter in response to step increases in renal perfusion pressure was impaired in Cx40 KO mice compared to the wild‐type littermates. These results suggest that Cx40 play a role in the autoregulation of renal blood flow.
Self‐Reported, Interview‐Assisted Diet Records Underreport Protein and Energy Intake in Maintenance Hemodialysis (MHD) Patients
Authors: Morrison Gillian Kye , Bross Rachelle , Kalantar‐Zadeh Kamyar , Kopple Joel D
Published: June 2021

Some studies state that MHD patients underreport energy intake (EI) although energy needs are normal and weight is stable over time. Body mass may not reflect changes in energy stores in MHD patients who cannot regulate water balance normally. Validity of dietary data was evaluated by comparing reported EI with actual dietary EI (aEI) in 13 MHD patients participating in a 93‐day N balance study in a research ward to determine protein needs. Resting energy expenditure (REE) was measured prior to admission to estimate total energy expenditure (TEE) and dietary needs to maintain stable weight. Protein intake was changed ~ every 19 days; aEI was constant throughout the study. Total energy requirements were estimated by aEI corrected for changes in body fat measured by DXA over the 93 day study. Underreporting of EI was determined by a EI:REE ratio <1.27 and EI:TEE ratio<1.0.

Age was 47.9±9.7 years; BMI, 25.7 ± 4.7 kg/m2.

Self‐reported Intake
REE (kcals/d ay)
TEE (kcals/d ay)
EI: RE E
EI: TEE

kcals/d ay
kcals/kg
Protei n (g/kg)

1712±4 98
25.4± 7.4
1.03± 0.3
1676±3 31
2134±3 54
1.0 ± 0.2
0.8±0 .2

Body mass and fat were essentially unchanged (−0.87 kg ± 2.02 kg; 0.74 kg ±1.62 kg) during the study suggesting that pre‐study EI was underreported by ~20%. Variance of reported protein and EI was twice that of aEI. Widespread energy underreporting severely compromises the validity of self‐reported, interview‐assisted diet records in MHD patients.
A transmurally and regionally heterogeneous fully coupled computational electro‐mechanics model of the canine left ventricle
Authors: Aguado‐Sierra Jazmin , Campbell Stuart G , Howard Elliot , Omens Jeff H , McCulloch Andrew D , Kerckhoffs Roy CP
Published: June 2021

The cardiac cell structure and function is heterogeneous. These heterogeneities are finely orchestrated to optimize the whole organ function in health. A computational model is used to analyze the stress, strain and haemodynamic effects of including a transmurally (endo, mid and epi) and regionally (apex to base) heterogeneous substrate to a fully coupled model of electromechanics in an idealized ventricular geometry. Campbell et al. (2009) developed a canine myocyte ionic current and cytosolic Ca2+ cycling model that was integrated to a myofilament model. This mathematical model was embedded element‐wise into a finite element mesh of an idealized geometry with fiber angles and haemodynamic boundary conditions to understand the effects of cell heterogeneity on systolic fiber mechanics and ventricular function.

Regional variation of action potential duration is mainly due to differences on the density of membrane ionic currents like IKs and Ito due to the regional heterogeneous density of KChIP2, Kv1.4, KvLQt1 and MinK. Multi‐scale computational models are useful to understand why such heterogeneities are important and the way in which they impact the whole organ function.
Stimulation of NFAT‐dependent transcription activity by agonists and hypoxia in pulmonary arterial smooth muscle cells
Authors: Yang Xiao‐Ru , Paudel Omkar , Sham James S.K.
Published: June 2021

Nuclear factors of activated T‐cells (NFATs) are Ca2+‐sensitive transcription factors regulated by calcineurin, and play critical roles in inflammation and cell proliferation. Several previous studies showed that calcineurin/NFAT pathway is involved in the pathogenesis of pulmonary hypertension. We have found recently that NFATc3 is predominantly expressed in rat pulmonary arterial smooth muscle cells (PASMCs), and hNFATc3‐EGFP fusion protein translocates to the nucleus upon stimulation in rat PASMCs. In this study, NFAT‐dependent transcription activity was further examined in primary cultured rat PASMCs using a 4xNFAT‐luciferasereporter. NFAT‐dependent luciferase acitivity was enhanced significantly by ionomycin+PMA, PDGF, ET‐1, and hypoxia (2% O2 for 24 hr). The stimulatory effects of PDGF and hypoxia could be blocked by SKF‐96365 and nifedipine, respectively. Moreover, the effects of ionomycin+PMA and PDGF were further potentiated in PASMCs exposed to hypoxia. Since many agonists and growth factors are involved in hypoxic pulmonary hypertension, these observations suggest that hypoxia may interact synergistically with agonists to enhance NFAT‐dependent gene transcription in PASMCs. (Supported by HL0771835, HL075134)
Estrogen alleviates enhanced airway reflex reactivity induced by increased lung reactive oxygen species in female rats
Authors: Ruan Ting , Chong Wei‐Ya , Ko Pin‐Ju , Lin Tzer‐Bin , Kou Yu Ru
Published: June 2021

Enhanced airway reflex reactivity (EARR) and increased lung reactive oxygen species (ROS) are two features of asthma. A deterioration of asthma symptoms is associated with a declining estrogen level in females with unknown mechanisms. Sensitization of lung vagal C‐fiber afferents (LVCFAs) has been implicated in the pathogenesis of asthma. We investigated the influence of estrogen on the LVCFA‐mediated EARR induced by increased lung ROS in anesthetized female rats. Apneic response to intravenous capsaicin, a selective stimulant of LVCFAs, before and after spontaneous inhalation of aerosolized H2O2 (a major type of ROS; 0.05 % for 90 s) was measured as an index of airway reflex reactivity. We found that H2O2 inhalation induced a LVCFA‐mediated EARR in intact rats and this EARR was more prominent during the metestrus/diestrus (low estrogen) phase than during the proestrus (high estrogen) phase. In ovariectomized rats, subcutaneous 17β‐estradiol replacement, at a dose that mimics its level during the proestrus surge, suppressed the H2O2‐induced EARR. The suppressive effect of 17β‐estradiol was reversed by pretreatment with ICI 182,780, an estrogen antagonist. These results suggest that endogenous and exogenous estrogen may alleviate ROS‐induced EARR in female rats possibly via suppression of sensitization of LVCFAs (NSC 98‐2320‐B‐010‐016‐MY3 and 98‐2628‐B‐040‐011‐MY3).
Heat shock protein 90 inhibitors reduce airway inflammation in a mouse model of allergic asthma.
Authors: Dimitropoulou Christiana , Snead Connie , Gallops Jenna , Catravas John D
Published: June 2021

Narrowing of the airways, the cardinal feature of asthma, is frequently a result of airway inflammation that produces mucous hyper‐secretion and airway smooth muscle proliferation and migration, leading to airway remodeling. Heat shock protein 90 (hsp90) inhibitors are a new class of chemotherapeutics that have also been reported to possess strong anti‐inflammatory activity. Thus, the present study was designed to test the hypothesis that hsp90 inhibitors may ameliorate the airway inflammation associated with allergic asthma. Female BALB/c mice were subjected to a standard 25‐day regimen of saline (control mice) or ovalbumin (OVA) sensitization (asthmatic mice). During the last week of OVA treatment, mice also received a daily dose of either vehicle or of the hsp90 inhibitor, 17‐AAG (5mg/kg/day, intranasally). On day 26, mice were anesthetized and bronchoalveolar lavage (BAL) was performed. Asthmatic mice exhibited pronounced leukocytosis in the BAL fluid (BALF) compared to controls. This increase in BALF leukocytes was prevented in asthmatic mice treated with 17‐AAG. BALF cell differential analysis showed that, compared to vehicle‐treated asthmatics, a significant decrease in the number of neutrophils occurred in 17‐AAG‐treated asthmatic mice. Similarly, histological examination of lungs fixed by tracheal instillation, demonstrated less inflammation, less cell infiltration and reduced mucus production in the airways of asthmatic animals treated with 17‐AAG, as compared to vehicle‐treated asthmatic mice. These data suggest that hsp90 inhibitors may be useful in the management of allergic asthma.
Prenatal nicotine exposure alters intrinsic properties of neonatal hypoglossal motor neurons in the rhythmic medullary slice preparation
Authors: Wakefield Hilary Ellen , Pilarski Jason Q , Fuglevand Andrew J , Fregosi Ralph F
Published: June 2021

Hypoglossal motor neurons (HMNs) participate in a host of oromotor behaviors including swallowing and breathing. Although these neurons receive input from diverse neurotransmitter systems, of critical importance is the central cholinergic system, which participates in chemosensitivity and state changes. Prenatal nicotine exposure (PNE) reduces efficacy of nicotinic acetylcholine receptors and is associated with a variety of neonatal respiratory abnormalities. Therefore, the purpose of this study was to determine the effects of PNE on intrinsic properties of HMNs which mediate respiratory output to tongue muscles. We obtained rhythmic medullary slice preparations from P1–P4 rats exposed to saline (n = 6) or nicotine (n = 5) in utero, and we recorded from HMNs using whole cell patch clamp electrophysiology. Based on current clamp protocols applied in between respiratory bursts, we found that HMNs of PNE animals had altered firing profiles compared to HMNs of control animals. Specifically, PNE cells required 31% less current to elicit firing and had 49% higher initial frequency‐current gain values compared to control. This change in responsiveness cannot be explained by differences in input resistance. These data suggest that PNE augments HMN intrinsic excitability, which could be a result of neuroplasticity caused by chronic nicotine exposure. This study was funded by American Heart Association #0855713G.
Localization of hypothalamic neurons that contain orexin or melanin concentrating hormone peptides and regulate diaphragm activity in cats
Authors: Badami Varun M , Rice Cory D , Lois James H , Madrecha Jayesh , Yates Bill J
Published: June 2021

A variety of studies have shown that the lateral hypothalamus (LH) participates in controlling breathing. The LH also contains many neurons that each produce one of two peptides that modulate homeostasis and emotional states: orexin and melanin concentrating hormone (MCH). The present study tested the hypothesis that hypothalamic neurons that influence diaphragm activity contain either orexin or MCH. For this purpose, rabies virus was injected into the diaphragm of cats to transneuronally infect hypothalamic neurons with multisynaptic connections to phrenic motoneurons; immunohistochemistry was performed to dual‐localize rabies and either orexin or MCH. In animals with early rabies infection of LH, a large fraction of the infected hypothalamic neurons contained orexin: 19–75%, median of 30%. However, in animals with advanced infections where rabies had passed through additional synapses, only 8–10% of the infected cells were orexinergic. In contrast, just 6–33% (median 11%) of the early‐infected LH neurons were immunopositive for both MCH and rabies. These data show that an appreciable number of LH neurons with the most direct influences on diaphragm activity contain either orexin or MCH, although most of these cells are orexinergic. These findings therefore suggest that orexins play a central role in conveying signals from the diencephalon to brainstem and spinal regions that regulate breathing.
Pattern of Inspiratory Intercostal Muscle Activation during High Frequency Spinal Cord Stimulation (HF‐SCS)
Authors: Kowalski Krzysztof E. , DiMarco Anthony F.
Published: June 2021

Objective

HF‐SCS is a novel technique of inspiratory muscle activation involving spinal cord pathways. The purpose of the present study was to compare the distribution of motor drive to the intercostal muscles during spontaneous breathing with that occurring during HF‐SCS.

Methods

In 4 anesthetized dogs, electrical stimulation was applied via a stimulating electrode on the ventral surface of the upper thoracic spinal cord at the T2 level. Fine wire recording electrodes were used to assess single motor unit (SMU) pattern of activation of the parasternal (PA, 2nd interspace) and external [dorsal portions of the 3rd (EI‐3D) and 5th (EI‐5D) and ventral portion of the 3rd (EI‐3V) interspaces] intercostal muscles.

Results

Mean maximum discharge frequency of PA was 10.6±0.3 Hz. Mean maximum discharge frequency of EI‐3D (13.7±0.5Hz) was significantly greater than that of the EI‐3V (8.8±0.3Hz) and the EI‐5D (9.9±0.3Hz) (p<0.05). Importantly, these values were not significantly different than those occurring in the same animals during spontaneous breathing.

Conclusion

This study demonstrates that HF‐SCS results in the same differential pattern of activation of the various intercostal muscles as that which occurs during spontaneous breathing. Moreover, the neural circuitry resulting in physiologic activation of the rib cage muscles exists within the spinal cord.

Support: NIH‐NINDS (R01NS064157)
Astrocytes play an essential role in reversing respiratory disturbances in a mouse model of Rett syndrome
Authors: Bissonnette John , Lioy Daniel T. , Hirrlinger Petra G. , Kirchhoff Frank , Mandel Gail
Published: June 2021

Recent in vitro studies have shown that astrocytes lacking methyl‐CpG‐binding protein 2 (Mecp2) have a non‐cell autonomous effect on neuronal properties such as dendrite arborization and morphology. Here we show that activation of Mecp2 in astrocytes of symptomatic null animals restores a normal breathing pattern. Mice carrying a loxP‐stop cassette in intron 2 of the Mecp2 gene were crossbred to animals expressing a fusion protein of Cre DNA recombinase and a mutated estrogen receptor under the control of the human glial fibrillary acidic protein (GFAP) promoter. Male offspring are Mecp2 null, but the transcription factor can be restored in astrocytes by tamoxifen injections at any postnatal age. At P45 and older these mice developed apneas (~50/h) and an elevated irregularity score (>0.4). Within 10 days of completing tamoxifen apnea declined, by 3 weeks it reached wild type levels and irregularity improved (~0.25). Immunohistochemistry showed co‐localization of Mecp2 in GFAP‐positive cells in the preBötzinger complex of tamoxifen‐treated mice. The results indicate the important role of astrocyte Mecp2 that functions to control neuronal network activity essential for normal respiration.

Support: MOD & IRSF (JB); DFG & EC (FK); HHMI & NIH (GM)
Neural activation associated with chronic tracheal obstructions (TO) in conscious rats
Authors: Pate Kathryn M , Davenport Paul W
Published: June 2021

The neural pathways mediating respiratory load compensation in conscious rats are unknown. The current study determined brainstem and suprapontine neural activation associated with TO. TO were performed in conscious rats instrumented with an abdominal blood pressure telemetry probe, diaphragm electromyography electrodes, and a tracheal cuff. In experimental animals, the trachea was obstructed 60 times for 3–8 seconds per episode over the course of a 20 minute time period. This was repeated once a day for 10 days. Control animals were instrumented but received no obstructions. Animals were sacrificed after the 10th day of TO and brains were immediately removed, blocked, and placed in paraformaldehyde for later cytochrome oxidase (CO) staining. Respiratory‐related brainstem and suprapontine nuclei were stained intensely for CO in experimental animals. These results indicate that TO conditioning in conscious rats modulates respiratory brainstem and suprapontine discriminative and affective neural pathways.
Quantification of perfusion in mouse ovarian grafts for fertility preservation
Authors: Trujillo Anna , Kim Eujin , Theodoropoulos Catherine , Gosden Roger G
Published: June 2021

A serious side effect for some young women undergoing cancer treatment is premature ovarian failure and sterility. To preserve fertility ovarian cortex may be harvested, cryogenically preserved and re‐implanted in patients who return to full health. In the absence of vascular anastomosis, tissue is ischemic and the grafting will only be successful if there is rapid reperfusion to minimize loss of follicles. As a model of the clinical procedure, mouse ovaries were grafted to skeletal muscle and monitored non‐invasively using MicroMarker contrast agent and high‐frequency ultrasound (Vevo770, VisualSonics). Vascular perfusion and relative blood volume approached the levels of intact ovaries after seven days. The results demonstrate that microultrasound can monitor reperfusion in small ischemic grafts and is potentially useful for studying the effects of drugs and hormones as well as measuring the progress of angiogenesis. The study was funded by VisualSonics Inc.
The GABAA agonist muscimol encapsulated in liposomes as a tool for the chronic inhibition of the central nervous system
Authors: Silva Luiz Gonzaga , Oliveira Aline Cristina , Santos Robson Augusto , Frezard Frederic , Fontes Marco Antonio
Published: June 2021

The GABAA agonist muscimol is widely used as inhibitor of central nervous system (CNS). Although the effects of muscimol microinjections are well known, there is a lack of information regarding the long‐term effects of the inhibition of regions in the CNS. Liposomes are a potential tool for sustained release of short half‐life compounds. The aim of this study was to encapsulate the GABAA agonist muscimol into liposomes and evaluate the effect of chronic release of muscimol into the DMH on the inhibition of stress‐induced cardiovascular changes. Liposomes with encapsulated muscimol were prepared by the dehydration‐rehydration method. The lipids used were DSPC, cholesterol and PEG2000‐DSPE. Liposomes were injected into the DMH and rats were submitted to the air stress paradigm after 15, 180 and 420 minutes after microinjection. Empty liposomes and free muscimol were used as control. Microinjection of liposome‐entrapped muscimol was found to inhibit the tachycardia induced by air stress paradigm and this inhibition lasted for at least 420 minutes after microinjection (79, 92 and 71% of tachycardia inhibition after microinjection at 15, 180 and 420 minutes respectively). Free muscimol inhibited the air stress‐induced cardiovascular changes for only 15 minutes after microinjection. Empty liposomes were not able to block the tachycardia induced by the air stress paradigm. Muscimol‐containing liposomes produced a long‐term attenuation in the cardiovascular response to emotional stress in rats. This technique will emerge as a potential tool for the chronic intervention on the central nervous system with the neurotransmission mediated by GABA and its agonists. Support: Fapemig/CNPq
A flow chamber to study the performance of artificial heart valves
Authors: Yin Wei , Ngwe Ching , Rubenstein David A.
Published: June 2021

The goal of this study was to build a flow chamber to study the hemodynamic performance of a novel artificial heart valve made from cross‐linked aerogels. This low density new material has a significantly improved mechanical strength and a promising biocompatibility with the cardiovascular system. The housing of the flow chamber was made of Teflon and clear PVC. Two aerogel heart valves were fixed opposing in the chamber, to control the flow direction. Through a polyurethane diaphragm, flow was driven by a reciprocating pump. The flow rate of the chamber was fixed at 5L/min with a stroke volume of 80 mL and stroke rate of 72 min−1. The systolic/diastolic ratio was 0.375. The performance of the system was examined using whole blood, platelet rich plasma (PRP) and washed platelets, under static and dynamic conditions. Hemolysis was measured with whole blood using a spectrophotometer. Platelet activation was measured by platelet surface P‐selectin expression after PRP contact with the system. Platelet thrombogenecity was examined using a prothrombinase assay after washed platelets were placed into the chamber. Preliminary results indicate that the flow chamber does not induce hemolysis or platelet activation under static conditions, while its dynamic performance depends on the valves used. These results indicate it is feasible to use the flow chamber to study the hemodynamic performance of artificial heart valves.
Quantitative analysis of oxygen transport and cellular metabolism in working skeletal muscle
Authors: Lai Nicola , Spires Jessica , Grassi Bruno , Gladden L Bruce , Saidel Gerald
Published: June 2021

Oxygen uptake dynamics of skeletal muscle (VO2m) in response to exercise slow with aging and disease states. Whether O2 delivery or metabolic changes are limiting the VO2m response has not been quantitatively distinguished. Previous humans studies found that intracellular O2 (iPO2) reached a plateau at 60% of the maximal work rate, while another study found iPO2 linearly related to work rate. Based on these apparent contradictory results, there are alternative hypotheses relating muscle work rate to O2 diffusion rate from capillary to myocytes. In this study, mechanisms regulating VO2m response to muscle contraction were analyzed by computational modeling and simulation of experimental data. A mechanistic mathematical model was developed to simulate O2 transport and cellular metabolism in skeletal muscle. The metabolic processes include anaerobic glycolysis, which plays an important role in ATP homeostasis at higher intensity exercise. The model was validated by comparison of simulated outputs to VO2m dynamics from canine muscle electrically stimulated in situ under various conditions (O2 content, blood flow, and stimulus intensity) to mimic human exercise. This model quantitatively related iPO2 changes to increases of ATP demand under different experimental conditions. This analysis suggests that VO2m response to high intensity exercise is limited by O2 diffusion rather than metabolic processes. Research Support: NIH‐NIDDK, GM088823‐012 and NASA, NNJ06HD81G.
Muscle ring finger‐1 (MuRF1) inhibits spontaneous cardiac hypertrophy induced by the PPARα agonist fenofibrate in vivo
Authors: Willis Monte S , Li Luge , Schisler Jonathan C. , Lockyer Pamela , Patterson Cam
Published: June 2021

The cardiac ubiquitin ligase MuRF1 specifically binds with and inhibits PPARα activity in vitro. To determine the role of MuRF1 in regulating PPARα activity in vivo, MuRF1 −/− and wild type control mice (N=16) were fed the PPARα agonist fenofibrate 0.5% w/w in chow (or sham diet) for 3 weeks. Fenofibrate has been utilized therapeutically to induce cardiac hypertrophy regression in both human and animal models. After 3 weeks of treatment, MuRF1 −/−, but not wild type controls, developed a spontaneous cardiac hypertrophy as evidenced by a 45.2% increase in MuRF1 −/− left ventricular mass/tibia length (8.7 +/− 0.2 vs. 6.0 +/− 0.5 mg/mm) and a 20% increase in both anterior and posterior cardiac wall thickness. M‐mode determination of systolic function in MuRF1 −/− hearts (FS% 53.1+/−1.6) did not identify differences from wild type or baseline cardiac. MuRF1 −/−, but not wild type mice, had increased levels of βMHC (~4 fold), BNP (~1.5 fold), and smooth muscle α‐actinin (~2 fold) by Real Time PCR after fenofibrate treatment, suggesting that pathologic cardiac hypertrophy was induced in MuRF1 −/− hearts. In the absence of MuRF1, enhanced PPARα signaling leads to a spontaneous pathologic cardiac hypertrophy. These findings identify for the first time the interplay between MuRF1, PPARα, and signaling pathways that mediate pathologic cardiac hypertrophy in vivo, giving insight into PPARα's role in regulating cardiac hypertrophy.
Mice Heterozygous for the Lipid Transporter ABCA3 Have Increased Susceptibility to Acute Lung Injury
Authors: Savani Rashmin C. , Liao Jie , Mija Dan , Al‐Saadi Rajiha , Cheong Naeun
Published: June 2021

Mice with targeted deletion of the lipid transporter ABCA3 have surfactant deficiency and develop lethal neonatal respiratory failure. In contrast heterozygous ABCA3+/− mice have altered lamellar bodies, but no respiratory difficulties and survive the perinatal period. Since subtle lamellar body abnormalities might precipitate surfactant deficiency under stressed conditions, we hypothesized that ABCA3+/− mice would have greater susceptibility to acute lung injury, and we examined their response to intratracheal (IT) bleomycin injury. Eight to twelve week old ABCA3+/− and WT mice were given either 0.25U/kg bleomycin IT or sterile saline as a control. ABCA3+/− mice had 40% mortality between d10 and d21, whereas WT mice had no mortality. IT bleomycin caused an earlier and greater (d3–d7) increase in respiratory rate and decrease (PN7‐10) in body weight in ABCA3+/− mice. Lavage hyaluronan (HA), a lung injury biomarker, was increased in ABCA3+/− mice, and significantly correlated with the change in body weight (r2=0.89, p<0.01). We conclude that ABCA3+/− mice are more susceptible to lung injury than WT controls. These findings are relevant to humans with ABCA3 mutations that develop chronic lung disease.
NOX2 OXIDASE AS A THERAPEUTIC TARGET IN INFLUENZA A VIRUS‐INDUCED LUNG INJURY
Authors: Selemidis Stavros , Stambas John , Drummond Grant , Vlahos Ross
Published: June 2021

Continuing threats of new pandemic strains of influenza A virus highlight an urgent need for novel pharmacological strategies that reduce viral replication and the lung pathology, irrespective of the infecting strain. The pathology to pandemic influenza consists of a persistent infiltration of inflammatory cells into the airways leading to excessive reactive oxygen species (ROS) production. We investigated the primary source of inflammatory cell ROS, Nox2 oxidase, as a pharmacological target against influenza A viruses of varying virulence. Wild‐type (C57BL/6) and Nox2−/− mice were infected with influenza A virus of mild (H3N2; 104 PFU/mouse) or high (H1N1; 50 PFU/mouse) virulence. Lungs of Nox2−/− mice displayed a substantial reduction in viral titer, airways inflammation and oedema compared to WT mice. Airway macrophages, neutrophils and T cells of Nox2−/− mice produced minimal superoxide compared to controls. The magnitude of airway and spleen influenza‐specific DbNP366+ and DbPA224+ CD8+ T cells were similar in WT and Nox2−/− mice indicating that the adaptive immune response that clears virus is preserved in Nox2−/− mice. In vivo treatment with the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide following H3N2 or H1N1 infection. In conclusion, Nox2 inhibition should be considered for seasonal and pandemic control of influenza A virus.
Mice With Targeted Deletion of Receptor for Hyaluronan‐Mediated Motility (RHAMM, CD168) Are Protected From Bleomycin‐Induced Lung Injury
Authors: Savani Rashmin C. , Cheong Naeun , Mitchell Jason , Al‐Saadi Rajiha , Liao Jie
Published: June 2021

Acute lung injury results in inflammation, Surfactant Protein B (SPB) deficiency and respiratory distress. Hyaluronan (HA) and its receptor RHAMM have been implicated in the inflammatory response to lung injury. We hypothesized that RHAMM KO mice would be protected from bleomycin‐induced lung injury. Littermate wild type (WT) and RHAMM KO mice were treated with IT 1U/kg bleomycin in 25μl saline or with 25μl saline alone. IT bleomycin in WT controls resulted in decreased body weight, and increased respiratory rate by d7‐10. RHAMM KO mice given IT bleomycin had significantly less weight loss and respiratory distress than WT mice. Compared to injured WT animals, RHAMM KO mice had lower lavage concentrations of HA, as well as lower neutrophil and macrophage enzyme activities. In injured WT mice on d7, western blot for SPB showed decreased expression of the mature 16‐kDa dimer, but no change in SPB expression was seen in injured RHAMM KO mice. We conclude that RHAMM KO mice have less inflammation, SPB deficiency and respiratory distress after acute lung injury. We speculate that these data are important for future development of novel therapeutic approaches to limit the severity of lung disease.
Transglutaminases locate to calcified areas in advanced human atherosclerotic lesions
Authors: Matlung Hanke , Groen Harald C , de Vos Judith , van Walsum Theo , van der Lugt Aad , Niessen Wiro J , Wentzel Jolanda J , van Bavel Ed , Bakker Erik NTP
Published: June 2021

We investigated the involvement of the two major transglutaminases (TGs), tissue transglutaminase (TG2) and factor XIIIa (FXIIIA), in human atherosclerotic calcification.

Methods

Cross‐sections of human carotid atherosclerotic plaques were stained for TG2, FXIIIA, the TG‐induced cross‐link and macrophage marker CD68. The correlation of these staining patterns to calcified areas throughout the specimens was tested by multi‐scale 3D mapping of the microscopic images to μCT images.

Results

The TG‐induced cross‐link was found in 64 ± 8% (mean ± SEM) of the analyzed areas inside the plaques. Calcified areas correlated with this cross‐link (p<0.001). FXIIIA was found to be the dominant transglutaminase (56 ± 10% of the cross‐link‐positive areas), as compared to 11 ± 3% for TG2. Both transglutaminases correlated with the cross‐link (p=<0.001 and p=0.002 respectively). Staining for FXIIIA correlated with CD68 (p<0.001) on tissue level and also co‐localized with CD68 positive cells as shown by double immunofluorescent staining.

Conclusion

Areas of calcification locate to the presence and activity of transglutaminases in human atherosclerotic arteries. FXIIIA is dominantly present over TG2 in these plaques and may be derived from local macrophages.

Supported by the Netherlands Heart Foundation (NHS 2001T038).
Adeno‐associated Virus 9 Vector in the Gene Therapy of Occlusive Vascular Diseases
Authors: Agrawal Devendra Kumar , Pankajakshan Divya , Del Core Michael , Pipinos Iraklis , Hatzoudis George
Published: June 2021

We analyzed the efficiency and duration of transfection of AAV9 vector containing SM22α promoter with either eGFP or LacZ as reporter genes. The vectors (1 × 1012 pfu) were incorporated into coronary and carotid arteries of swine using microporous balloon catheter‐based gene delivery approach. Segments from all vessels were excised at various time points and immediately frozen for mRNA/protein expression or were processed for frozen sections for X‐Gal, GFP or H&E staining. GFP was visualized distinctly in the medial layer. Frozen sections were stained and β‐galactosidase gene transfer was assessed by blue‐stained cell nuclei. The endothelial and adventitial layers were carefully scraped off from the harvested vessel and total RNA was isolated from the remaining medial layer for qPCR. In the medial layer of coronary and femoral arteries LacZ mRNA expression was visualized 7 days after vector administration. The GFP mRNA expression was detected at 7 days and lasted for at least 2 months showing the prolonged expression of the AAV9‐vector. The GFP expression was confirmed by Western blot. Control arteries did not show any expression of GFP or LacZ. These findings support the use of AAV9 as a vector to effectively transduce a gene in coronary arteries.

(Supported by LB692 Nebraska Tobacco settlement Funds and NHLBI‐sponsored Penn Vector Core, University of Pennsylvania)