bluuebunny/crossref_metadata_2025 huggingface dataset:
Further characterization of the allosteric site of action of amiodarone at Gq‐linked muscarinic receptors (LB616)
Authors: Ellis John , Jayasuriya Gihan , Elmslie Gwendolynne , Burstein Ethan
Published: June 2021

We have previously reported that amiodarone interacts with a novel allosteric site on muscarinic receptors. Amiodarone’s most striking effect is to enhance the maximal response elicited by muscarinic agonists at the M1, M3, and M5 receptors. Furthermore, the quaternary analog N‐ethylamiodarone (NEA) is inhibitory at these receptors and appears to compete with amiodarone at that allosteric site. In the present studies, we show that dronedarone (DRON) also modulates Gq‐mediated responses, although in a more discriminating manner. For example, DRON markedly enhances pilocarpine‐stimulated release of arachidonic acid from CHO cells, via the M3 receptor subtype, but does not affect the acetylcholine‐stimulated response. In comparison to these effects at M3, DRON is strongly inhibitory toward both pilocarpine and acetylcholine at the M1 subtype. The effects of DRON are consistent with an interaction at the amiodarone site: DRON inhibits the enhancement of acetylcholine’s response produced by amiodarone at the M3 subtype; and, NEA reverses the enhancement of pilocarpine’s response at M3 produced by either DRON or amiodarone. In studies with the M1‐selective allosteric agonist AC260584, amiodarone enhanced the maximal response observed, whereas DRON was inhibitory. On the other hand, BQCA, the well‐known PAM that dramatically enhances the potency of acetylcholine at the M1 subtype, had no effect on the response profile of AC260584. In summary, DRON acts at Gq‐linked muscarinic receptors in a manner that complements amiodarone and provides an additional tool with which to investigate this novel allosteric site.

Grant Funding Source: Supported by PHS R01 005214 to JE
Prolonged Access to Cocaine Results in Distinct Epigenetic Changes in the Prefrontal Cortex (LB622)
Authors: Ploense Kyle , Carr Amanda , Baker‐Andresen Danay , Li Xiang , Woodward Nick , Sun Yi , Bredy Timothy , Kippin Tod
Published: June 2021

DNA methylation is a key determinant of gene expression and is implicated in neuroplasticity relevant to addiction. Here, we examine DNA methylation and mRNA expression for two genes, Homer2 and NPAS4, within the dorsal medial prefrontal cortex (dmPFC) following saline, short‐access (1h), or prolonged‐access (6h), to cocaine during self‐administration. As reported previously, only 6h rats exhibited escalated cocaine intake across sessions. Initial MeDIP‐CHIP analyses of dmPFC tissue by ChIPMonk software revealed dramatic and extremely gene‐specific changes in DNA methylation/demethylation within promoter regions and flanking the transcription start site (TSS). The Homer2 and NPAS4 promoters showed active changes in DNA methylation following 6h and 1h cocaine self‐administration compared to saline. A follow‐up analysis, via sodium bisulphite conversion followed by mass spectrophotometry interrogating a 1000bp sequence of the Homer2 promoter, confirmed increased methylation of CpG7 in the 6h rats, which contains putative consensus sequences for GATA‐1 and p300. Additionally, MeDIP followed by qPCR revealed an increase in DNA methylation for both 1h and 6h rats within the 457‐645bp upstream of the NPAS4 transcription start site (TSS).qPCR revealed an increase in Homer2 mRNA levels in the 6h versus 1h and saline conditions, and a decrease in NPAS4 mRNA in 1h and 6h versus saline conditions. These results demonstrate that prolonged cocaine self‐administration alters DNA methylation of the Homer2 and NPAS4 gene promoters within the dmPFC in a distinct manner. Additionally, the altered DNA methylation leads to decreases in NPAS4 mRNA and increases in Homer2 mRNA expression within the dmPFC. Given the known roles of Homer2 as a scaffolding protein for the metabotropic glutamate 1 and 5 (mGluR1/5) receptors and NPAS4 as a transcription factor promoting inhibitory synapse formation, these data indicate that excessive cocaine intake induces changes in gene regulation for inhibitory and excitatory synapses. Thus, excessive cocaine intake induces a distinct change in DNA methylation which may contribute the long‐term alterations in brain function associated with addiction.

Grant Funding Source: NIH R21DA027115
Baicalein exerts neuroprotective effects in 6‐hydroxydopamine induced experimental Parkisonism (LB625)
Authors: Mu Xin , Lu Yang , Du Guanhua
Published: June 2021

Baicalein, a flavonoid with potent antioxidant and anti‐inflammatory properties, has been shown to have neuroprotective effects. But baicalein has a pool dissolvability and scarcely dissolve in water. Thus,our topic group studied the crystal form of the chemical composition of baicalein. The result showed that baicalein possessed polymorphism. Moreover, β crystal form of baicalein was determined to be a superiority drug crystal because of its dominant position in stability and absorbance by constancy and biological test. So β crystal form of baicalein was used in this experiment. In rats, the behavioral and immunohistochemical manifestations after unilateral 6‐OHDA lesion were determined. Baicalein could significantly attenuate muscle tremor of 6‐OHDA lesioned rats (the burst frequency and amplitude are 13.43%, 35.18% compared to 6‐OHDA group), but could not reduce apomorphine (APO)‐induced rotations. Moreover, baicalein treatment could also increase tyrosine hydroxylase (TH)‐positive neurons (265.52% compared to 6‐OHDA group) and ameliorate the severe increase of glial fibrillary acidic protein (GFAP) immunoreactivity (70.23% compared to 6‐OHDA group) in the substantia nigra. Therefore, baicalein can be a promising candidate for prevention or treatment of Parkinson’s disease, owing to its anti‐apoptotic, pro‐differentiation and anti‐inflammatory action.

Grant Funding Source: This work was supported by the Research Special Fund for Public Welfare Industry of Health (No. 200802041) and National Significant Projects of New Drugs Creation (No. 2009ZM09501‐021).
MNG promotes neurogenesis against cerebral ischemic injury (LB626)
Authors: Wang Wen , Sun Fang‐ling , Ai Hou‐xi , Zhang Li
Published: June 2021

MNG is an active component of sarcocarp of C. officinalis that have shown neuroprotective effects. The aim of the present study is to test whether MNG promote neurogenesis via Wnt/β‐catenin signaling pathway for brain recovery in a rat model of focal cerebral ischemia. MNG were administered intragastrically once daily at the concentrations of 30, 90 and 270 mg/kg respectively for 7 days post‐ischemia. Ludmila Belayev score tests was performed to detect neurological function. Immunofluorescence staining of Nestin was used to identify the endogenous neural stem cells proliferation in the subventricular zone. The expression of proteins involved in and related to Wnt/β‐catenin signaling pathway was detected by western blotting analysis. MNG significantly promoted neurogenesis for brain recovery 7 days post ischemia. Increased expression of Wnt 3a and β‐catenin, along with activation of downstream transcription factors, indicated that the neurorestorative effects of MNG may be associated with Wnt/β‐catenin signaling pathway. These data provide support for understanding the mechanisms of MNG in neurorestorative effects and suggest a potential new strategy for stroke treatment.

Grant Funding Source: Supported by the National Science and Technology Major Project 2012ZX09102201‐106.
Oral toxicity of a lectin‐protease inhibitor fraction from Tepary bean (Phaseolus acutifolius) in rats (LB632)
Authors: Noriega‐Girón Héctor , Andrade‐Portillo Verónica , López‐Martínez Francisco , Moreno‐Celis Ulisses , Blanco‐Labra Alejandro , Garcia‐Gasca Teresa , Ferriz‐Martínez Roberto
Published: June 2021

Our previous studies showed that a lectin fraction from Tepary bean exhibits low toxicity but antinutritional effects in rats. The aim of this study was to determine the in vivo effect of a lectin‐protease inhibitor fraction (LIP‐60). Three administration schedules were tested for intragastric administered LIP‐60 in Sprague Dawley rats: 1) acute toxicity was determined using a single dose of LIP‐60 per group (10, 100, 1000 mg/body weight kg), 2) for the subchronic toxicity a daily LIP‐60 dose (100 mg/body weight kg) was administered for 28 days, 3) for the long‐term scheme, LIP‐60 (100 mg/body weight kg) was administered twice a week for 6 weeks. The animals were sacrificed to obtain blood samples in order to determine markers for renal, hepatic and pancreatic damage and also the nutritional status. Acute toxicity did not show adverse effects, only the highest dose provoked lethargy for the first 12 h. Subchronic test showed piloerection, yellow hair pigmentation, diarrhea, abdominal distention, irritability, partial food intake decrease and stationary body weight gain with a 10% decrement, blood markers for toxicity or nutritional status did not show significant alterations. The long‐term scheme did not show adverse effects neither before sacrifice nor blood markers. Our results suggest that LIP‐60 present a low toxicological profile, further studies will focus on effects on gastrointestinal tract and against colon cancer.

Founding: CONACYT FOMIX (QRO‐2011‐C02‐175340)
Protective effects of dipotassium glycyrrhizinate against snake venom muscle damage (LB633)
Authors: Ziegler Jussara , Gracia Marta , de Oliveira Thais , Nascimento Laís , Rocha Thalita
Published: June 2021

Myonecrosis is a serious consequence of Bothrops snake bites that may be due to destabilization and rupture of sarcolemma and organelles, inflammation, leading to permanent loss of muscle. Antivenom has been ineffective to neutralize this tissue damage and plant extracts have being studied as an associative therapy. In this research we analyzed the protective effect of Dipotassium Glycyrrhizinate (DPG) against myotoxic effects of Bothrops jararacussu snake venom using in vivo experimental protocols. DPG, isolated from Glicirriza glabra, is an anti‐inflammatory agent employed in treating dermatitis. The myotoxic process was followed in male Swiss mice gastrocnemius muscle at early (3 hours), intermediate (24 hours), late (3‐7 days) and final (21 days) phases after intramuscular injection of B. jararacussu snake venom (100µg/µL), followed by 2% DPG injection 30 minutes later. Control muscles were injected with DPG only. Muscles sections (10µm) were processed for light microscopy and immunohistochemistry for TNFα, IFNγ, myoD and miogenin. At early and intermediate phases, the muscle damage was characterized by delta lesions, densely‐clumped myofibrils and empty‐looking sarcoplasmic areas, inflammatory infiltrate and intense TNFα and IFNγ expression. Satellite cells and myoblasts, followed by myotubes were observed, from late to final phases, by myoD and miogenin expression. Cytokines expression coincided with the intense muscle proteolysis caused by the venom and the climax of regeneration, whereas cytokines decline, corresponded to periods of tissue remodeling and intense myofibril synthesis. The results show that DPG was able to decrease the myotoxic effects of this venom by inducing the inflammatory response and muscle regeneration.

Grant Funding Source: Supported by FAPESP 2013/10163‐4
Tissue effect of lens at leukergy of patients who received low doses of ionizing radiation (LB635)
Authors: Eltokova Manshuk , Kazymbet Polat , Bakhtin Meirat , Hoshi Masaharu , Altaeva Nursulu
Published: June 2021

Background |Aims: to explore the possibility to use the lens in the extract as a stimulant in vitro by the stimulated leukergy reaction at the liquidators of the Chernobyl Nuclear Power Plant (CNPP) with cataracts in the long term.

Materials and Methods: the response of white blood cells in the presence of agglomeration of lens antigen in vitro in 60 men ‐ the liquidators of the Chernobyl accident, at the age of 42 to 65 years old, with a cataract. The average radiation dose from 3.25 ± 0.31 to 18.2 ± 0.58 cGy. Comparison group was consisted of 60 men with cataracts, the identical age, who had no exposure to ionizing radiation. The control group was consisted of 60 men, aged from 42 to 58 years old without lens pathology.

Results: leukergy index of 1.03 ± 0.05% is referent at leukocyte stimulation by lens extract in the control group . The phenomenon of stimulated leukergy was identified in individuals exposed to ionizing radiation at a dose of 18,2 ± 0,58cGy., and ranged from 5.7% to 8.05% (p ˂ 0.001). This supposes a continued high autoaggression to the lens antigens and the strength of cell‐mediated immunity.

Conclusion: the proposed method of leukergy in the presence of lens antigens in vitro is quite informative and resource useful for the empowerment of the differential diagnosis of cataract of radiation genesis.
Reduction of α‐5 HbNO by dimethylsulfoxide in septic shock rats (LB638)
Authors: Arellano Ahumada Stephany , Villanueva López Cleva , Méndez Chávez Francisco , Zamorano Ulloa Rafael , Kross Robert , Ramírez Rosales Daniel
Published: June 2021

Septic shock is characterized by an excess of reactive oxygen and nitrogen species (ROS and NOS) like Nitric Oxide (NO). It is well known that the change in the physiological function of NO in shock is due to oxidative stress and the interaction of NO with hemoglobin (HbNO). We have formerly demonstrated that alpha‐pentacoordinate HbNO (α‐5 HbNO) specie is detected very early in hearts of rats with septic shock, and the amount of this specie increases in pace with shock. The goal of this study was to evaluate the effect of dimethylsulfoxide (DMSO, a ROS scavenger) on systemic α‐5 HbNO formation in septic shocked male Wistar rats, by E. coli lipopolysaccharide (LPS) infusion. α‐5 HbNO was clearly detected by electron paramagnetic resonance (EPR) in venous blood. It is concluded that ROS play an important role on α‐5 HbNO formation in shock. It remains to be determined if modulation of α‐5 HbNO formation has any impact on the evolution of septic shock.
Fetal Biometry of Pregnant Women in Khartoum State, Central Sudan (LB64)
Authors: Mokhtar Ahmed , Mustafa Wegdan , Ali Qurashi
Published: June 2021

Introduction: Ultrasonic measurement of fetal Femur Length (FL) and Bi‐Partial Diameter (BPL) is a precise tool for estimation of fetal growth and development. Accurate estimation of gestational age is vital for efficient obstetric planning and for detection of fetal malformation.

Objectives: Assist to construct new reference charts and equations for fetal biometry in ethnic Sudanese population, and to compare them with existing references from different international populations.

Methods: This was a cross sectional study done in Khartoum ‐ Sudan involving 256 of healthy pregnant women aged between 18‐40 years that were examined by ultrasound for FL and BPL. Women selected were with singleton pregnancy in the third trimester (30‐40 weeks). Regression models were used to estimate the mean and standard deviation of both the FL and the BPD at each gestational age.

Results: The results demonstrate a linear relationship between the FL and BPD measurements and the gestational age throughout the third trimester (P<0.001). The mean BPD values of Sudanese fetuses measured between 30 to 40 weeks showed that they are lower than the mean BPD values of Caucasian fetuses measured during the same period. The mean of FL measured between 30 to 40 weeks of Sudanese fetuses was higher when compared to Chinese and Singaporean fetuses, but was not significantly different when compared to fetuses from European countries.
Increased NO‐synthase activity and beneficial effect of PDE‐5 inhibition by sildenafil in pulmonary hypertension caused by pulmonary embolism (LB646)
Authors: Mizera Roman , Hodyc Daniel , Herget Jan
Published: June 2021

Background Two mechanisms that contribute to pulmonary hypertension after acute pulmonary embolism (PE) are vascular obstruction and vasoconstriction. Vasoconstriction is influenced by NO release. We focused on NO synthase activity, production of NO oxidative products (NOx) and effect of phosphodiesterase‐5 (PDE‐5) blockade by sildenafil on pulmonary hypertension after PE.

Methods Using the model of in vivo pulmonary embolism we measured changes of perfusion pressure in isolated rat lungs after blockade of NO‐synthase by L‐NAME and of PDE‐5 by Sildenafil. The NOx concentration was measured before and after PE in venous blood.

Results The increase in pulmonary perfusion pressure after L‐NAME administration was higher in rats after PE. NOx plasma concentration was higher after PE than before. Perfusion pressure after PE in rats treated with sildenafil was lower (12.9 ± 0,79 mmHg) than after PE in control group (21.4 ± 3,20 mmHg).

Conclusion Experimental PE in rats increases NO‐synthase activity and production of NOx. Administration of sildenafil inhibits pulmonary hypertension after PE. Our study indicates that similarly as in other forms of pulmonary hypertension sildenafil may be beneficial in the treatment of acute pulmonary embolism.

Grant Funding Source: Supported by GAUK n. 634112/2012
Characterization of conserved noncoding regulatory regions by flow cytometric analysis (LB66)
Authors: Brown Amber , Pira Charmaine , Howard Kristy , Oberg Kerby
Published: June 2021

Spatial and temporal regulation of developmental gene transcription often involves regulatory sequences found in noncoding DNA separate from the gene’s promoter. Conservation of a noncoding region (CNR) across divergent species may indicate a regulatory region critical to basic morphogenesis. CNR‐green fluorescent protein (GFP) constructs can demonstrate spatial and temporal activity; however, characterization of regulatory modules within a CNR requires quantitative, in addition to qualitative, analysis. Here we describe a novel application of flow cytometry in the characterization of CNRs.

CNR‐reporter constructs were generated using a limb‐specific Shh regulatory region (LSSRR) linked to a minimal HSV‐tk driven GFP. CNR‐reporter constructs were co‐electroporated with β‐actin driven red fluorescent protein (RFP) into the presumptive limb of chicks. Adequately transfected limbs were analyzed using the 7‐Color MACSQuant Analyzer and FlowJo software to evaluate changes in CNR activity. Data was normalized to nontransfected limbs.

Limbs electroporated with the β‐actin driven construct demonstrated transfection in 65.1% ± 7.9 of cells. Electroporation of the basal HSV‐tk‐GFP construct produced minimal fluorescence (relatively intensity 0.1 ± 0.04) in a small subset of cells (14.2% ± 4.4). Limbs electroporated with LSSRR‐GFP construct demonstrated robust activity in 29.8%± 6.0 of cells, with a relative intensity of 1.2 ± 0.2. A restriction enzyme generated LSSRR fragment showed intermediate activity (relative intensity 1.0 ± 0.2) in 20% ± 8.2 of cells.

Our data indicate that flow cytometric analysis can identify changes in the distribution and intensity of CNR activity, which enhances our ability to characterize CNR‐related regulatory sequences.
Isometric handgrip (IHG) training‐induced reductions in resting blood pressure: reactivity to a 2‐minute handgrip task identifies responders and non‐responders in young normotensive individuals (LB661)
Authors: Somani Yasina , Hanik Sarah‐Anne , Malandruccalo Amanda , Freeman Shane , Caruana Nic , Badrov Mark , Baross Anthony , Swaine Ian , Milne Kevin , McGowan Cheri
Published: June 2021

In people with hypertension, systolic blood pressure (SBP) reactivity to an isometric handgrip task (IHGT), but not a cold pressure task (CPT), is predictive of IHG training‐induced reductions in resting SBP. To investigate whether these findings could be extended to include young normotensives, resting BP and BP reactivity to an IHGT (2‐minute sustained contraction at 30% maximal voluntary contraction, MVC) and a CPT (2‐minute hand immersion in a cold water bath) were measured prior to and following 10 weeks of IHG training (4, 2‐minute IHG contractions at 30% MVC, using alternating hands, interspersed with 1‐minute rest periods, 3X/week) in 7 normotensive individuals (3 females, 4 males; age=25 ± 5.7 years). BP reactivity was derived by calculating the difference between peak stress BP and mean baseline resting BP. Significant training‐induced reductions in resting SBP (p<0.001) were strongly correlated with pre‐training SBP reactivity to the IHGT (r=‐0.8, p=0.03), but not the CPT (r= 0.2, p=0.6). These preliminary findings suggest that SBP reactivity to a short and simple handgrip task can be used as a predictive tool to identify who will respond best to IHG training. It might be possible to further explore how the IHG training stimulus could be optimized in non‐responders, to ensure that they too, experience reductions in resting BP.

Grant Funding Source: Supported by University of Windsor Kinesiology seed grant
Heart rate variablity indices are poor predictors of exercise capacity in healthy type 1 diabetics (LB664)
Authors: Peebles Karen , Baldi J, Chris , Hoye Neil , Murray Angus , Sheat Catherine , Williams Michael , Wilson Genevieve , Wilson Luke
Published: June 2021

People with diabetes often have chronotropic incompetence and reduced exercise tolerance. Chronotropic incompetence has been associated with autonomic dysfunction, thus we determined the ability of resting heart rate variability (HRV) to predict an altered exercise response in healthy individuals with type 1 diabetes (T1D). Baseline measures were taken followed by quantification of HRV with power spectral analysis during controlled breathing (15 breaths·min‐1) in 20 T1D (10 male & 10 female) screened for ischaemic cardiac disease and 12 age‐, weight‐, and training‐matched non‐diabetics (CON: 7 male & 5 female). These measurements were followed by a VO2max test (with ECG). Resting HR (73 ± 10 vs. 64 ± 10 beats·min‐1; P = 0.01) was higher and mean arterial blood pressure (92 ± 12 vs. 90 ± 11 mm Hg; P = 0.68) was not different between T1D and CON respectively. HR reserve (104 ± 17 vs. 121 ± 11 beats·min‐1; P < 0.001), VO2max (31 ± 9 vs. 41 ± 9 mL·min‐1·Kg‐1; P =0.005), HRV total power (1317 ± 1146 vs. 7493 ± 15080 ms2; P = 0.024), and HRV high frequency power (441 ± 491 vs. 3840 ± 9883 ms2; P = 0.029) were lower in T1D vs. CON respectively. Absolute max HR (177 ± 15 vs. 185 ± 10 beats·min‐1; P = 0.09), % of age predicted max HR (95 ± 6 vs. 97 ± 2 %; P = 0.07), and HRV low frequency power (328 ± 279 vs. 1672 ± 3433 ms2; P = 0.09) were not different in T1D vs. CON respectively. Despite group differences in HRV, linear correlations showed that resting HRV total, high frequency, and low frequency power accounted for < 20% of the variability in resting HR, HR reserve, max HR, and VO2max in T1D. Resting HRV indices are poor predictors of cardiovascular exercise response in healthy T1D.

Grant Funding Source: Supported by Anderson's trust
Effect of chronic quercetin supplementation on dystrophic cardiac pathology in mdx mice (LB672)
Authors: Ballmann Christopher , Hollinger Katrin , Quindry John , Selsby Joshua
Published: June 2021

Duchenne Muscular Dystrophy (DMD) includes cardiac complications with 10‐40% of deaths resulting from cardiac pathologies. In absence of a cure, we are exploring clinically viable interventions to counter cardiac complications associated with DMD. Quercetin (Q) is a SIRT‐1/PGC‐1α activator that up‐regulates antioxidant genes, mitochondrial biogenesis, and decreases inflammation when taken by nutraceutical means. Thus, we investigated 0.2% Q dietary enrichment to counter cardiac abnormalities in mdx mice, a widely used model for DMD. Mice were randomly treated from either 3 weeks or 3 months for a 6 month intervention (3w6m or 3m6m, n=10/group). Control (C) mice consumed rodent chow for equal time periods (n=10/group). At the end of the study, hearts were excised, weighed, and frozen (‐80 C°), cut into 10µm sections for immunohistochemistry, or prepped for western blotting (WB). Body weight did not change. Relative and absolute heart weights were decreased in Q3m6m mice (p=.03,p=.021). WB targets for mitochondrial biogenesis (Cytochrome C, p=.045) and antioxidant expression (SOD2, p=.012) were increased in Q3w6m mice. Indications of remodeling and fibrosis (MMP9, p=.043, hematoxylin and eosin, p=.001) were improved in Q‐fed mice while TGFβ‐1 expression was unchanged. 6 months of Q feeding improved long term markers and appears to effectively attenuate dystrophic cardiac pathology. Based on findings from younger mice, improvement in mitochondrial content and antioxidant activity may precede these outcomes. Results reveal a promising intervention to complications of dystrophic cardiac pathology. Additional experiments are needed to confirm these findings with more reductionist approaches.
Involvement of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase in Nω‐Nitro‐L‐arginine methyl ester (L‐NAME)‐induced hypertension (LB676)
Authors: Leo M. Dennis , Kandasamy Kathirvel , Subramani Jaganathan , Tandan Surendra , Kumar Dinesh
Published: June 2021

Chronic administration of L‐NAME in rats is an excellent model to study the induction and progression of a NO deficiency‐induced endothelial dysfunctional state. This study was performed to determine how different vascular beds compensate for the loss in nitric oxide. Male Wistar rats received L‐NAME (50mg/kg/day in drinking water) or no drug for 6 weeks. Aorta, pulmonary and renal arteries were isolated for determination of basal cGMP production, PDE5 activity, dimethylarginine dimethylaminohydrolase (DDAH) and hemeoxygenase (HO) activity. mRNA expression studies were done by real time PCR. Plasma IL‐1β level was measured by ELISA. Results: L‐NAME‐induced hypertension was associated with an increase in mean arterial pressure and plasma IL‐1β. The treatment had varied effects on mRNA expression levels of eNOS, soluble guanylyl cyclase, PDE5 and HO1 but showed an increased iNOS and decreased DDAH2 expression in all three tissues studied. Basal cGMP levels were decreased in all tissues, but PDE5 activity was increased only in renal and pulmonary artery. DDAH activity was significantly reduced, whereas HO activity was significantly increased in all the tissues. This study showed that different vascular beds do not react similarly to chronic NO inhibition and that vascular inflammation triggered by an increase in iNOS and indirectly by a decrease in DDAH activity might be the causative factors for the progression of vascular dysfunction in this model.
Vascular dysfunction induced by organ culture is dependent of male sex hormones (LB677)
Authors: Andres Monica , Flynn Daniel , Abdu Semah , Gomez Juan , Portilla María , de Bethencourt Fermin , Ferrer Mercedes
Published: June 2021

Androgens exert beneficial effects on vascular function by regulating the release and function of NO, prostanoids and reactive oxygen species. Since organ culture induces phenotypic changes in endothelial cells similar to those observed in some cardiovascular diseases, the aim of this study was to analyze the preventive role of male sex hormones on the vascular damage induced by organ culture. We used aortic segments from control and orchidectomized rats incubated for 20 hours at 37ºC in serum free culture medium. In this system we studied: i) the prostanoids and H2O2 production in the culture medium; ii) the hyperpolarizing mechanisms on the responses induced by acetylcholine (Ach) and by the NO donor, sodium nitroprusside (SNP) and iii) the effect of TXA2‐mimetic U‐46619 on vasodilator responses. The results showed that organ culture: i) increased the production of prostanoids and H2O2 in aorta from both groups of rats; ii) decreased the Ach‐induced relaxation in aorta from orchidectomized rats, while increased that induced by SNP and iii) decreased the Ach‐ and SNP‐induced responses after incubation with U‐46619 only in orchidectomized rats. These findings suggest a preventive role of male sex hormones on organ culture‐induced vascular damage.

Grant Funding Source: Supported by grants (to M. F.) from the Fondo de Investigaciones Sanitarias (PI0011406) and (to F. R. B.) from the Sociedad Española de Urología (2013‐14).
Diet induced obesity promotes aortic stiffness by impairing elastin functionality (LB695)
Authors: Garner Tyler , Fleenor Bradley
Published: June 2021

Cardiovascular diseases (CVD) are the leading cause of mortality in the United States. Obesity is a primary risk factor for CVD resulting in greater large elastic artery stiffness. Importantly, stiffening of the aorta is an independent predictor of CV events and is a novel therapeutic target for attenuating CVD risk with obesity. The purpose was to determine if a high fat diet promotes aortic stiffening in mice that would result in a significant increase in aortic pulse wave velocity (aPWV) and intrinsic mechanical stiffness. Mice were randomly assigned into two groups receiving either a low fat (LF, 10% kcal fat) or high fat (HF, 60% kcal fat) diet. HF mice had greater aortic stiffness as assessed by aPWV (401 ± 26 vs. 304 ± 17 cm/s, P < 0.05) and in vitro aortic mechanical stiffening. Specifically, the elastic modulus of the elastin region was decreased in HF compared with LF mice (398 ± 6 vs. 615 ± 42 kPa, P < 0.05), whereas the collagen elastic modulus showed no difference between groups (3707 ± 694 vs. 3732 ± 403 kPa, P<0.05). Collectively, these data suggest that the decrease in aortic elastic modulus of the elastin region is due to loss of functionality of the elastin protein fiber. These results support the notion that high fat diets promote aortic stiffening thus increasing CVD risk.
Establishment and validation of a pre‐clinical, multi‐center, multi‐species consortium for the assessment of infarct size‐reducing agents: CAESAR (Consortium for the preclinicAl assESsment of cARdioprotective therapies) (LB698)
Authors: Jones Steven , Steenbergen Charles , Lefer David , Tang Xian‐Liang , Kukreja Rakesh , Li Qianhong , Kong Maiying , Bhushan Shashi , Stowers Heather , Kondo Kazuhisa , Hunt Gregory , Goodchild Traci , Orr Adam , Chang Carlos , Ockaili Ramzi , Salloum Fadi , Guo Yiru , Bolli Roberto
Published: June 2021

Despite decades of effort and staggering financial support, the cardioprotection field failed to deliver a drug that effectively reduces infarct size in patients. To address the limitations to such preclinical studies and promote progress in this critical area of medical research, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR). The first step for CAESAR’s validation involved testing the “gold standard” for infarct size limitation: ischemic preconditioning (IPC). CAESAR tested the capacity for IPC to reduce infarct size in three species (at two sites/species): mouse (n=22‐25/group), rabbit (n=11‐12/group), and pig (n=13/group). During this validation phase, we confirmed that IPC significantly (p<0.05) reduced infarct size in all species, and, successfully established a multi‐center structure to support CAESAR’s operations, including: two distinct surgical centers for each species, a pathology core (to assess infarct size), a biomarker core (to assess cardiac protein release into blood), and a data coordinating center ‐ all with the oversight of a protocol review and monitoring committee of distinguished experts. Such an infrastructure promotes careful, blinded, randomized, statistically sound, and rigorous evaluation of cardioprotective agents. Thus, CAESAR is operational and represents a new approach to pre‐clinical cardioprotection studies.

Grant Funding Source: Source of Support: National Institutes of Health (NIH)‐Heart Lung and Blood Institute (NHLBI) CAESAR Consortium‐U24 HL094373
Effect of mTOR Antagonism on Endothelial Function in the Human Cutaneous Vasculature (LB699)
Authors: Kellogg Dean , Zhao Joan , Wu Yubo , Roman Linda
Published: June 2021

Inhibition of the mechanistic Target of Rapamycin (mTOR) for 1 week causes endothelial dysfunction in rodents. We hypothesized that a similar effect would be observed in humans. 4 middle‐aged persons participated. mTOR was antagonized by topical application of 8% rapamycin (RAPA) ointment to skin. RAPA was applied twice daily for 7 days to an 8x8 cm area of skin on the abdomen while the ointment vehicle only was applied to a second untreated control site. We examined the effect of these treatments on skin blood flow (SkBF) vasodilatory responses to 5 increasing doses of methacholine (MCH) given by intradermal microdialysis. Responses were monitored by laser‐Doppler flowmetry (LDF) at the RAPA treated and untreated control sites. After delivering the 5 MCH doses, 58mM nitroprusside (SNP) was given at both sites to effect maximal vasodilation for data normalization (%maxLDF). Results: At baselines and in response to MCH doses of 10microM, 100microM, 1mM, and 100mM no differences were found between responses at RAPA and control sites. At 10mM MCH, the vasodilation at RAPA treated sites exceeded that at untreated sites (RAPA 85±6 vs Control 68±9%maxLDF, p<0.05). Absolute value (mV) responses to SNP did not differ between RAPA and vehicle treated sites (p>0.05). We conclude that chronic RAPA treatment improves endothelium dependent vasodilation in middle‐aged persons in contrast to causing endothelial dysfunction in rodents. (supported by NIH Grant AG041365)

Grant Funding Source: NIA
Epiglottis cross‐sectional area diminishes with increased severity of obstructive sleep apnea (LB70)
Authors: Ma Melinda , Yadav Santosh , Harper Ronald , Kumar Rajesh
Published: June 2021

Obstructive sleep apnea (OSA) is a condition characterized by episodic upper airway collapse with continued diaphragmatic efforts during sleep. Significant cardiovascular and neurophysiological defects may result from brain injury accompanying repeated epochs of hypoxia. The majority of occlusions in OSA patients occur at the soft palate and uvula of the oropharynx. However, the extent of cross‐sectional area at the level of the epiglottis, between the oropharynx and hypopharynx, remains unclear. Morphological aspects at lower regions of the oropharynx, with different cranial nerve innervation from upper airway regions, may also contribute to obstruction. Using a 3.0 Tesla brain magnetic resonance imaging (MRI) scanner, we collected two high‐resolution T1‐weighted image series and measured axial cross‐sectional areas and oropharyngeal airway lengths for 13 mild or moderate OSA (mean age ± SD: 48.24 ± 8.28; AHI 5‐29.9 events/hr; 6 male), 11 severe OSA (48.81 ± 8.31; AHI 蠅 30 events/hr; 8 male) and 50 control subjects (47.17 ± 9.06; 28 male). Epiglottis cross‐sectional areas were significantly reduced in OSA over control subjects (p=0.005) and collapsible oropharyngeal airway lengths were significantly increased in OSA over control subjects (p <0.001). The diminished epiglottis cross‐sectional areas and increased airway lengths in OSA patients may contribute to a greater vulnerability for airway collapse.

Supported by National Institutes of Health R01 HL‐113251.
Effect of estradiol on MAPK p44/42 expression in the brain induced by angiotensin II (LB709)
Authors: Almeida‐Pereira Gislaine , Coletti Ricardo , Mecawi André , Elias Lucila , Antunes‐Rodrigues José
Published: June 2021

This study aims to investigate the influence of estradiol on the MAPK p44/42 expression in the lamina terminalis and hypothalamic nucleus induced by central stimulation angiotensinergic.

Wistar rats (~250g) were submitted to ovariectomy and on the following day they were treated with estradiol cypionate (10µg/rat, sc) or vehicle (corn oil, 0.1mL/rat, sc) for eight days. On the eighth day, these rats were submitted to icv (lateral ventricle) administration of angiotensin II (ANGII, 25ng/2μL/rat) or vehicle (0.9% saline, 2μL/rat). Ten min after the animals were decapitated for brain collection and MAPK p44/42 analysis by western blotting. Data were analyzed using Student t test and the level of significance was set at 5%.

The pretreatment with estradiol attenuated the MAPK p44/42 total expression in the lamina terminalis and paraventricular and supraoptic nucleus (t(12)=3.8; t(10)=3.5, respectively) in comparison with rats treatment oil and saline icv. Both ANG II and estradiol increased the phosphorylation of the MAPK p44/42 in lamina terminalis and hypothalamic nucleus in comparison with rats treatment oil and saline icv (t(12)=‐3.75, t(13)=‐2.81, t(14)=‐3.85, t(14)=‐4.15, respectively). However ANG II did not further increase the phosphorylation of MAPK p44/42 induced by estradiol in all structures analyzes.

These result suggest that the known effect of estradiol on the responses behaviors and neuroendocrine induced by ANG II can be due to change of MAPK p44/42 phosphorylation this structures central involved with control body fluids.

Grant Funding Source: FAPESP, CNPq and CAPES
Effect of renal ablation on blood pressure and muscle sympathetic nerve activity in chronic kidney disease patients (LB722)
Authors: Wilson Genevieve , Hoye Neil , Wilson Luke , Shoemaker J, Kevin , Walker Rob , Jardine David , Schollum John , Wilkins Gerard , Baldi J, Chris
Published: June 2021

Chronic kidney disease (CKD) is associated with renal sympathetic hyperactivity and uncontrolled hypertension. Catheter‐based renal ablation may reduce arterial blood pressure and muscle sympathetic nerve activity (MSNA) in patients with drug resistant hypertension. A cohort of 4 dialysis patients are reported who had uncontrolled hypertension despite two agents and clinical normovolemia, and in whom successful MSNA and BP recordings were obtained in both pre and post ablation tests. These patients were tested to determine whether renal ablation would reduce blood pressure and whether changes in blood pressure would be associated with changes in MSNA. This was achieved by comparing burst activity (incidence and frequency) and action potential (AP) composition (APs/burst, APs/min and APs/100 heart beats) pre‐ and one month post‐ablation. Analyses were conducted using software that detected AP content of the multi‐unit recording in human neural signals. Compared to the pre session, these patients had reductions in average ambulatory blood pressure (Systolic 178 ± 18 to 158 ± 25 mmHg p=0.04 and diastolic 95 ± 13 to 82 ± 7 mmHg p=0.046) post‐ablation. Average burst frequency (64 ± 15 to 61 ± 18 p=0.58) and burst incidence (84 ± 12 to 82 ±19 p=0.6) were unaffected by ablation. However, ablation increased average APs/burst (4.5 ± 1.3 to 7 ± 2.3 p= 0.03), APs/min (223 ± 103 to 423 ± 202 p=0.039) and APs/100 heart beats (297 ± 117 to 582 ± 280 p=0.04). These data suggest that the observed decrease in blood pressure was not associated with changes in burst activity (frequency and incidence): However, the observed increases in sympathetic AP content/burst and per minute suggest an affect of ablation on AP recruitment and synchronization.

Grant Funding Source: Supported by The Kidney and Health and Disease reserach theme Univeristy of Otago
Serum cystatin C in early prediction of risk of chronic kidney disease progression in type 2 diabetes (LB727)
Authors: Wijayatunga Nadeeja , Perera Pradeep , Wanigasuriya Kameni , Wijeysiri Ananda , Moustaid‐Moussa Naima , Peiris Hemantha
Published: June 2021

Serum cystatin C (CysC) is superior to serum creatinine (SCr) in diagnosing mild diabetic nephropathy. We hypothesized that, CysC can predict the risk of CKD progression at an early stage in T2DM. Our objective was to compare levels of CysC, SCr and eGFR in T2DM patients in no/low risk with moderate risk of CKD progression according to Kidney Disease Initiative Global Outcomes (KDIGO) guideline’s CKD risk stratification. Sixty one non‐obese, T2DM patients aged 30‐60 years, suspected of mild to moderate CKD were recruited. Creatinine was measured using Jaffe method in serum and urine. Serum CysC and urine albumin were measured using immunoturbidimetry. eGFRs were calculated using Modification of Diet in Renal Disease equation (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equations. Moreover, Albumin to Creatinine Ratio (ACR) was calculated. Patients were categorized, to CKD risks categories using eGFR CKD‐EPI based on SCr and ACR. Our results demonstrate that patients in the moderately increased risk group had significantly higher CysC than those in the no/low risk group. However, SCr, eGFR based on CKD EPI and MDRD equations were not significantly different between the two groups. Thus, CysC is valuable in detecting moderately increased risk in CKD progression in T2DM patients than SCr and SCr based eGFR. These observations may be useful for early prediction of CKD progression and clinical management of patients with early diabetic nephropathy.

Grant Funding Source: Research award from the University of Sri Jayewardenepura (Grant number : ASP/06/R/MED/2011/21), and Texas Tech University
Contribution of the MnPO Angiotensin Receptors to Brain Stem Activity and Hypertension (LB734)
Authors: Shell Brent , Cunningham J.
Published: June 2021

Repeated bouts of hypoxia during sleep apnea and chronic intermittent hypoxia (CIH) are associated with persistent blood pressure elevation. Neurological mechanisms that drive this maladaptive blood pressure increase are not well understood. Knockdown of the Angiotensin type 1a (AT1a) receptor in a forebrain nucleus, the median preoptic nucleus (MnPO), prevents normoxic hypertension during CIH. In the current study, adult male rats were exposed to CIH after MnPO injections with adeno‐associated viruses containing shRNA‐AT1a or scrambled shRNA. We examined the activity of downstream nuclei by performing immunohistochemistry for ∆FosB, a marker for neuronal activity. We hypothesize that knockdown of AT1a in MnPO results in decreased ∆FosB expression in downstream nuclei such as the rostral ventrolateral medulla (RVLM), and the nucleus tractus solitarius (NTS). ∆FosB staining was significantly lower in the subpostremal region of the NTS and in the RVLM following CIH in AT1a knockdown treated rats versus scramble treated rats. This suggests that CIH mediated ∆FosB staining in the NTS and RVLM is at least partially dependent on AT1a receptors in MnPO. These results, coupled with the prevention of the normoxic hypertension, indicate that angiotensin acting at the MnPO is affecting the activity of neurons in brainstem area that regulate blood pressure.

Grant Funding Source: Supported by P01 HL‐88052 and National Institute of Aging, Training in the Neurobiology of Aging, T31 AG020494
Catecholaminergic (CA) neurons modulate hypoxic ventilatory response in newborn rats (P7‐8) (LB737)
Authors: Patrone Luis , Bícego Kênia , Gargaglioni Luciane
Published: June 2021

CA neurons are involved in autonomic and respiratory regulation during low O2 conditions in adult mammals. We evaluated the participation of central CA neurons of neonate male and female rats (P7‐8) in the hypoxic ventilatory response (HVR) by specifically lesioning them with antidopamine beta‐hydroxylase‐saporin (DBH‐SAP, 42ng / 100nL) injected via the 4th ventricle. Minute ventilation (VE) of newborn was recorded by pressure‐plethysmography from the body chamber during normoxia and hypoxia (10%O2). HVR was reduced significantly (~23%) in male and female lesioned newborn rats (male‐ control group: 137.6±6.4 (% of baseline) vs lesioned group: 104.3±1.4 (% of baseline), p<0.01; female‐ control group: 131±1.0 (% of baseline) vs lesioned group: 102.6±2.6 (% of baseline) p<0.02). These results suggest that catecholaminergic neurons exert an excitatory modulation in newborn rats in O2 chemosensitivity.

Grant Funding Source: Fapesp and CNPq
Ca2+/CaMKII‐dependent ERK1/2 signaling mediates serotonin 5‐HT3 receptor‐mediated vomiting (LB742)
Authors: Zhong Weixia , Hutchinson Tarun , Chebolu Seetha , Darmani Nissar
Published: June 2021

The 5‐HT3R agonist 2‐Me‐5‐HT induces vomiting. Its downstream signaling remain unknown. 5‐HT3R has high permeability to extracellular Ca2+ and upon stimulation allows Ca2+ influx. We examined the contribution of Ca2+/CaMKIIα, interaction of 5‐HT3R with calmodulin, and ERK1/2 signaling to 2‐Me‐5‐HT‐induced emesis in least shrews. 2‐Me‐5‐HT augmented intracellular Ca2+ levels in the brainstem slices and the 5‐HT3R antagonist palonosetron, abolished the response. Pre‐treatment with either: i) amlodipine, an antagonist that blocks L‐type Ca2+ channels; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs); iii) a combination of their less‐effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose‐dependently suppressed emesis. 2‐Me‐5‐HT also: i) enhanced the interaction of 5‐HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, and their colocalization in the area postrema and intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and enterochromaffin cells as shown by western blot and immunocytochemistry. These effects were suppressed by the 5‐HT3R antagonist, palonosetron. 2‐Me‐5‐HT treatment also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER IP3Rs by 2‐APB, had no significant effect on these parameters. Thus, Ca2+ mobilization via extracellular Ca2+ influx through 5‐HT3Rs/L‐type Ca2+ channels and Ca2+ release via RyRs on ER, initiate a Ca2+‐dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 2‐Me‐5‐HT‐evoked emesis.

Grant Funding Source: COMP/WesternU startup funds
The precipitation of behavioral defects due to inflammation in the DSS mouse model of colitis (LB748)
Authors: Emge Jacob , Gareau Melanie , Barrett Kim
Published: June 2021

Background: Anxiety, depression, and altered memory have been previously associated with intestinal diseases, including inflammatory bowel disease (IBD). Understanding the association between these behavioral changes and IBD may provide clinical significance seeing as concomitant mood disorders often increase a patient’s risk of developing secondary functional gastrointestinal diseases (FGIDs).

Aims: The aim of this study was to determine whether active intestinal inflammation precipitates behavioral defects in memory and anxiety and to determine if these changes persist through the resolution of inflammation.

Methods: Dextran Sodium Sulfate (DSS) was administered for 5 days via drinking water followed by either 3 days for active inflammation studies or 8 days for resolution of inflammation studies. Mice were weighed daily and colon lengths were measure at the time of sacrifice in order to assess the degree of sickness. Anxiety‐like behavior (light/dark box) and memory (novel object test) were assessed in the DSS mice vs. controls (WT;C57BL/6).

Results: DSS mice at the peak of inflammation demonstrated impairments in non‐spatial memory with an exploration ratio of 54.06 +/‐ 1.84 while controls had a mean exploration ratio of 57.91 +/‐ 1.29 (p<.01; n=13). Anxiety‐like behavior was also evident in the DSS mice, which spent 133.91 +/‐10.17 seconds in the light area of the light/dark box while the controls spent 174 +/‐ 14.51 seconds in the light area (p<.04; n=11). These behavioral defects did not persist at the resolution of intestinal inflammation. However, physiological changes, such as a shortening of the colon, were equally evident at the peak of inflammation as well as at the resolution phase.

Conclusions: At the peak of inflammation, the DSS mouse model of colitis displays altered baseline behaviors in regards to memory and anxiety. Although physiological changes persist at the resolution of colitis as seen with persistent shortening of the colon, behavioral defects are not evident post inflammation. Taken together, these findings indicate and underlying connection between the brain and the gut relevant to the context of Inflammatory Bowel Disease.