bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological Profile of BIA 10‐2474, a Novel FAAH Inhibitor, in the Rat
Authors: Bonifacio Maria‐Joao , Aires Cátia , Loureiro Ana Isabel , Moser Paul , Soares‐da‐Silva Patrício
Published: November 2023

BIA 10‐2474 (3‐(1‐(cyclohexyl (methyl)carbamoyl)‐1H‐imidazol‐4‐yl)pyridine 1‐oxide) is a novel fatty acid amide hydrolase (FAAH) inhibitor. In this study, the pharmacodynamic effects of BIA 10‐2474 in the rat were evaluated as well as its interaction with FAAH.

The interaction of the compound with the enzyme was evaluated by determining the potency of the compound in inhibiting rat brain FAAH in vitro at various pre‐incubation periods. The IC50 for BIA 10‐2474 decreased with pre‐incubation time, from 1134 (745; 1726) nM with no pre‐incubation to 400 (265, 488) nM with 1 h pre‐incubation, suggesting a time‐dependent interaction with the enzyme.

The pharmacodynamic effects of BIA 10‐2474 in Wistar rat were explored by evaluating hepatic and brain FAAH activity and levels of fatty acid amides in the brain. BIA 10‐2474, at 0.1 mg/kg, completely abolished hepatic FAAH activity in the first 8 hours post‐administration, with the enzyme slowly recovering activity and reaching 79 % control values after 6 days. The ED50 determined at 1 h was 5 (4; 7) μg/kg. In the brain, the effect of BIA 10‐2474 was less marked at 1 h with an ED50 of 96 (36; 255) μg/kg. At a dose of 0.1 mg/kg the maximum inhibition of brain FAAH (92 % inhibition) was achieved at 8 h post‐administration with the enzyme slowly recovering activity and reaching 73 % control levels after 6 days.

A PK/PD study in the rat using 10 mg/kg BIA 10‐2474 showed that anandamide (AEA), palmitoeylethanolamide (PEA) and oleoylethanolamide (OEA) levels peaked at 4 h (respectively 75.10±14.2 pmol/g, 4.8±0.5 nmol/g and 2.1±0.2 nmol/g) and slowly declined up to 24 h. The Cmax of BIA 10‐2474 was achieved at 1 h both in plasma (4266±352 ng/ml) and brain (840±51) and, at 8 h levels were almost undetectable in brain and had decreased to 278±60 ng/ml in plasma.

In conclusion, these data show that BIA 10‐2474 acts as a time‐dependent FAAH inhibitor, with a potent inhibitory effect upon FAAH and prolonged action in vivo and modulates brain AEA, PEA and OEA levels.
Long Term Treatment with Empagliflozin Attenuates Renal Damage in Obese Zucker Rat
Authors: Ginjupalli Gautam kumar , Manne Nandini D. P. K , Rice Kevin M , Blough Eric R
Published: November 2023

Empagliflozin, a known sodium‐glucose cotransporter type 2 (SGLT2) inhibitor is currently prescribed as an antihyperglycemic drug in adult Type 2 diabetic patients. Empagliflozin reduces hyperglycemia by promoting urinary excretion of glucose through inhibition of SGLT2 in proximal renal tubules. Data from the three year Empa‐Reg‐Outcome clinical study demonstrated that empagliflozin treatment was associated with improvements in blood glucose levels, decreased A1C, improved kidney function and significantly lower rates of CV death, all‐cause mortality and hospitalization for heart failure. In an effort to better understand the mechanistic basis of these findings, we investigated the long term effects of empagliflozin on kidney function in the diabetic Obese Zucker animal model. Obese Zucker rats were randomly assigned to one of four different groups; lean control, lean treated, obese control, and obese treated. The treated animals received empagliflozin (10 mg/kg body weight) for 25 weeks. Preliminary results suggest that treatment was associated with significant reductions in blood glucose and in the urinary levels of NGAL and Cystatin C. Consistent with these data, histological examination of renal sections from the obese control and treated animals demonstrated that empagliflozin treatment attenuated the loss of brush border, proximal tubule dilatation and glomerular sclerosis. Taken together, our findings suggest that the long term use of empagliflozin in the diabetic Obese Zucker rats is associated with improvements in glucose tolerance, attenuation of renal damage and improved kidney function.

Support or Funding Information

The study was funded by Boehringer Ingelheim to Eric R. Blough
Resveratrol‐Mediated Repression of a Liver Cancer Cell Line
Authors: Zhang Baochun , Lakshmanan Jaganathan , Motameni Amirreza , Harbrecht Brian G
Published: November 2023

Among the polyphenols, resveratrol (trans‐3,5,4′‐trihydroxystilbene) is a natural product with potential for the treatment of several diseases, including cancer. The aim of present study was to investigate the ability of resveratrol (RES) to inhibit HepG2 liver cancer cell growth, and examined the underlying mechanisms.

HepG2 cells were cultured in MEM with 5% FBS, the cells were treated with vehicle, 50 micro Molar, or 100 micro Molar of RES for 48 hours. Cell viability measured by MTT ((3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) stain were decreased approximately 26% and 32% respectively among the cells treated with RES. To understand whether RES – induced cell death and/or cell proliferation contributed the loss of cell viability, Bromodeoxyuridine (BrDU) and apoptosis gene screen assay were performed. HepG2 cells treated with 50 micro Molar and 100 micro Molar RES exhibited a marked decrease in BrDU incorporation rate (18% and 5%); indicating that suppressed cell proliferation played a major role of the effects of RES on HepG2 cell viability. Multi‐Target apoptosis paths scan showed that 100 micro Molar RES induced a significantly increase of both phosphorylated‐ and total‐p53 protein level. Meanwhile, no significant changes were observed on level of dephosphorylated and total Bad, cleaved caspase 3, and cleaved PARP on cells treated with RES.

In this study, we found that RES induced inhibition of HepG2 cell viability by suppressing their proliferation. Increased phosphorylation of p53 may contribute the effect of RES and may trigger apoptosis at long time. Thus, these findings suggest that RES has potential as a candidate agent against human liver cancer.
A Rat Model of Ischemic Enteritis: Pathogenic Importance of Enterobacteria, iNOS/NO, and COX‐2/PGE2
Authors: Takeuchi Koji , Komatsu Yoshino , Kotani Tohru
Published: November 2023

We investigated the roles of prostaglandins (PGs), nitric oxide (NO) and enterobacteria in a model of ischemic enteritis. Under ether anesthesia, ischemic enteritis was induced in 18 h‐fasted rats by a stenosis in the superior mesenteric artery (SMA) that was produced by placing a needle (23 guage) on the vessel, ligating both the vessel and needle, and then removing a needle from the ligature. After operation, the animals were fed normally and killed 3 days after surgery. Various drugs were given once daily for 2 days after the operation. Stenosis of the SMA produced hemorrhagic enteritis on the anti‐mesenteric side of the small intestine. The occurrence of enteritis was accompanied by enterobacterial invasion and the upregulated expression of iNOS/NO as well as COX‐2/PGE2 in the mucosa. The enteritis was prevented by ampicillin, L‐NAME or aminoguanidine, while aggravated by indomethacin, rofecoxib or an EP4 antagonist. The deleterious effect of indomethacin was antagonized by the co‐administration of PGE2 or an EP4 agonist. These results suggest that enterobacteria played a major pathogenic role in this model of ischemic enteritis, and also that iNOS/NO was deleterious in the pathogenesis of these lesions, while COX‐2/PGE2 prevented the development of ischemic enteritis by activating EP4 receptors.

Support or Funding Information

none
Omega‐3 polyunsaturated fatty acids rescued from indomethacin‐induced gut damages via GI mucosal membrane stabilization of lipid raft organization
Authors: Han Young‐Min , Park Jong‐Min , Kang Jing X , Hahm Ki Baik
Published: November 2023

Non‐steroidal anti‐inflammatory drugs (NSAIDs) damage GI epithelial cell membranes by inducing several signals through lipid raft organization after membrane incorporation, whereas ω‐3 polyunsaturated fatty acids (PUFAs) can relieve inflammation, reduce oxidative stress, and provide cytoprotection, consequent to lipid raft disorganization. Therefore, we hypothesized that ω‐3 PUFAs can protect the GI from NSAID‐induced damages by initiating the gatekeeper action of cell membranes, subsequent to anti‐inflammatory and anti‐oxidative actions. Administration of indomethacin (IND) leads to the formation of lipid rafts and activation of caveolin‐1; however, no such observations were made upon co‐administration of eicosapentaenoic acid (EPA) and IND. In addition, the EPA‐induced lipid raft disorganization, caveolin‐1 inactivation, and cellular cytotoxicity were inhibited when target cells were knocked‐out using GPR120. EPA significantly attenuated IND‐induced oxidative damage and apoptosis. IND administration induced significant ulceration, bleeding, and oedema in the stomach or small intestine of wild‐type (WT) mice; whereas GI mucosal damages significantly decreased in fat‐1 transgenic (TG) mice (p < 0.001), accompanied with significantly decreased cyclooxygenase‐2 expression and apoptosis, decreased interleukin‐1b and FAS concentrations, and increased HO‐1 concentration. Among common sources of plant oils containing the ω‐3 PUFAs ALA fatty acid include walnut which also showed that walnut‐phenolic‐extract had similar effects in IND‐induced gastrointestinal damage via anti‐oxidant and anti‐inflammatory actions. Our study indicates that the gatekeeper function of ω‐3 PUFAs improves GI safety when administered with NSAID.
β‐Adrenergic Function is Attenuated in Double Transgenic Mice Overexpressing 5‐HT4 Receptors and PP2A in the Heart
Authors: Gergs Ulrich , Gerigk Timo , Wache Hartmut , Neumann Joachim
Published: November 2023

Serotonin (5‐HT) can exert positive inotropic and chronotropic effects in humans via 5‐HT4 receptors. We have generated transgenic mice (5‐HT4) which overexpress the human 5‐HT4 receptor specifically in cardiomyocytes via the α‐myosin heavy chain promoter. In these mice, but not in wild type mice (WT), heart rate and ejection fraction (EF) are increased by 5‐HT. We crossed these mice with mice overexpressing the catalytic subunit of protein phosphatase 2A (PP2A) leading to double transgenic mice (5‐HT4 × PP2A = DT). In PP2A mice alone, reduced cardiac protein phosphorylation, cardiac hypertrophy, impaired cardiac contractility, and a diminished response to β‐adrenergic stimulation were noted compared to WT littermates. Aim of the present study was to investigate whether 5‐HT signaling is able to rescue the attenuated β‐adrenergic response in the PP2A overexpressing heart. PP2A and DT mice exhibited elevated levels of the catalytic subunit of PP2A (=PP2Ac) in Western blotting of cardiac samples compared to WT and 5‐HT4. In vivo, 5‐HT (100 μl of 10 mM i.p.) increased EF (via echocardiography) in 5‐HT4 from 60.7 ± 2.1 % to 97.0 ± 2.2 % (p < 0.05, n = 8) and in DT from 39.7 ± 2.3 % to 61.7 ± 5.5 % (p < 0.05, n = 9) but not in WT (69.5 ± 2.8 % versus 77.5 ± 4.1 %, n = 7) and PP2A (40.0 ± 3.0 versus 45.3 ± 3.1 %, n = 8). As expected from previous studies, basal EF was reduced in PP2A compared to WT. Interestingly, the diminished basal contractility caused by PP2A overexpression is still preserved in DT. Moreover, β‐adrenergic stimulation by isoproterenol (100 μl of 1 mM i.p.) increased EF less in DT (to 67.1 ± 6.1 %, n = 9) than in WT (to 98.7 ± 0.8 %, p < 0.05, n = 7) or in 5‐HT4 (to 97.8 ± 1.2 %, p < 0.05, n = 8). Relative heart weight was higher in all transgenic groups compared to WT. We tentatively conclude that β‐adrenergic function is compromised in DT like in PP2A and thus 5‐HT4 receptor overexpression does not rescue the phenotype of PP2A in this regard.
Cardioprotective Effects of the Ethanol Leaf Extract of Andrographis Paniculata in Isoproterenol‐induced Myocardial Infarction in Rats
Authors: Adedapo Adeolu Alex , Adeoye Bisi Olajumoke , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Yakubu Momoh Audu
Published: November 2023

Andrographis paniculata commonly known as king of bitters is an annual ‐ branched, erect ‐ running 1/2 to 1 meter in height. The aerial parts of the plant (leaves and stems) are used to extract the active phytochemicals. It grows abundantly in south eastern Asia: India (and Sri Lanka), Pakistan and Indonesia ‐ but it is cultivated extensively in China and Thailand, the East and West Indies, and Mauritius. Modern pharmacological studies have demonstrated its hepatoprotective, antithrombotic, anti‐inflammatory, immunostimulant, antimalarial, antihyperglycemic and cardioprotective properties. Because of its well‐known medicinal properties, this study was designed to evaluate the cardioprotective effect of the Nigerian species of Andrographis paniculata (AP) ethanol leaf extracts in isoproterenol‐induced myocardial infarction in rats using electrocardiogram, blood pressure changes as well as anti‐oxidant status.

Male rats were randomly divided into 7 groups of 7 animals per group. While group A served as control, group B was administered with Isoproterenol (ISO), group C was administered with Enalapril (10 mg/kg), but groups D, E and F were pretreated respectively with EEAP in three different doses (100, 200 and 400 mg/kg) orally for 7 days and group G was administered with 200 mg/kg EEAP post treatment with ISO. Myocardial infarction was induced intraperitoneally (i.p.) using isoproterenol (85 mg/kg) at interval of 24 h on 8th & 9th day in pretreated groups while on 1st & 2nd day in post treated group. The electrocardiogram, heart rate and blood pressure measurements were carried out on the 10th day in all groups and all animals were sacrificed afterwards and heart tissues were collected, homogenized, and centrifuged for anti‐oxidant studies.

ISO injection (85 mg/kg i.p.) produced changes in ECG pattern including prolonged QT interval and notches. ISO injection also produced a significant decrease in SAP, DAP and MAP levels. Although the extract did not amend the changes in ECG to normal but significantly increased the hemodynamics parameters. The extract increased antioxidant enzymes (SOD, GPX, GST, and GSH) and reduced oxidative stress markers (MDA using lipid peroxidation, H2O2, PT and NPT).

It could be concluded that the ethanol leaf extract of Andrographis paniculata exhibited cardioprotective effect in rats showing potent anti‐oxidant properties as well as improving the haemodynamic changes in the rats. This cardioprotective effect was similar to that of enalapril, an ACE inhibitor used in this study.

Support or Funding Information

National Research Foundation of the Tertiary Research Fund, Nigeria.
Heme oxygenase byproducts variably influences myocardial and autonomic dysfunctions induced by the cyclosporine/diclofenac regimen in female rats
Authors: Ibrahim Karim S , El‐Yazbi Ahmed F , El‐Gowelli Hanan M , El‐Mas Mahmoud M
Published: November 2023

We recently reported that simultaneous exposure to cyclosporine (CSA) and diclofenac (DIC) causes hypertension and left ventricular (LV) and arterial baroreceptor dysfunction in conscious female rats. In this study we tested the hypothesis that these CSA effects could be mitigated by facilitated heme oxygenase (HO) signaling. Experiments were performed in female rats to assess the effects of 10‐day treatment with the CSA (25 mg/kg/day)/diclofenac (1 mg/kg/day) regimen on cardiovascular functions in the absence and presence of pharmacologic maneuvers that upregulate HO or its enzymatic products. The hypertensive effect of CSA/DIC and associated shift in cardiac sympathovagal balance towards parasympathetic dominance (i.e. reduced low‐frequency/high‐frequency spectral ratio) were blunted upon concurrent treatment with hemin (HO‐1 inducer), tricarbonyldichlororuthenium (II) dimer (CORM‐2, carbon monoxide‐releasing molecule), or bilirubin. Whereas none of the latter treatments affected the CSA/DIC‐evoked decreases in the isovolumic relaxation constant (Tau, τ, a measure of cardiac diastolic function), the increases in LV contractility (dP/dtmax) in CSA/DIC‐treated rats were abolished by bilirubin only. Further, measurement of arterial baroreflex activity by the vasoactive method showed that bilirubin reversed the attenuated reflex bradycardia in contrast to no effect for hemin or CORM‐2. Paradoxically, the attenuated reflex tachycardia caused by CSA/DIC treatment was improved in presence of hemin and CORM‐2, but not bilirubin. All the favorable effects of hemin disappeared in rats treated simultaneously with the NOS inhibitor L‐NAME. These finding highlights key and NOS‐dependent modulatory roles for HO and the products of its enzymatic activity in improving the worsened cardiovascular profile in CSA/DIC‐treated female rats.

Support or Funding Information

Supported by the ALEX‐REP Grant Fund, Alexandria University, Egypt (Grant HLTH‐13‐01).
High Fat Feeding in Rats Alters Respiratory Parameters by a Mechanism that is Unlikely to be Mediated by Carotid Body Type I Cells
Authors: Rakoczy Ryan , Pye Richard L , Fayyad Tariq H , Santin Joseph M , Barr Barbara L , Wyatt Christopher N
Published: November 2023

The carotid bodies (CB), the primary peripheral chemoreceptors, respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin caused perturbations of intracellular calcium and membrane ion movement in isolated CB Type I cells (Pye et al, 2015) and blunted the response of the intact CB to hypoxia (Pye et al, 2016). This study's aim was to examine, in‐vivo, if elevated leptin modulated CB function and breathing.

Rats were fed high‐fat chow or control chow for 16‐weeks. High‐fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals (n=18; p<.001; 512.56 g ± 14.70 g vs. 444.11 g ± 7.09 g). HFF animals also had significantly higher serum leptin levels compared to CF (n=18; p<.0001; 3.05 ng/mL ± 0.24 ng/mL vs. 1.29 ng/mL ± 0.12 ng/mL). Whole‐body plethysmography was used to test the acute hypoxic ventilatory response (HVR) in unrestrained, conscious animals. HFF animals had an attenuated 2nd‐phase of the HVR when compared to CF (n=18; p<.05; 710.1 ± 41.9 mL kg−1 min−1 vs. 855.4 ± 44.05 mL kg−1 min−1). CB Type I cells were isolated and intracellular calcium measured; no significant differences in the cellular hypoxic responses between groups were observed.

These data show differences in the 2nd‐phase of the HVR caused by high fat feeding are unlikely to be caused by an action of leptin on the Type I cells. However the possibility remains that leptin may have in‐vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated.

Support or Funding Information

This work was funded by NIH grant RO1HL091836
Actin Polymerization Contributes to Augmented Basal Pulmonary Arterial Tone and Endothelin‐1‐Induced Vasoconstriction Following Chronic Hypoxia
Authors: Cross Laura Weise , Sands Michelle A. , Sheak Joshua R. , Snow Jessica B. , Gonzalez Bosc Laura V. , Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: November 2023

Chronic hypoxia increases pulmonary vascular resistance and promotes pulmonary hypertension through mechanisms that stimulate vasoconstriction and vascular remodeling. We have previously shown that chronic hypoxia augments pulmonary vasoconstrictor reactivity to endothelin‐1 (ET‐1) through reactive oxygen species generation and Rho kinase (ROCK)‐dependent Ca2+ sensitization.

Because ROCK promotes actin polymerization and the actin cytoskeleton is important for tension generation in smooth muscle, we hypothesized that actin polymerization is required for both basal pulmonary arterial tone and enhanced ET‐1 dependent vasoconstriction following chronic hypoxia. To test this hypothesis, we applied the actin polymerization inhibitors cytochalasin B (10−6 M) and latrunculin A (5 × 10−7 M) to isolated, endothelium‐disrupted pulmonary arteries from control and chronically hypoxic (4 wk at 0.5 atm) rats that were exposed to increasing intraluminal pressure, and we monitored the development of tone. Arteries from each group were also exposed to increasing concentrations of ET‐1 (10−10 – 10−7 M) in the presence and absence of cytochalasin B. Consistent with our hypothesis, inhibition of actin polymerization reduced pressure‐dependent tone and attenuated ET‐1‐induced vasoconstriction in arteries from chronically hypoxic animals, while having no effect on arteries from control rats.

To test whether chronic hypoxia directly alters the actin profile in pulmonary arteries, we measured filamentous (F) actin to globular (G) actin ratios by two methods: fluorescent labeling of F‐actin and G‐actin in fixed pulmonary artery segments, and actin sedimentation assays on homogenized pulmonary artery lysates. Results from both sets of experiments indicated that there is no significant difference in actin polymerization between pulmonary arteries from control and chronically hypoxic rats under baseline conditions, but that ET‐1 (10−8 M) stimulation significantly enhances actin polymerization in pulmonary arteries from rats exposed to chronic hypoxia. We conclude that actin polymerization contributes to pulmonary vasoreactivity following chronic hypoxia, under both pressure‐induced and ET‐1 stimulated conditions, but enhanced levels of actin polymerization following chronic hypoxia are only detected after stimulation with ET‐1.

Support or Funding Information

NIH R01 HL132883

AHA 16GRNT27700010

K12 GM088021

AHA 15GRNT25090039
PKCβ Signals Through Mitochondrial Reactive Oxygen Species Production in Pulmonary Arterial Smooth Muscle Cells of Neonatal Rats
Authors: Sheak Joshua R. , Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: November 2023

Chronic hypoxia (CH)‐induced vasoconstriction has been implicated in the pathogenesis of pulmonary hypertension in infants with chronic cardiorespiratory disorders. Although mechanisms of pulmonary vasoconstriction resulting from neonatal CH are not well understood, preliminary studies from our group support a role for both PKCβ and reactive oxygen species (ROS) in this response. However, neither the signaling relationship between PKCβ and ROS nor the cellular source of ROS involved in this response has been previously addressed. Considering the importance of PKCβ in mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, we hypothesized that PKCβ signals to induce mitochondrial ROS generation in neonatal pulmonary arterial smooth muscle cells. To test this hypothesis, we transiently cultured primary pulmonary arterial smooth muscle cells (PASMC) collected from neonatal normoxic control rats. The production of mitochondrial ROS was assessed using the mitochondrial selective fluorescent O2− indicator, mitoSOX (5 μM). We treated PASMC with PMA (10 μM), a phorbol ester known to activate PKC isoforms, including PKCβ, and observed PMA‐dependent increases in mitoSOX fluorescence that were prevented by selective PKCβ inhibition with LY‐333,531 (10 nM, p<0.05 vs. PMA alone, n=6 rats). Selectivity of mitoSOX for mitochondrial‐derived O2− was verified using the mitochondrial‐targeted O2− scavenger, mitoTEMPO (200 μM), which prevented PMA‐induced increases in mitoSOX fluorescence. Western blot analysis confirmed the expression of PKCβI and PKCβII splice variants in homogenates of isolated pulmonary arteries from neonatal normoxic control rats. We conclude that PKCβ stimulation leads to mitoROS production in PASMC from neonatal rats. Future studies are planned to evaluate the contribution of mitochondrial ROS to enhanced vasoconstrictor reactivity and associated pulmonary hypertension resulting from neonatal CH, and the cellular signaling mechanisms involved.

Support or Funding Information

Awarded to Thomas C. Resta, PhD: NIH R01 HL132883 and AHA 16GRNT27700010. Awarded to Joshua R. Sheak: NIH F31 HL131334.
Regulation of Endothelial Cell Barrier Disruption in Acute Lung Injury by Autophagy
Authors: Spencer Spencer , Leonard Antony , Grose Valerie , Fazal Fabeha , Rahman Arshad
Published: November 2023

Recent studies have implicated autophagy in many disorders including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remains uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of autophagy inhibitor 3‐methyladenine (3‐MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. Inhibiting autophagy by this approach also reduced the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Pretreatment of cells with 3‐MA inhibited thrombin‐induced barrier disruption by inhibiting the cleavage and loss of VE‐cadherin at adherens junctions. 3‐MA was also effective in protecting against thrombin‐induced actin stress fiber formation. Together, these data reveal a novel role of autophagy in causing EC barrier dysfunction and lung microvascular permeability associated with ALI.

Support or Funding Information

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL116632 and HL130870.
Methionine sulfoxide reductase as a neuroprotective mechanism in a model of anoxia tolerance: Trachemys scripta
Authors: Reiterer Melissa , Milton Sarah L.
Published: November 2023

The damaging effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) have been linked to several neurodegenerative diseases as well as aging and senescence. Mammals and especially humans are extremely susceptible to this damage as can be observed following an ischemic stroke when blood flow is restored and oxygen is reintroduced. ROS damage lipids, proteins, and DNA, and thus can induce further cell death beyond that caused by the ischemia. However, some organisms can withstand extended anoxia and reoxygenation without damage, including the freshwater turtle Trachemys scripta.

To cope with potential oxidative stress, the freshwater turtle possesses high endogenous levels of antioxidants and can also inhibit ROS production. One potential antioxidant mechanism is the Methionine Sulfoxide Reductase System (Msr) that catalyzes the reduction of oxidized Methionine (Met) residues. Methionine residues are easily oxidized amino acids; oxidized Met is associated with decreased protein activity and Msr may restore function to damaged proteins; it has also been shown to scavenge ROS and thus ameliorates cellular damage.

The present study investigated the role and regulation of MsrA in turtle neuronal cultures exposed to 4 hours of anoxia/4 hours of reoxygenation by altering the expression of MsrA. As FOXO3a has been shown to regulate MsrA levels in other animal models, we also induced its expression by transfecting a FOXO3a plasmid into the cells. We found that knock down of MsrA expression with target specific siRNA resulted in decreased cell viability; and induction of FOXO3a showed protection against cell death. Furthermore, treatment of cells with Epigallocatechin gallate (EGCG), an extract found in green tea that has antioxidant properties, results in increased expression of FOXO3a. These results suggest that MsrA may play an important role in protection against anoxia and oxidative stress in the turtle brain, and may prove to be a target to treat diseases of ischemia.

Support or Funding Information

Florida Atlantic University GRIP Grant (2015–2016)

John Nambu Scholarship for Molecular Biology (FAU‐ 2016)

Andrew Todd Auster Alumni Scholarship (FAU‐2016)
Comparative physiology of hypoxia tolerance in intertidal sculpins
Authors: Richards Jeffrey , Lau Gigi
Published: November 2023

The goal of our work is to characterize the underlying factors that result in variation in hypoxia tolerance among organisms. At that heart of hypoxia tolerance is the ability to effectively acquire and utilized environmental oxygen at the mitochondria to sustain aerobic function even when exposed to oxygen limitations. In order to elucidate the factors that explains variation in hypoxia tolerance among organisms, we use a well‐described, phylogenetically informed comparative system involving several species of marine fish from the family Cottidae (sculpins) that show a strong habitat zonation along the near‐shore environment. Sculpins from the intertidal zone experience daily fluctuations in O2 with species located in the higher intertidal oscillating between ~0 to 400% air saturation and are hypoxia tolerant, whereas subtidal species rarely experience hypoxia and are not as hypoxia tolerant. These interspecific differences in hypoxia tolerance is reflected in modifications to various steps in the oxygen transport cascade, whereby hypoxia tolerant sculpins show higher gill surface area, better hemoglobin O2 binding affinity, and an overall lower oxygen consumption rate. In the present study, we focus on the mitochondria and the terminal protein of the oxygen transport cascade, cytochrome c oxidase (COX), to determine if oxygen use by the mitochondria differs among sculpin species that vary in hypoxia tolerance. Hypoxia tolerant species showed higher COX oxygen binding affinity (measured as a lower apparent Km for O2) in both brain and liver, and a higher overall COX capacity. In order to provide a mechanistic hypothesis to explain the putatively adaptive changes in COX Km for O2, we performed in silico protein analyses of the deduced amino acid sequence of the mitochondrial‐encoded COX3 subunit, which is postulated as the entry point for O2 and protons into the COX protein. Our analysis suggests that variation in two amino acid residues (positions 55 and 224) on COX3 may affect cardiolipin binding and O2 movement, thus contributing to the variation in COX Km for O2. As such, we provide evidence for adaptive variation in COX function across sculpins that vary in hypoxia tolerance.

Support or Funding Information

This work was funded by an NSERC Discovery Grant to Jeffrey Richards.
Metabolic Rate and Hypoxia Tolerance Are Effected by Group Interactions and Sex in the Fruit Fly (Drosophila melanogaster): New Data and a Meta‐Analysis
Authors: Burggren Warren , Souder BriAnna , Ho Dao
Published: November 2023

Population density and associated behavioral adjustments are potentially important in regulating physiological performance in many animals. In r‐selected species like the fruit fly (Drosophila), where population density rapidly shifts in unpredictable and unstable environments, density‐dependent physiological adjustments may aid survival of individuals living in a social environment. Yet, how population density (and associated social behaviors) affects physiological functions like metabolism is poorly understood in insects. Additionally, insects often show marked sexual dimorphism (larger females). Thus, in this study on D. melanogaster, we characterized the effects of fly density and sex on both mass‐specific routine oxygen consumption (VO2) and hypoxia tolerance (PCrit). Females had significantly lower routine VO2 (~4 μl O2·mg−1·h−1) than males (~6 μl O2·mg−1·h−1) at an average fly density of 28 flies·respirometer chamber−1. However, VO2 was also inversely related to respirometer chamber fly density in males, with values ranging from 4 to 11 μl O2·mg−1·h−1 at a density of 10 and 40 flies·chamber−1, respectively (r2=0.58, P<0.001). Female flies showed a similar but less pronounced effect, with a VO2 of 4 and 7 μl O2·mg−1·h−1 at a density of 10 and 40 flies·chamber−1, respectively (r2=0.43, P<0.001). PCrit (~5.5 to 7.5 kPa) varied significantly with density in male (r2=0.50, P<0.01) but not female (r2=0.02, P>0.5) flies, with higher fly densities having lower PCrits. A meta‐analysis of ~30 metabolic studies on fruit flies revealed a similar inverse relationship between fly density and oxygen consumption. We conclude that future studies on insect metabolism should control for, and report on, both of these factors.

Support or Funding Information

National Science Foundation Grant IOS1543301
Oxytocin Receptors are expressed on Neurons within the Prefrontal Cortex that Control Preference for Social Novelty
Authors: Tan Yalun , Singhal Sarthak , Hiller Helmut , Nguyen Dan‐Tam , Colon‐Perez Luis M. , Febo Marcelo , Wang Lei , de Kloet Annette D. , Frazier Charles J. , Krause Eric G.
Published: November 2023

Oxytocin is a promising therapeutic for social impairments associated with neuropsychiatric conditions; however, the precise brain circuits influenced by oxytocin remain unknown. The prefrontal cortex (PFC), which exerts top‐down control over various subcortical regions, densely expresses oxytocin receptors (OTR) that may mediate the effects of oxytocin on social behavior. Here, we use the Cre‐LoxP system in mice with anatomical, electrophysiological, imaging and optogenetic approaches to discern how ‘oxytocin sensitive’ neurons in the PFC may influence social behaviors. Male mice with expression of Cre recombinase driven by the promoter for the OTR gene (OTR‐Cre mice) were delivered Cre‐inducible adeno associated virus producing channel rhodopsin‐2 and enhanced yellow fluorescent protein (AAV‐ChR2‐eYFP) into the PFC. Viral delivery revealed that 54% and 27% of OTR‐expressing neurons in the PFC were glutamatergic or GABAergic, respectively. Retrograde tract‐tracing, in conjunction with in vitro optogenetics, found that OTR‐expressing PFC neurons sent glutamatergic projections to the nucleus accumbens, bed nucleus of the stria terminalis and basolateral amygdala (BLA). Functional magnetic resonance imaging confirmed that in vivo photo‐stimulation of OTR‐expressing neurons evoked increased activation of the PFC and downstream subcortical regions including the BLA. To assess the function of OTR‐expressing PFC neurons, OTR‐Cre mice were administered AAV‐ChR2‐eYFP or control virus and chronically implanted with fiber optics targeting the PFC. Subsequently, mice were evaluated in the social interaction (SI) and social novelty paradigms, novel object recognition test, elevated plus maze (EPM) and open field (OF). In vivo optogenetic stimulation of OTR‐expressing PFC neurons abolished preference for social novelty (n=6 per group; Control: P<0.01; ChR2: P=0.4), but had no effect on anxiety‐like behavior in the EPM, OF, or SI test. Locomotor activity and novel object recognition were also not affected. Collectively, our results suggest that excitation of OTR‐expressing neurons in the PFC exerts top‐down control over brain circuits controlling preference for social novelty.

Support or Funding Information

National Heart, Lung, and Blood Institute Grant HL122494 (Eric G Krause); National Institute of Mental Health Grant MH104641 (Charles J Frazier)
Exposure to Homocysteic Acid Early in Postnatal Development Leads to a Mixed Depressive/Manic Behavioral Phenotype and Changes in NMDA Receptor Expression
Authors: Simko Stephanie , Johnson Jacob , Flores Guillermo , Barney Christopher C. , Chase Leah
Published: November 2023

Homocysteic acid (HCA), a NMDA receptor agonist, is an endogenous metabolite formed from the oxidation of homocysteine. Since hyperhomocysteinemia is a risk factor for several neuropsychiatric disorders, including bipolar disorder and major depressive disorder (MDD), we tested the hypothesis that elevated HCA levels in developing rats may induce alterations in NMDA receptor expression and the development of behaviors associated with MDD and/or bipolar disorder. Twenty postnatal male and twenty female rats were injected daily with either HCA or saline from day P3 to P21. HCA‐treated rats displayed increased risk‐taking behavior, reduced social behavior, novelty‐induced hyper‐locomotion, anhedonia in the saccharine preference test, and reduced spatial learning in the Morris water maze, consistent with a depressive state with manic tendencies. Interestingly, female rats were more sensitive to the effects of HCA than male rats. As expected, HCA treatment had no effect on motor coordination (Rotarod) or startle behavior (paired‐pulse inhibition). In addition to these behavioral changes, we observed that HCA led to an increase in expression of the GABAergic marker, GAD‐67, in the cortex, but not the hippocampus, of both male and female rats. We also observed a significant change in the NMDAR2b:NMDAR2a subunit expression ratio in the cortex. Specifically, HCA triggers an increase in the NMDAR2b:NMDAR2a ratio in male rats, while female rats exhibit a decrease in this ratio. We also observed that HCA triggered an increase in NMDAR2a expression in the hippocampus in both males and females. Finally, HCA also led to a decrease in NMDAR2b expression in females, but an increase in NMDAR2b expression males in the hippocampus. We are currently examining the effect of HCA exposure on NMDAR1 subunit expression. Collectively, these data suggest that early postnatal exposure to HCA may lead to a mixed manic/depressive phenotype that may also be accompanied by changes in GABAergic signaling in the cortex. Given the role that NMDA receptors are proposed to play in regulating GABAergic interneuron activity and the regulation of mood, we suggest that this may serve as a novel animal model for studies of complex mood disorders such as bipolar disorder or major depressive disorder.

Support or Funding Information

This research was supported by the Hope College Neuroscience Program, Biology Department and Chemistry Department.
NPY‐Induced Stress Resilience and Downregulation of HCN1 Channels Modifies Expression of Conditioned Fear
Authors: Bompolaki Maria , Rosenkranz J. Amiel , Colmers William F. , Urban Janice H.
Published: November 2023

The anxiolytic and stress‐buffering properties of neuropeptide Y (NPY) in the basolateral amygdala (BLA) have long been established in animal models as well as in humans. Repeated injections of NPY into the BLA promote long‐lasting resilience to restraint stress (Sadjyk et al., 2008), which is associated with downregulation of HCN1 channels. In addition to inducing stress resilience, we hypothesize that repeated injections of NPY and downregulation of HCN1 subunits in the BLA will ameliorate freezing behavior in a cued fear conditioning paradigm. Male rats received bilateral infusions of 10pmol NPY or vehicle (100nl; 0.1M PBS) into the BLA repeated daily for 5 days. Two weeks after the injections, animals underwent a cued fear conditioning protocol: On day 1, animals were placed in chamber 1 and received 4 pairings of a tone (1500Hz, 85dB) with a foot shock (0.4mA; fear acquisition). The following day the rats were placed in novel chamber 2 and received 15 trials of the tone without a foot shock at 60s intertrial intervals (fear extinction) and freezing behavior was monitored (Anymaze software). Both vehicle‐ and NPY‐treated rats displayed similar rates and amplitudes of fear acquisition on day 1. On day 2, NPY‐treated rats exhibited significantly lower freezing in the first five sessions of the extinction phase. Additionally, at the time point we performed the fear conditioning experiment, we observed an induction of LC3B expression (an autophagy marker) and downregulation of brain‐derived nerve factor (BDNF), however downregulation of the HCN1 protein is not seen until 4 weeks after the NPY treatment. Lastly, to test the involvement of HCN1 subunits in fear conditioning, HCN1 protein was knocked‐down in the BLA using lentiviral delivery of HCN1‐shRNA. Four weeks after the surgery, the rats underwent the same cued fear conditioning protocol as described above and freezing behavior was monitored. HCN1‐knockdown animals had reduced freezing on the acquisition day. This work indicates that the induction and maintenance of NPY‐induced stress resilience is associated with differential changes in protein expression. NPY‐induced resilience is correlated with BDNF downregulation and induction of autophagic pathways at early time points, while HCN1 downregulation later consolidates a new stress‐relieving baseline in NPY‐treated animals. These findings can be of great importance for the way anxiety disorders like post‐traumatic stress disorder are viewed and treated.

Support or Funding Information

Work supported by MH090292 (JHU; WFC)
Passive Leg Cycling Comprehensively Restores Cerebrovascular Health After Experimental High‐thoracic Spinal Cord Injury
Authors: Jia Mengyao , Phillips Aaron A. , Matin Nusrat , Yung Andrew , Zheng Mei Mu Zi , Lee Amanda , Monga Aaron , Kozlowski Piotr , Dorrence Anne , Krassioukov Andrei V.
Published: November 2023

Individuals living with chronic spinal cord injury exhibit a 3–4 fold increased risk of stroke. Reduced cerebral blood flow following is associated with cognitive decline, increased risk of stroke, and development of white matter irregularities. Previously, we have reported impairment in cerebrovascular health following experimental high‐thoracic spinal cord injury, including cerebrovascular reactivity declines, as well as inward remodelling and profibrosis of the middle cerebral artery. Passive hind‐limb exercise after spinal cord injury has been shown to restore systemic cardiovascular function, however it has never been explored for improving cerebrovascular health.

Objective

To preclinically investigate the effect of passive hind‐limb exercise in preventing cerebrovascular decline following high‐thoracic spinal cord injury.

Design/Method

Male Wistar rats (n=45) were uninjured, T3‐level complete transected or T3‐transected with passive exercise. Six days after surgery, the hind‐limb passive cycling intervention was initiated and lasted for five weeks. Six weeks after surgery, Magnetic resonance imaging was performed to evaluate baseline cerebral blood flow. Middle cerebral arteries were then harvested for functional and structural assessments, including in vitro pressure myography and immunohistochemistry.

Conclusion

There was a 44% reduction in global cerebral blood flow (p=0.001) after spinal cord injury, which was restored with passive exercise (Figure 1). Passive exercise also prevented the endothelial dysfunction seen after spinal cord injury (p=0.046). Profibrotic remodelling of middle cerebral arteries demonstrated by increased collagen (95%, p=0.004), was also reversed by passive exercise. As such, passive hind‐limb cycling is effective in restoring healthy brain blood flow, endothelial function, and preventing profibrosis in the cerebral vasculature. Clearly, passive exercise is a promising pre‐clinical therapy to restore cardiovascular health after spinal cord injury, and the present data extend these benefits even to the brain.

Support or Funding Information

Support: AAP: Killam Trust, Craig H. Neilsen Foundation Postdoctoral Fellowship 337427, Heart and Stroke Foundation of Canada Research Fellowship, Michael Smith Foundation for Health Research Postdoctoral Fellowship AVK: Canadian Institute of Health Research, Craig H. Neilsen Foundation.
Aortic Augmentation Index and Age in Persons with SCI and Intact Controls
Authors: Katzelnick Caitlyn G , Weir Joseph P , Chiaravalloti Nancy , Bauman William A , Wylie Glenn R , Dyson‐Hudson Trevor A , Wecht Jill M
Published: November 2023

Objective

Arterial stiffness (AS) has been identified as an independent risk factor for cardiovascular disease (CVD) progression. Increases in AS are associated with advancing age and resting blood pressure (BP) in the general population; however, these associations have not been described in the spinal cord injury (SCI) population. The objective of this investigation was to quantify the impact of age and seated systolic BP (SBP) on AS in persons with SCI by estimating aortic augmentation index (AI) compared to healthy controls.

Participants/methods

Three groups of age‐matched subjects (25–48 years) were recruited for study; 29 individuals with HIGH‐SCI (C3‐T5), 17 LOW‐SCI (T7–T12), and 27 age‐matched controls (CONT).

Design

Participants were asked to remain seated for instrumentation and five minutes of beat‐to‐beat SBP was assessed at the finger arteriolar using photoplethysmography (Finometer PRO, Finapres Medical Systems B.V., Netherlands). After the seated data collection, participants transferred to supine position and radial artery pulse wave analysis (SphymoCor, AtCor Medical) was used to estimate aortic AI, which was normalized to a heart rate of 75 bpm (AI@75). ANOVA models were used to determine group differences in AI@75, age and SBP and multiple regression models were constructed to determine the influence of age and seated SBP on aortic AI@75 among the groups.

Results

The groups did not differ for age; however the group main effect for AI@75 was significant (F=11.618; p<0.0001) suggesting that AI@75 was significantly increased in the HIGH‐SCI (19.6±9.3%) compared to the CONT (6.2±11.0%; p<0.0001) and the LOW‐SCI group (9.8±12.2%; p<0.001). The group by age interaction effect was statistically significant (F=3.188; p<0.05); follow‐up procedures using separate bivariate regressions between AI@75 and age were performed for each group. Age was directly related to AI@75 in CONT (F=67.46, r2=0.60; p<0.0001), but did not contribute to AI@75 in either the HIGH‐SCI (r2 <0.01) or LOW‐SCI groups (r2 <0.06). Seated SBP was significantly lower in the HIGH‐SCI (97.2±16.9 mmHg) compared to the CONT and LOW‐SCI groups (122.1±14.3 and 119.0±18.7 mmHg, respectively; p<0.0001), and there was a significant inverse association between AI@75 and SBP in the HIGH‐SCI group (F=5.703, r2=0.174; p<0.05).

Conclusion

These preliminary data suggest that, although age does not contribute to AS in individuals with SCI, level of lesion appears to influence vascular stiffness, which may be dependent on resting SBP. We speculate that the inverse association between AI and SBP in the HIGH‐SCI group may relate to an increased dependency on the renin‐angiotensin‐aldosterone system for orthostatic BP maintenance.

Support or Funding Information

Supported by the New Jersey Commission for Spinal Cord Research [Grant # CSCR13IRG018]