bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐Contrast Ultrasound Perfusion Imaging of Human Brown Adipose Tissue
Authors: George Mark K. , Weeks Abbie E. , Byram Brett C. , Walker Greg , Damon Bruce M. , Coolbaugh Crystal L.
Published: April 2020

Brown adipose tissue (BAT) is a highly vascularized tissue that uptakes and oxidizes fatty acids from the circulation in response to a cold stimulus, resulting in thermogenesis. Tissue perfusion has been proposed as an approach to understand BAT metabolism, but current imaging techniques require invasive contrast or ionizing radiation. Power Doppler ultrasound imaging enables sensitive, high temporal resolution measures of the movement of blood as it perfuses a tissue without the need for contrast injections. The purpose of this study was to explore the utility of non‐contrast ultrasound perfusion imaging of human BAT during personalized cooling. Five healthy subjects [4 men; age: 31.8 ± 5.6 yrs.; body mass index: 22.5 ± 3.3 kg/m2; total body fat % (dual‐energy x‐ray absorptiometry scan): 22.9 ±7.0 %] underwent an individualized, perception‐based cooling protocol to stimulate BAT. Infrared thermography, a surrogate measure of BAT activity, and power Doppler ultrasound images were acquired over the right and left supraclavicular space, respectively, every five minutes in thermoneutrality (TN; duration: 15 min) and during cold exposure (CE) to the participant’s shiver threshold (duration: 58.3 ± 10.8 min; cooling dose: 319.7 ± 140.4 °C*min). Ultrasound images were post‐processed with a block‐wise, independent component analysis filter to analyze signal changes related to perfusion. BAT regions of interest were defined, and TN and CE conditions were compared as the mean ± standard deviation of the difference in the 95th percentile skin temperatures (infrared thermography) and the mean power Doppler signal (ultrasound). Supraclavicular skin temperature increased by 0.56 ± −0.21 °C (95% bootstrap confidence interval (CI): −0.57 to 1.72 °C), indicating a potential thermogenic response of BAT to individualized cooling (TN: 33.8 ± 1.1 °C vs. CE: 34.3 ± 0.9 °C). Similarly, the mean power Doppler signal increased by 11.6 ± 3.8 dB (95% CI: 3.2 to 19.4 dB) following cold exposure (TN: 46.8 ± 5.3 dB vs. CE: 58.3 ± 9.1 dB). These preliminary data demonstrate the feasibility of non‐contrast ultrasound perfusion imaging to detect the microvascular response of BAT to a cold stimulus in healthy adults. Power Doppler ultrasound imaging could prove useful when combined with existing noninvasive modalities (e.g. magnetic resonance imaging) to assess both the perfusion and metabolic substrate uptake response of BAT to potential obesity‐targeted therapies.

Support or Funding Information

NIDDK/NIH R01‐DK‐105371, NCATS/NIH UL1‐TR000445
LIPOXINS SELECTIVELY CHANGE M2‐LIKE PHENOTYPE OF TUMORASSOCIATED MACROPHAGES BY CHANGING THEIR UNIQUE METABOLIC PROFILE AND IMPAIRING TUMOR PROGRESSION
Authors: Barja-Fidalgo Christina , de Brito Nat á lia Mesquita , da Costa Hayandra Cunha , Moretti Julia Duncan , Simões Rafael Loureiro
Published: April 2020

In tumor microenvironments, most tumor‐associated macrophages (TAM) exhibit a characteristic M2 phenotype that favors tumor development, with deficient production of NO and ROS, key features of pro‐inflammatory M1 cytotoxic macrophages. Evidences have shown a positive correlation between high TAM density and poor prognosis. The balance between the different tumor derived mediators may modulate the outcome of the Mϕ response and consequent tumor development. Lipoxins (LX) are specialized pro‐resolving lipid mediators, able to shift inflammatory monocytes and macrophages towards M2‐like profile, but their effects on TAMs remained non‐elucidated. We have shown that, in vitro, LX selectively induce a switch of TAM M2‐like phenotype towards an M1‐like profile, increasing ROS and NO production, and enabling these cells to promote tumor cell apoptosis. LX did not affected the M2 profile of IL‐4‐stimulated macrophages. In vivo, the treatment of tumor‐bearing mice with LX, diminished the population of LY6Chi monocytes (TAM precursors) in spleen, blood and bone marrow, decreasing macrophage infiltration into the tumor, reducing the M2 markers expression on TAMs, and impairing tumor progression, increasing mice survival. The differential activation of macrophages is supported by profound intracellular metabolic changes, being well accepted that the pro‐inflammatory M1/M(LPS+IFN‐γ) phenotype rely on aerobic glycolysis, while anti‐inflammatory M2/M(IL‐4) macrophages depend on oxidative metabolism. However, little is known about TAM metabolism. We investigated the metabolic profile and requirements of TAMs, analyzing the lipoxin effects on their metabolic pathways. We show for the first time that despite TAMs present high expression of M2 markers, similarly to the M1, but distinct from M2 cells, have high glycolytic activity, with high lactate secretion, mTOR/Akt activity and increased GLUT‐1 expression. Inhibition of glycolysis, but not the oxidative phosphorylation or PPP, decreased M2 markers expression in TAMs. The inhibition of PPP impairs lipoxin to shift the M2‐profile and restore cytotoxic properties in TAMs. In conclusion, TAMs although phenotyped as M2, are metabolically distinct from these cells, being rather similar to M1, depending on the glycolytic metabolism to support their profile and functions, that lipoxin effects rely on PPP activation.

Support or Funding Information

by: FAPERJ # E‐26/202.782/2017; CAPES: Finantial code # 001; CAPES/PROEX # 0574/2018; CNPq # 302413/2017‐0; 118566/2017‐2, Brazil.
A Gene Expression Profile of Metabolic Dysfunction in the Aorta of Male Sprague‐Dawley Rats Following a Short‐Term High‐Fat Diet
Authors: Mohr Alex E. , Reiss Rebecca A. , Sena Johnny , Naik Jay S. , Walker Benjimen R. , Sweazea Karen L.
Published: April 2020

Diet‐induced metabolic dysfunction precedes multiple disease states, including diabetes, heart disease, and atherosclerosis. The critical role of the vasculature in disease progression is established, yet the details of gene expression remain an enigma. The objective of the current project was to demonstrate that a quantitative assessment of gene expression within the vascular wall (aorta) of healthy rats compared to those exhibiting metabolic dysfunction symptoms could reveal potential mediators of vascular dysfunction. RNA was extracted from the aorta of four rats from a larger experiment; two animals that were maintained on a high‐fat diet (HFD) exhibited symptoms of metabolic dysfunction (including hypertension, weight gain, glucose intolerance, and insulin insensitivity) and two healthy animals that were fed a standard chow diet (CHOW) served as age‐matched controls. Poly‐A libraries were constructed and subjected to Illumina sequencing protocols. The post‐sequencing workflow was conducted with the software package Lumenogix® and sequencing reads were mapped to the Rat Genome Assembly version Rnor_5.0 using the Genomic Mapping and Alignment Program (GMAP). The program Empirical Analysis of Digital Gene Expression data in R (EdgeR) was used to evaluate statistical significance. Genes with a P adjusted value of ≤ 0.05 were passed to Cytoscape App ClueGO/CluePedia for enrichment analysis using Gene Ontology (GO) Biological Process terms, network construction and visualization. Preliminary analyses indicated 93 genes were significantly differentially expressed in the aorta of HFD rats. Enrichment analysis and the resulting network contained 54 genes relevant to physiological processes including fat and protein metabolism, oxygen transport, hormone regulation, vascular regulation, thermoregulation, and circadian rhythm. The majority of differentially regulated genes were downregulated (82.5%) including Nicotinamide Phosphoribosyltransferase (visfatin), which plays a role in the modulation of the circadian clock function. In contrast, Leptin and 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (Hmgcs2) were notably upregulated. Leptin is involved in several major energy balance signaling pathways and Hmgcs2 is a mitochondrial enzyme that catalyzes the first reaction of ketogenesis. Together, these data describe the changes in gene expression within the aortic wall of HFD rats with early metabolic dysfunction and highlight potential pathways and signaling intermediates that impact vascular disease.
Norepinephrine‐Alpha 2 Adrenergic Receptor Axis is Critical to Long‐Term Sequelae of Ischemic Acute Kidney Injury
Authors: Jang Hee-Seong , Noh Mi Ra , Ha LiGyeom , Kim Jinu , Padanilam Babu J.
Published: April 2020

Objective

Upregulated levels of kidney norepinephrine (NE) have been recognized as a risk factor of development and progression of chronic kidney disease. Our reports showed renal denervation can prevent renal fibrosis and inflammation in renal fibrogenesis models through inhibition of NE‐alpha 2 adrenergic receptors (α2‐ARs) axis. However, the molecular mechanism by which NE‐α2‐ARs axis elicits long‐term sequelae of acute kidney injury (AKI) remains to be elucidated.

Methods

Based on our published data, we hypothesized that NE‐α2‐ARs axis mediates its effect through macrophages recruited into ischemic kidneys. In this study, we investigated whether in vivo and in vitro NE‐α2‐AR axis regulates macrophage recruitment, activation and differentiation during the development of chronic kidney disease after ischemia/reperfusion injury (IRI).

Results

Renal bilateral IRI resulted in severe kidney fibrosis in medullary region over 2 weeks with enhanced tissue NE level, persistent tubular injury and massive macrophage infiltration. Inhibition of α2A‐ and/or α2C‐AR subtypes by pharmacological or genetic intervention prevented macrophage accumulation with downregulation of M1 and M2 macrophage markers, reduced levels of macrophage‐released pro‐inflammatory and/or fibrogenic cytokines, resulting in suppression of persistent renal tubular injury and fibrosis progression. In in vitro study using Raw264.7 cells, pharmacological inhibition of α2‐AR blocked macrophage migration to proximal tubule cells exposed to NE. In addition, we demonstrated that NE results in upregulation of both M1 and M2 macrophages.

Conclusions

Collectively, NE‐α2‐ARs axis induces long‐term sequelae of ischemic AKI through macrophage recruitment and subsequent activation. Our data suggest that targeting NE‐α2‐ARs axis can be a potential therapeutic strategy to prevent long‐term sequelae of ischemic AKI.

Support or Funding Information

This work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).
Leucine Supplementation Does Not Enhance Translation Initiation of Low Birth‐Weight Neonatal Pigs Despite Greater Sestrin2 Expression in Skeletal Muscle
Authors: Yonke Joseph A. , Seymour Kacie A. , El-Kadi Samer W.
Published: April 2020

Lean growth is impaired in low birth‐weight (LBWT) neonatal pigs and correlates with a reduction in protein synthesis rate in skeletal muscle. Translation initiation via mTOR signaling pathway is the rate limiting step in protein synthesis. Leucine binds and inactivates Sestrin2, an inhibitor of the mTORC1 complex. The objective of this study was to determine whether dietary branched‐chain amino acids (BCAA) stimulate translation initiation in skeletal muscle of LBWT pigs. LBWT and normal birth weight (NBWT) were defined when weight at birth was 2 SD below or within 0.5 of the corresponding litter average. Three day old LBWT and NBWT pigs (n=11‐13/group) were assigned to a Control or BCAA sow milk‐replacer formula in a 2×2 factorial arrangement. Formulas were isonitrogenous and contained either 1% BCAA (1:0.68:0.5 leucine:isoleucine:valine) or 0.7% alanine, and were offered at 250 mL·kg body weight−1·day−1 in 5 equal meals. Carotid and jugular catheters were surgically placed on day 21. On day 29, blood samples were taken over 4 h following a meal. Pigs were euthanized 1 h after a final meal. Data were analyzed using MIXED procedure of SAS version 9.4. BCAA supplementation increased BCAA concentration in plasma (P<0.0001) and in muscle free amino acid pool (P<0.001) compared to the Control diet, which similarly increased alanine concentration in plasma and muscle (P<0.0001). Although BCAA supplementation decreased circulating Pro, Ser, Asp, and Lys (P<0.001, 0.05, 0.05, 0.06, respectively), concentrations were not different in skeletal muscle. LBWT pigs expressed less BCAT2 mRNA in the longissimus dorsi than NBWT pigs, but expression was enhanced to normal levels with BCAA supplementation (P<0.05). BCKDHA expression was greater in the liver of LBWT compared to NBWT pigs irrespective of diet (P<0.05). Circulating branched‐chain α‐keto acids increased only in response to BCAA supplementation (P<0.0001). LBWT pigs expressed less Sestrin2 in skeletal muscle (P<0.01), suggesting enhanced sensitivity to leucine. However, despite lower expression of Sestrin2 and increased intramuscular BCAA concentrations, 4EBP1 and S6K1 phosphorylation were not affected by BCAA supplementation. These data suggest that branched‐chain amino acid supplementation did not enhance translation initiation in muscles of neonatal pigs regardless of birth weight.
Effect of Acute Creatine Supplementation on Arterial Stiffness and Muscle Oxygen Saturation
Authors: Pellinger Thomas K. , Gimblet Colin J. , Vance Morgan M. , Shepherd Meghan , Ortlip Austin T. , Staudmyer Timothy B. , Lamanca John J. , Werner Timothy J.
Published: April 2020

Objective

To determine the effects of acute creatine monohydrate supplementation on arterial stiffness (AS) and skeletal muscle oxygen saturation (SmO2). There is a void in our knowledge on the impact of acute creatine monohydrate supplementation on AS in the major elastic arteries. In addition, there is a dearth of findings on the effects of creatine supplementation on SmO2 in the lower leg. Data have indicated that creatine supplementation can result in an increase in lower leg anterior compartment pressure at rest and post exercise. Although the increased pressures seen during these studies were not pathological, this and additional factors associated with creatine supplementation could possibly effect SmO2 during exercise and recovery.

Methods

20 male, physically active participants were randomized in a double‐blind fashion to placebo (PL) (n=10, 22.9±3.1 yrs.) or creatine (CM) (n=10, 21.3±1.8 yrs.) groups. Subjects received 0.3 g/kg/day creatine monohydrate or placebo in gelatin capsules for 7 days. Ultrasonography of the carotid artery, applanation tonometry, submaximal exercise tests (10 minute treadmill activity at 3.7 mph and 9% incline), SmO2, and lower leg pain (analog visual scale and pain test algometer) assessments were conducted at baseline and on day 7 of the study period.

Results

There were baseline differences in central systolic blood pressure (cSBP) (mean difference, 109; 95% confidence interval (CI) 105 to 113, vs. mean difference, 130; 95% CI 114 to 146, p < 0.05) between the PLA and CM groups, respectively. Following the intervention, there were no significant differences between PL and CM in arterial stiffness indices between the groups. There was a significant group effect (P<0.03) but no significant effect of supplementation (P>0.05) on the % change in SmO2 during the exercise tests (CM: pre 66.49 ± 30.54; post 59.61 ± 23.87 vs. PL: pre 39.87 ± 16.72; post 38.51 ± 26.95 % change SmO2; M ± SD). Percent fat free mass (FFM) was significantly increased (mean, 68.5±7.5%; 95% CI 62.1% to 72.9%, to mean 69.3±7.5% 95% CI 62.9% to 73.6%, P<0.05) in the CM group only. There were no other significant changes in anthropometric measurements.

Conclusions

Using a randomly controlled, double‐blind trial with validated measurements of AS and SmO2, acute creatine supplementation does not appear to impact vascular compliance or oxygen saturation in skeletal muscle in young, healthy males.
Respiratory Muscle Activity during Maximal Efforts in ALS Patients and Healthy Controls
Authors: Sajjadi Elaheh , Ehrbar Jessica G. , Seven Yasin B. , Dickinson Patrick G. , Mitchell Gordon S. , Smith Barbara K.
Published: April 2020

Amyotrophic lateral sclerosis (ALS) is a type of neurodegenerative disease involving upper and lower motor neuron loss. ALS patients usually die of respiratory failure due to breathing muscle weakness from motor neuron death. It is important to better understand how breathing changes in ALS to design effective treatments to target breathing insufficiency. Breathing capacity is preserved until late stages of disease in the SOD1G93A rodent model of ALS. Possible mechanisms preserving breathing in ALS rats include: 1) spinal synaptic plasticity; 2) neuromuscular junction plasticity; and 3) transition in balance from more affected muscles (eg. diaphragm) to less affected accessory respiratory muscles. If these mechanisms also exist in humans with ALS, we predict that characteristic patterns of altered respiratory muscle activity would precede ventilatory failure, assessed via traditional pulmonary function tests. Thus, using surface EMGs (sEMG) in ALS patients, we tested the hypothesis that ALS patients exhibit differential recruitment patterns of inspiratory muscle activity during a maximal inspiratory pressure (MIP) test versus healthy age‐ and sex‐ matched controls. EMG electrodes (Bagnoli surface electrode, Delsys, Massachusetts, USA) were bilaterally placed on the diaphragm, 2nd parasternal, scalene and sternocleidomastoid muscles. Subjects (8 patients and 8 controls) were instructed to perform the MIP test. The test was repeated until 3 measurements were obtained within 10% variability. RMS EMG amplitude of MIP testing was calculated using a 100ms sliding window with MATLAB v.9.2., and relationships between MIP and EMG activity were evaluated using a linear regression. A moderate relationship (r=0.65) was seen between parasternal muscle activity and MIP in ALS patients, while the controls showed moderate correlations between neck muscle activity (sternocleidomastoid and scalene) and MIP (rSCM= 0.56 and rscalene=0.69). Moreover, MIP‐generated parasternal (r=0.76) and diaphragm (r=0.7) activity corresponded strongly with disease severity of patients, as measured by the ALS‐FRS scale. Interestingly, we did not see a clear relationship between diaphragm surface EMG activity and MIP in either patients or controls. Several factors could account for this, including the use of surface electrodes and the mass‐effort nature of the MIP test. Further analyses are underway to assess respiratory muscle activity during other inspiratory challenges and compare the amount of sEMG output distribution between controls and ALS patients. These preliminary results support the idea that ALS patients use a different pattern of muscle activity during maximal breathing effort from controls.

Support or Funding Information

Supported by: UF Clinical and Translational Science Institute (supported byNIH UL1TR001427), UF “Moonshot” grant initiative, and UF McKnight Brain Institute
Impact of Nrf2‐deficiency on vasoreactivity of posterior cerebral arteries is sex‐specific.
Authors: Caldwell Jacob T. , Reid-Foley Emily , Ballehr Kelsey , Restrepo Alexis , Dewell Isabella , Wahl John , Maraj Joshua , Park Hyerim , Delp Michael , Muller-Delp Judy
Published: April 2020

Introduction

Increases in cellular oxidative stress lead to the activation of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and the NRf2 response element, which are responsible for maintaining cellular redox homeostasis. Activation of Nrf2 may be important in adaptations that occur in the vasculature in response to exercise training. We have previously shown that the exercise training increases Nrf2 protein in skeletal muscle; however, the role of Nrf2 in cerebral vascular adaptation remains unknown. Additionally, the role of Nrf2 in sex‐specific vascular adaptations to exercise training has not been explored in any vascular bed. We hypothesized that Nrf2 deletion would differentially impact cerebral adaptations to exercise training in male and female rats.

Methods

We exercise trained male and female wild type Sprague‐Dawley (WT) rats, and their littermates lacking the Nrf2 gene (Nrf2KO). Rats were exercise trained (EX) on a motor‐driven treadmill 5 days/wk for 10–12 weeks. A mixed moderate aerobic and high‐intensity interval training protocol was utilized. At the end of the training period, posterior cerebral arterioles were isolated and cannulated for assessment of vascular reactivity. Myogenic responsiveness (pressure 0cm H20 – 130cm H20) and the responsiveness to potassium chloride (KCl) (10–100mM), and Diethlyamine‐NONO‐ate (DEA) (1e‐9 to 1e–4M) were evaluated.

Results

Myogenic responsiveness was significantly reduced in arteries from Nrf2KO rats (p<0.01). Myogenic responsiveness was significantly greater in arteries from male Nrf2KO as compared to arteries from Nrf2KO females (p<0.01). Contractile responses to KCl (30–100 mM) responses were blunted in arteries from Nrf2KO rats, but responses were not different between males and females (p=0.13). Endothelium‐independent vasodilation to the NO donor, Dea‐NONO‐ate was reduced in arteries from Nrf2KO rats as compared to WT rats, regardless of sex.

Conclusion

In conclusion, Nrf2 deficiency reduces posterior cerebral vasoreactivity; however, myogenic responsiveness is better preserved in arteries from Nrf2 males compared to females. These results may have important implications for regulation of brain blood flow and vascular adaptability to exercise training in males and females.

Support or Funding Information

Supported by NIH R15AG055029
Effect of the Exercise of Low, Medium and High Intensity in the Profile of Lipids and the Contraction and Resistance to the Fatigue of Skeletal Muscle of Rats
Authors: Gómez-Barroso Mariana , Vargas-Vargas Manuel Alejandro , Sánchez-Duarte Elizabeth , Cortés-Rojo Christian , Montoya-Pérez Rocío
Published: April 2020

Exercise has been implemented as an efficient method to maintain an optimal state of health, also, has been used as a non‐pharmacological complementary treatment in numerous pathologies. It has been documented that the application of exercise protocols improves muscle functioning, prevents lipids accumulation and cell damage, as well as the delay of onset of fatigue. The objective of this work was to evaluate the effect of three different intensities of exercise, low, medium, and high, on the development of muscle strength, fatigue resistance, and lipid profile in rats this, to identify the protocol with greater efficiency. Male Wistar rats (250–300 g) were divided into four groups: control, exercised low intensity, exercised medium intensity, and exercised high intensity. The soleus muscle and the extensor digitorum longus (EDL) of an extremity were extracted to perform muscle tension tests, while the serum was obtained to determine the lipid profile. Results showed an increase in maximum and total tension in each of the three exercise protocols respect to control, an increase of 20.12% for maximum tension, and 14.16% for the total tension in low‐intensity exercise protocol group. While in medium intensity group was observed an increase of 40.02% for the maximum tension and 28.54% for the total tension. Finally, the high‐intensity group showed an increase of 60.25% in maximum tension and 42.52% in total tension. Regarding lipid profile, rats submitted to exercise protocols obtained lower cholesterol levels compared to control. Low intensity protocol showed a decrease of 20% (ẋ = 60 mg/dL), while the average protocol intensity a decrease of 23% (ẋ = 58 mg/dL), finally the high intensity protocol presented a decrease of 53% (ẋ = 35 mg/dL), with respect to the control (ẋ = 75 mg/dL), observing greater efficiency in the high intensity group. On the other hand, triglyceride levels showed the same trend, presenting a decrease in each of the protocols, low (ẋ = 75 mg/dL), medium (ẋ = 70 mg/dL) and high intensity (ẋ = 65 mg/dL), with respect to control (ẋ = 87 mg/dL), this decrease was more evident in the group subjected to the high intensity protocol. These results prove that the implementation of an exercise protocol improves the functioning of skeletal muscle, by increasing its contraction and fatigue resistance time. In the same way, the use of these protocols decreases the lipid profile in the serum of rats. Besides, it was found that high‐intensity exercise proved to be more efficient in reducing cholesterol and triglycerides.
Repurposing Losartan as Dry Powder Inhalation (DPI) for Asthma Related Cardiovascular Complications
Authors: Pinninti Anusha S. , Ponnoth Dovenia S. , Dave Rutesh
Published: April 2020

Our previous work published suggests that systemic administration of losartan is effective against asthma and asthma related implications. Dry powder inhalation (DPI) formulations as dosage forms are gaining momentum because of their propellant‐free nature, improved physical and chemical stability, low drug dose requirements and high systemic absorption. We developed a dry powder inhalation (DPI) formulation of losartan to treat the symptoms of asthma‐related cardiovascular complications. In this study, we evaluated the compatibility of some common DPI excipients with losartan and their ability to improve critical aerodynamic properties of the formulation to make it suitable for pulmonary drug delivery. There are different manufacturing techniques available for developing DPI formulations including milling, freeze‐drying, and spray drying (SD). We used SD in the current study to develop our formulation. The screening studies were performed by following one factor at a time approach (OFAT). We used amino acids and sugars, either alone or in combination to develop the DPI formulation by using SD technique. The SD conditions were kept constant at 110°C inlet temperature, 100% aspiration, 10% (approx. 3ml/min) pump rate, and 601 liters/hour gas flow rate. The developed formulations were characterized for their physicochemical, aerodynamic, and solid‐state properties. Particle size (<5μm) is critical to understand the deposition behavior of the DPI. Our formulations have the particle size distribution of the 50% of the particles (Dx50) below the respirable range that is <6μm. The fine particle fraction (2.2 – 3.4μm) of our formulation is 53.72%, which is the amount of dose that is actually reaching the lungs and shows therapeutic activity. The respirable fraction (3.4 – 5.3 μm) of our formulation is 70.90%, which is the amount of drug that is within the inhalable range. The solid‐state characterization studies, including differential scanning calorimetry, powder X‐ray diffraction studies indicate the drug in the formulation is amorphous, and the excipients and carrier used help to keep the formulation stable. The drug‐excipient compatibility was proved by quantitatively and qualitatively analyzing the samples using High‐Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared Spectroscopic (FT‐IR). The developed formulations showed good aerosol performance and can be used for preclinical work for further evaluation of therapeutic efficacy of the losartan in asthma models.
Intestinal MiR194 Regulates Hepatic Bile Acids through Intestinal FXR‐FGF15 Pathway in Alcoholic Liver Disease in Mice
Authors: Jiang Mengwei , Liu Yunhuan , Gu Zelin , Li Fengyuan , Zhang Lihua , McClain Craig , Feng Wenke
Published: April 2020

Background

Alcoholic liver disease (ALD) is characterized by gut dysbiosis, higher hepatic and circulating levels of bile acids (BAs) and intestinal epithelial dysfunction. BA homeostasis is modulated by hepatic and intestinal nuclear farnesoid X receptor (FXR, Nr1h4), a ligand activated transcription factor, through regulating key genes involved in bile acid synthesis and metabolism. BA synthesis regulation by intestinal FXR is through induction of fibroblast growth factor 15 (FGF15) that inhibits hepatic Cyp7a1, the key gene in BA de novo synthesis. Previous studies showed that FXR expression is suppressed miR194 in various tissues. In this study, we investigated the regulation of FXR by miR194 in intestinal tissues in mice with ALD.

Methods

C57BL6 and intestinal specific FXR knockout (FXRΔIEC) mice were subjected to the Lieber DeCarli liquid diet containing 5% alcohol for 10 days, and one bonus of alcohol (5 g/kg) was gavaged on last day 9 hours before tissue collecting. Control mice were fed isocaloric maltose dextrin. A group of mice was supplemented with Lactobacillus rhamnosus GG (LGG) at a dose of 109 CFU/day for 10 days before alcohol exposure.

Results

Alcohol feeding significantly increased hepatic fat accumulation and serum ALT and AST levels. Interestingly, the alcohol‐induced effects were associated with an increased expression of miR194 and a decreased expression of FXR and FGF15 in the intestine, and with an upregulation of hepatic Cyp7a1 and BA concentration. When the mice were supplemented with probiotic LGG, intestinal miR194 expression was suppressed, FXR‐FGF15 signaling pathway was upregulated, and hepatic BA levels were decreased. Alcohol induced hepatic steatosis and injury were markedly prevented by LGG. More importantly, the beneficial effects of LGG on hepatic BA homeostasis in ALD mice were diminished when the intestinal epithelial FXR was deleted. In vitro study showed that alcohol treatment increased miR194 and decreased FXR mRNA, and inhibition of miR194 attenuated the suppression of FXR mRNA by alcohol.

Conclusions

Taken together, our results demonstrate that intestinal FXR‐FGF15 signaling plays a pivotal role in maintaining hepatic BA homeostasis in ALD mice and probiotic LGG protects against ALD through maintaining BA homeostasis regulated by intestinal miR194‐FXR‐FGF15‐mediated signaling pathways.

Support or Funding Information

Supported by grants from NIH.
Vena Caval Abnormalities Discovered During Dissection in the Anatomy Lab: Two Cadaveric Case Reports
Authors: Steiner Nicholas J. , Sgro Jonathan M. , Dowhayko Alexander P. , Raffoul Adam J. , Farias Anna
Published: April 2020

Intro

Congenital abnormalities of the inferior vena cava (IVC) are rare in the general population with the incidence of IVC duplication being between 0.2 – 3%. Persistent left superior vena cava (SVC) has an incidence of 0.3 – 0.7%. It is important to identify such variations through imaging prior to various thoracic and abdominal surgeries to minimize peri‐operative complications.

Methods

Two cadavers with vena caval abnormalities were identified in a medical school cadaveric anatomy lab. The donor’s age, sex and cause of death were identified through bequeathal services. The pathway of the aberrant structures was identified through dissection and any tributaries along their courses were isolated. Surrounding structures were examined to identify any other concurrent abnormalities.

Results

We identified two cadavers with vena caval abnormalities. The first case was a 75‐year old female whose cause of death was a bowel obstruction with comorbid dementia. On this cadaver we identified a persistent left SVC that originated from the brachiocephalic vein on the left side that measured 19 mm in diameter. The aberrant structure drained into the right atrium via the coronary sinus, which was noticeably enlarged. The second case was a 76‐year old female whose cause of death was multiorgan failure with comorbid end stage liver failure, pulmonary edema and hypothyroidism. We identified a duplicated IVC with a diameter of 12 mm originating at the left iliac vein and draining into the left renal vein. Along the duplicated IVC were multiple branches originating from the posterior abdominal cavity.

Conclusion

We present here two cases with rare vena caval abnormalities found in cadavers in our anatomy lab. The embryological origin of a duplicated IVC stems from the persistence of both left and right supracardinal veins during development. A persistent left SVC occurs when the left superior cardinal vein fails to regress inferior to the brachiocephalic vein. Although rare, recognizing these anatomical variations bears clinical relevance, especially in a surgical setting. These abnormalities are important to identify pre‐operatively to avoid unexpected peri‐operative complications.
α‐Synuclein’s Role in Platelet Exocytosis
Authors: Smith Alexis Nicole , Joshi Smita , Whiteheart Sidney W.
Published: April 2020

Platelets use SNARE‐mediated exocytosis to maintain hemostasis and thrombosis. These processes are maintained by the exocytosis of platelet releasate from the three types of granules in platelets: dense, alpha, and lysosomal. Understanding how the process of exocytosis is regulated in secretion, we look for potential SNARE regulators and found the protein α‐synuclein. α‐Synuclein appears to be the only member of the synuclein family present in platelets and is very abundant. To address the role of α‐synuclein in platelets, we examined the hemostatic phenotype of α‐synuclein−/− mice. Using a tail‐bleeding time assay, we observed that α‐synuclein−/− mice bleed for a slightly longer time compared to the wild‐type mice, suggesting a platelet granule secretion defect. Secretion kinetic and dose‐response assays showed that platelets from α‐synuclein−/− mice have defective release from the dense granules and less so from lysosomal granules. To understand the mechanism of this defect, we asked whether α‐synuclein is acting as a VAMP‐chaperone in platelets and thus modulates secretion by controlling V‐SNARE levels. The two dominant T‐SNARES SNAP‐23 and Syntaxin‐11 are not altered in α‐synuclein−/− mice. However, the dominant V‐SNARE VAMP‐8 was reduced. Other V‐SNARES in the α‐synuclein−/− mice VAMP‐2, VAMP‐3, and VAMP‐7 were normal. Additional western blotting experiments demonstrated the presence of the α‐synuclein‐interactor Cysteine String Protein, further identifying elements of the platelet secretory machinery. These experiments demonstrate a role for α‐synuclein in platelet exocytosis and hemostasis and will further fill a gap in our knowledge on α‐synuclein’s physiological function and understanding how the process of exocytosis is regulated.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health, National Heart, Lung, and Blood Institute (HL56652) and a Veterans Affairs Merit Award to S.W.W.
Corticosterone at physiological dose accelerates the healing of experimental gastric erosions in rats
Authors: Filaretova Ludmila , Komkova Olga , Morozova Olga
Published: April 2020

The data of literature about the action of glucocorticoids on the gastric ulcer/erosion healing mainly concern the effects of lasting glucocorticoid therapy or single injection of glucocorticoids at pharmacological doses. These data clearly indicate that glucocorticoids delay the natural healing of experimental gastric injury. However, the role of endogenous glucocorticoids in the gastric mucosal healing processes remained unknown for a long time. In our previous studies we tested this question and the results obtained argue the contribution of endogenous glucocorticoids to the healing of injured gastric mucosa. In the present study we verified whether natural exogenous glucocorticoids (such as corticosterone in rodents) injected at physiological doses similar to endogenous ones may contribute to the healing of gastric erosions accelerating the healing process. For this, in experiments in male SD rats we studied the effects of corticosterone injected at physiological dose 4 mg/kg or dexamethasone injected at the dose 1 mg/kg on the healing of injured gastric mucosa. The glucocorticoid receptor antagonist RU‐38486 (20 mg/kg) was injected to one half of each group 2 h before the glucocorticoid administration. Glucocorticoid or RU‐38486 vehicle (propylene glycol) was injected as a control. Indomethacin (IM) at the dose 35 mg/kg and 3.5 h gastric ischemia‐reperfusion (IR, 30 min occlusion of celiac artery followed by 3 h of reperfusion) were used as ulcerogenic stimuli in preliminary fasted rats. To estimate IM‐ or IR‐induced gastric erosion formation (erosion area) two groups of rats were sacrificed in 4 h after IM injection or 3.5 h after IR. Corticosterone or dexamethasone as well as their vehicle were injected 4 h after IM administration or 3.5 h after IR (0 time point of healing) to other groups to estimate their effects on the healing of injured gastric mucosa in 6 h and 24 h after the hormonal or vehicle administration. IM‐ and IR‐induced erosions healed with time. In control (vehicle treated rats) an average area of erosions in 6 h time point did not differ from that in 0 time point. At the same time an average area of erosions in 24 h time point was significantly lower than that in 0 time point (healing). Corticosterone but not dexamethasone injection accelerated the healing: in corticosterone treated rats significant decrease in an average area of erosions was already observed in 6 h time point. The preliminary glucocorticoid receptor antagonist RU‐38486 injection prevented accelerating effect of corticosterone on the healing process. The findings suggest that natural glucocorticoid corticosterone injected at physiological dose may accelerate the healing of experimental gastric erosions in rats.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) N 19‐15‐00430.
SPARC: Spinal cord epidural stimulation and microstimulation of dorsal root ganglion with penetrating microelectrode arrays enables selective activation of micturition and continence reflexes.
Authors: Gopinath Chaitanya , Jantz Maria K. , Kumar Ritesh , Nanivadekar Ameya C. , Ogren John I. , Chitnis Girish , Wong Liane , McLaughlin Bryan L. , Fisher Lee E. , Gaunt Robert A.
Published: April 2020

Lower urinary tract (LUT) dysfunction arising from injury and disease remain a clinical challenge due to unavailability of pharmacological treatments. Neuromodulation devices developed to tackle these disorders, such as sacral stimulators, are non‐selective for a single nerve with numerous off‐target effects while also stimulating the afferent and efferent population in a nerve. Here, we investigate whether epidural spinal cord stimulation (SCS) and microstimulation of dorsal root ganglion (DRG) through penetrating microarrays, can selectively activate and control reflexes relevant to LUT function. We demonstrate that custom high‐density epidural arrays and Utah arrays in the DRG can selectively access pelvic and pudendal innervation, and can modulate LUT function.

To determine the effects of stimulation on peripheral nerve recruitment, α‐chloralose anesthetized male cats (SCS: n=6, DRG: n=4) were instrumented with nerve cuffs on the pelvic nerve as well as the pudendal nerve and its branches. High‐density epidural electrodes were placed corresponding to the lumbosacral (L6 and L7 laminae) and sacral (S1 laminae) spinous processes. In separate DRG experiments, 32‐channel high‐density penetrating Utah arrays were implanted in the S1, S2 and S3 DRG. We stimulated epidurally and within the DRG while recording compound action potentials in nerve cuffs. Functional effects of stimulation were measured using intravesical and transurethral catheters to measure bladder and urethral pressures respectively across various stimulus amplitudes and frequencies.

All the instrumented nerves could be selectively recruited with both DRG and SCS stimulation, although not every nerve was selectivity recruited in every animal. The stimulation amplitude to evoke functional responses was higher (SCS: 600–1000 μA, DRG: 20–45 μA) than the minimum threshold to evoke antidromic activity in nerve cuffs. SCS and DRG stimulation both evoked bladder parasympathetic contractions similar to direct stimulation of the pelvic nerve. In several trials, stimulation through the same epidural electrode during cystometry, low frequency (3 Hz) stimulation evoked a continence reflex resulting in a suppression of detrusor activity, while high frequency (33 Hz) stimulation evoked a micturition reflex resulting in high intravesical pressures. Isovolumetric and filling cystometry was performed to confirm the action of these reflexes.

SCS and DRG stimulation can selectively activate pelvic and pudendal innervation, and elicit micturition and continence reflexes.

Support or Funding Information

R01 NS088184 and OT2OD024908. Craig H. Neilsen Foundation Award 476681
Age‐Related Sympathetic Dysregulation is Associated with Glial Senescence in the Brainstem
Authors: Herron Angela , Stansbury Samantha , Balasubramanian Priya , Subramanian Madhan
Published: April 2020

Aging is an independent risk factor for cardiovascular diseases such as hypertension, myocardial infarction, stroke and heart diseases. Although the specific mechanisms for age‐related increase in the incidence of cardiovascular diseases are not yet known, accumulating evidence suggests that sympathetic nervous system (SNS) dysregulation plays a crucial role. Senescent cells accumulate in the brain with age and are implicated in the pathogenesis of neurodegenerative diseases, possibly through secretion of pro‐inflammatory cytokines, growth factors, and proteases through acquisition of senescence associated secretory phenotype (SASP). Our laboratory studies previously established that aging causes cellular senescence in the key brainstem regions important for sympathetic nervous system activity. In our current study, we investigated which cell type undergoes senescence in the brainstem of aged mice. Neurons are post‐mitotic and do not undergo senescence, whereas glial cells can undergo senescence. Hence, we hypothesized that aging induces glial senescence in the brainstem. To address this hypothesis, we used 28‐month old (n=4) and 4‐month old (n=5) C57BL/6J mice. The animals were sacrificed, and the brainstems were collected. The freshly isolated brainstem was enzymatically digested (45 minutes, 37°C) and filtered through a 30μm mesh to form a cell suspension. Neuron isolation kit from Miltenyi Biotec was used to isolate glial and neuronal enriched cell population from the brainstem cell suspension. RNA was extracted from these sorted cells and used for RT‐PCR analysis. Senescence was assessed using gene expression of cell cycle inhibitors and SASP factors. Data was analyzed by unpaired student’s t‐test and a p‐value less than 0.05 was considered statistically significant. We observed an increase in the gene expression of cell cycle inhibitors, p16, p21 and p53 in the glial cell fraction isolated from the brainstem of aged mice compared to its young counterparts. In addition, there was an increase in the mRNA levels of SASP factors such as the MCP1, TNF‐α, and IL‐1β suggesting that aging induces senescence in the glial cells of the brainstem. We were not able to obtain viable neurons for senescence analysis. Glial cells are known to play a supporting role in neuronal function through regulation of neurotransmission. Future studies will address glial cell specific mechanisms and understand how glia‐neuron crosstalk contributes to SNS dysregulation in aging.

Support or Funding Information

NIH‐HL148844 and RED account funds
Optimized Protocol for Robust Differentiation of SH‐SY5Y Cells into Neuronal‐like Cells: An in vitro Model of Human Neuronal Cultures
Authors: Jun Jinwoo , Meyer Madeline R. , Cowles Landry E. , Hall Emma M. , Arnold Benjamin W. , Ambegaokar Surendra S.
Published: April 2020

The SH‐SY5Y cell line, derived from a human glioblastoma, grow and divide indefinitely under normal conditions, but have the potential to differentiate into neuronal‐like cells when treated with retinoic acid (RA). However, the extent of differentiation has been contested, leading to a variety of protocols to induce differentiation. We took a systematic approach to test the various conditions and treatments used in previously published studies to develop an optimized protocol that yields robust differentiation in an efficient manner. In morphological assays, robust and extensive processes from cell bodies and network development between cells in culture were established when brain‐derived neurotrophic factor (BDNF), B‐27, and potassium (K+) were supplemented to RA, that survived up to 10 days in culture. One‐day treatment of RA and BDNF during differentiation was as effective as prolonged post‐differentiation treatment of BDNF, thus reducing the amount of BDNF required. Conversely, supplements of vitamin B12, AraC, and dB‐cAMP did not provide any significant difference in differentiation. In RT‐qPCR analyses, our optimized protocol led to significant increased expression of microtubule associated protein tau (MAPT) and microtubule associated protein 2 (MAP2), both of which are markers of neuronal cells. We are validating the sub‐cellular localization of these proteins via immunohistochemistry. This optimized protocol thus differentiates SH‐SY5Y cells into robust and reliable neuronal‐like cells, which can be used as a model of human neuronal cells and neurodevelopment in a reproducible in vitro system, without the ethical concerns or practical limitations of using fetal cells for human neuronal cultures.

Support or Funding Information

Ohio Wesleyan University: Department of Botany and Microbiology, Neuroscience Program, Summer Science Research Program, and Theory‐to‐Practice Grant.
More than Words? An Analysis of the Sentiment Polarity of Anatomy Lectures in Medical and Allied Health Education
Authors: Muldoon Kathleen M. , Williams Avery A. , Grow Wade A. , Kaufman Jason A. , Plochocki Jeffrey H. , Dobson Seth D.
Published: April 2020

Previous research suggests that medical and allied health education can erode student empathy. The decline in student empathy seems to correlate with the “hidden curriculum” ‐ the attitudes and values conveyed, most often in an implicit, informal, and unintentional fashion, via the formal educational structures, practices, and culture of an educational institution. We typically think of education as simply the transmission of facts, but the words and images used to convey those facts can also shape attitudes, even if this impact is unintended. Our study examines variation in the sentiment polarity of lectures given within the anatomy curriculum at one institution. We tested the following hypotheses: 1) the sentiment polarity of first‐year anatomy lectures is significantly different from neutral and 2) the sentiment polarity of lectures is correlated with the percentage of total lecture time spent on clinical applications. Anatomy lectures (n = 120) recorded during the 2016–17 academic year across all programs (excluding veterinary medicine) at Midwestern University were anonymized and transcribed by work study students. We analyzed transcribed lectures using text mining and a published sentiment lexicon. We counted individual words that matched the lexicon with a positive or negative sentiment, in addition to total word count, and individual word frequency. Standard anatomical descriptors (i.e., “inferior”) were excluded from the analysis. The net sentiment score for each lecture was defined as the difference between the number of positive vs negative words. We quantified the clinical content of a lecture as the proportion of time (minutes) spent on clinical applications. We used a one‐sample Wilcox test to evaluate Hypothesis 1 and Spearman rank correlation to evaluate Hypothesis 2. We used the text mining package tidytext and ran all statistical tests in R. The median sentiment score across all lectures was −18.5, which was significantly different from zero (p < .001). Sentiment score was negatively correlated with the proportion of lecture time spent on clinical applications (rho = −0.316, p < .001). These results suggest that first‐year anatomy students might be at risk for empathy erosion especially in the context of clinical applications because of the relatively high frequency of words with negative sentiment. This study was approved by Midwestern IRB (#AZ 1111, Approved on 01/25/2018).