bluuebunny/crossref_metadata_2025 huggingface dataset:
High Fat Fed Nr4a1 Knock Out Mouse has Significant Modulation of Mitochondrial Respiration Across Various Tissues
Authors: Wynn Adam G. , Garland Kevin G. , Kener Kyle B. , Weber K. Scott , Bikman Benjamin T. , Hancock Chad R. , Tessem Jeffery Sivert
Published: June 2021

The orphan nuclear receptor Nr4a1 is critical for fuel utilization in various tissues. Studies have shown that Nr4a1 controls expression of genes necessary for mitochondrial respiration. Further studies have demonstrated that Nr4a1 knock out animals fed a high fat diet have significant shifts in fuel utilization profiles. We sought to define the changes in mitochondrial respiration and function across various tissues in wild type and Nr4a1 KO mice fed a normal chow or high fat chow diet for twenty weeks. Nr4a1 KO mice fed the high fat diet had significantly worse glucose tolerance than wild type animals fed the high fat diet. Furthermore, Nr4a1 deficient animals demonstrated significant changes in the macrophage population as compared to wild type animals. Mitochondrial respiration of muscle and liver from Nr4a1 KO animals fed normal chow or high fat diet were significantly impaired compared to the wild type controls. Interestingly, mitochondrial respiration from macrophages was significantly enhanced in the Nr4a1 KO animals. These data demonstrate systemic effects of Nr4a1 deletion, as well as demonstrating a mechanism by which Nr4a1 KO have shifts in fuel utilization during high fat feeding regimes.

Support or Funding Information

This study was funded by a BYU ORCA Grant to KG and a BYU MEG Grant to JST.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An In Situ Kidney Slice Model for Studying Angiotensin II‐ and TRPC5‐Mediated Calcium Signaling
Authors: Schleifenbaum Johanna , Hennig Grant W. , Nelson Mark T.
Published: June 2021

Current approaches to study calcium signaling in glomeruli use isolated glomeruli or cultured cells, however the structure and physiology of the tissue is likely to be perturbed by the isolation process in both models. We used kidney slices maintained in physiological solution to better preserve the macroscopic structure of glomeruli and measured the impact of angiotensin II receptor activation and blockade of Transient Potential Receptor Canonical 5 (TRPC5) to examine their possible interaction in a number of pre‐ and intraglomerular cell types using spinning‐disk confocal calcium imaging.

Calcium transients and tissue distortion in intact kidney slices of mice expressing genetically modified calcium‐sensors (GCaMP5) in Cx40‐positive cells were recorded and analyzed using particle analysis.

We observed spontaneous calcium transients in preglomerular cells located at the vascular pole that were blocked by the SERCA pump inhibitor, cyclopiazonic acid (CPA). Stimulation with angiotensin II increased calcium activity and contraction in these cells and induced calcium transients in distinct populations of intraglomerular cells that were also blocked with CPA. While removal of external calcium did not change angiotensin II‐induced calcium transients in intraglomerular cells, the frequency of calcium transients in preglomerular cells was significantly reduced. In angiotensin II‐preincubated kidney slices, spontaneous calcium transients in the preglomerular area were diminished by the specific TRPC4/5 inhibitor, ML204. If given alone, ML204 completely inhibited all preglomerular calcium transients.

Thick kidney slices maintained in vitro appear to better preserve the structure and responses of different cell types in glomeruli as measured using calcium transients and contractions. In our experimental set‐up, angiotensin II induced calcium transients in intraglomerular cells, while its impact on preglomerular cells appeared more indirect. As the TRPC5 antagonist was more effective at inhibiting calcium transients in preglomerular cells when the tissue was not prestimulated with angiotensin II, we suggest that TRPC5‐mediated calcium signaling is at least party modulated by angiotensin II.

Support or Funding Information

Supported by Deutsche Forschungsgemeinschaft (DFG)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal V1A Receptor Does Not Increase Na+ Excretion in the Rat Isolated Kidney Perfused at Constant Pressure
Authors: Lindstrom Beatriz , Kalina Sara , Wisniewski Kazimierz , Laporte Regent
Published: June 2021

The effect of arginine vasopressin (AVP) on Na+ excretion has been the subject of many studies and is still a matter of debate. While AVP given at physiological doses was shown to enhance Na+ excretion in water‐loaded rats, others have shown that AVP‐induced natriuresis occurred at doses associated with increases in mean arterial pressure, suggesting that pressure natriuresis may be the underlying mechanism. This natriuresis is thought to be mediated by the V1A receptor (V1AR). To investigate if the renal V1AR mediates natriuresis, we employed the rat isolated kidney perfused at constant pressure to assess the effect of the selective V1AR agonist, [Phe2, Ile3, Orn8]‐vasopressin, on various parameters. Specifically, we sought to test the hypothesis that selective activation of V1AR in the kidney increases urinary excretion of Na+.

Each isolated kidney was perfused for a total of 85 min, starting with 10 min for equilibration, and followed by five 15 min‐periods with ascending concentrations of the agonist (0, 30, 100, 300, and 1000 pM). Time‐control kidneys were perfused with media without the agonist for the entire 85 min.

We found that the agonist increased renal vascular resistance in a concentration‐dependent manner without changing GFR. At the highest concentrations, the agonist showed antidiuretic activity characterized by decreased urine flow rate and effective water clearance, and increased urine osmolality. Our results further show that although there was an increase in absolute Na+ excretion and fractional excretion Na+, no difference was observed between treated‐ and control‐kidneys, suggesting that the changes in Na+ handling were inherent to perfusion itself rather than activation of the renal V1AR.

We conclude that AVP‐induced natriuresis may not be mediated by the renal V1AR, and that AVP may induce natriuresis indirectly through extrarenal factors lacking in the isolated kidney.

Support or Funding Information

Ferring Research Institute, Inc., San Diego, CA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Chronic Lower Limb Heating on Cutaneous Microvascular Function in Aged Humans
Authors: Romero Steven A. , Gagnon Daniel , Adams Amy , Jaffery Manall , Crandall Craig G.
Published: June 2021

Cutaneous microvascular function is reduced with advancing age. Recent evidence suggests that chronic local heating of a limb improves cutaneous microvascular function in young healthy humans through nitric oxide‐dependent mechanisms. However, exposure to chronic heating of limb has not been studied in aged adults. The purpose of this study was to test the hypothesis that chronic lower limb heating improves nitric oxide‐dependent vasodilation in the cutaneous microvasculature of aged adults. Six healthy aged adults (5 females; age 67 ± 4 years; height 164 ± 3 cm; weight 70 ± 18 kg; mean ± SD) were exposed to 8 weeks of chronic lower limb heating. Subjects immersed their lower limbs ~33 cm into a heated (~ 42°C) and circulated water bath 4 days per week, for 45 min per session. Before and after the 8‐week regimen, cutaneous blood flow (laser Doppler red blood cell flux) was measured in response to 39 °C heating of a discreet area of skin exposed to chronic heating with and without (control) intradermal microdialysis administration of the nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methyl ester hydrochloride (L‐NAME; 20 mM). Estimated maximal cutaneous blood flow was assessed in response to combined local heating to 43 °C and intradermal infusion of sodium nitroprusside (56 mM). Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure and expressed as a percentage of maximal vasodilation (%CVCmax), where appropriate. Cutaneous vascular conductance during 39 °C local heating was unchanged following chronic heating (pre 78 ± 5 %CVCmax vs. post 85 ± 7 %CVCmax; P = 0.2). Likewise, nitric oxide‐dependent vasodilation (calculated as the difference between control and L‐NAME sites) did not differ (pre 68 ± 3 % vs. post 64 ± 3 %; P = 0.3). Interestingly, maximal cutaneous vascular conductance increased after chronic heating (pre 1.8 ± 0.1 flux mmHg−1 vs. post 2.4 ± 0.2 flux mmHg−1; P < 0.05). Taken together, chronic lower limb heating does not improve nitric oxide‐dependent cutaneous microvascular function during a local thermal provocation in aged adults, an effect that may be superseded by vascular remodeling as maximal vasodilator capacity was increased after the 8‐week regimen.

Support or Funding Information

National Institutes of Health (GM‐068865 & GM‐117693)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NO EFFECT OF HEAD UP TILT ON TUMOR PERFUSION IN A RAT PRE‐CLINICAL MODEL OF PROSTATE CANCER
Authors: Rand Taylor , Pyle Joseph , Baumfalk Dryden , Opoku‐Acheampong Alexander , Musch Timothy , Behnke Brad
Published: June 2021

Previous research has demonstrated an enhanced prostate tumor blood flow and oxygenation associated with the augmented mean arterial pressure during exercise. Further, prostate tumor arterioles lack functional smooth muscle and have a diminished myogenic response. Thus, we tested the hypothesis that with elevations in the heart‐to‐prostate tumor hydrostatic gradient 70° head‐up tilt (HUT) would result in an enhanced perfusion of the prostate tumor. An enhanced tumor perfusion may improve tumor oxygenation and radiation therapy outcomes.

Methods

Dunning AT‐1 tumor cells (1×104) were injected into the ventral prostate of male Copenhagen rats (n=7) and studies were performed ~ 6 weeks after cell injection. After 5 minutes in each body position, fluorescent microspheres (red and blue/green, ~250,000 spheres per label) were injected via the carotid artery in the level and HUT body positions. Blood flow was determined in the tumor, kidneys and soleus muscle. Vascular conductance was calculated as blood flow divided by mean arterial pressure (MAP). Blood flow and conductance rates were calculated and a two‐tailed paired t‐test was conducted to compare values in the level vs. HUT positions.

Results

Average tumor weight was 7.1 ± 0.5 g. MAP was not different between level and HUT (p = 0.15). Adequate blood flow distribution, as determined by a <15% difference in flow between the left/right kidney and/or soleus muscle, was achieved in all animals. Tumor blood flow was not different between level and HUT (12.0 ± 1.1 vs. 10.4 ± 0.8 ml/100 g/min, respectively). No difference was observed between body positions in tumor vascular conductance (p>0.05).

Discussion

Contrary to our hypothesis, we did not see any significant differences in either blood flow or vascular conductance in the tumor with HUT. With HUT, the prostate shows an increase in vascular resistance; thus, the combination of immature and co‐opted host tissue arterioles present in tumors may have resulted in an unchanged perfusion of the tumor with tilt. Specifically, there would be an enhanced vascular resistance of the host‐tissue (prostate) that may counter hydrostatic differences in perfusion pressure. These findings suggest that elevations in the hydrostatic gradient via HUT may not be sufficient to enhance prostate tumor perfusion.

Support or Funding Information

American Cancer Society RSG 14‐150‐1‐CCE (BJB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelium Mediated Dilation Does Not Blunt α1‐adrenergic Vasoconstriction in First Order Arterioles
Authors: Ferguson Brian S. , Kozina Erik , Hill Michael A. , Clifford Philip S.
Published: June 2021

In contracting skeletal muscle, several vasoactive compounds have been proposed to override sympathetic vasoconstriction, a phenomenon termed functional sympatholysis. Adenosine triphosphate (ATP) has emerged as a candidate molecule capable of mediating these effects, however, the precise source and mechanisms have not been fully elucidated. We tested the hypothesis that endothelium mediated mechanisms are responsible for attenuating α‐adrenergic vasoconstriction. Experiments were conducted on 1A arterioles surgically dissected from the rat cremaster muscle. Sequential sections of arteriole were cannulated on glass micropipettes, pressurized to 70 mmHg, and warmed to 34°C in physiological saline solution. We measured changes in isolated vessel diameter in response to logarithmic concentrations of the α1‐adrenergic receptor specific agonist phenylephrine following dilatory responses to intraluminal flow (flow mediated dilation, n=12), intraluminal adenosine (n=10), or intraluminal ATP (n=11). All vessels tested spontaneously developed myogenic tone (>40% from initial diameter) and displayed functioning endothelium (>40% dilation to acetylcholine). Vessels dilated equally 177±19 μm, 183±10 μm, and 181±22 μm during flow mediated dilation, adenosine, and ATP, respectively. There were no significant differences between treatments following concentration‐response curves generated by phenylephrine administration and the EC50 was not different −5.90±0.02, −6.12±0.18, and −6.00±0.06 for flow mediated dilation, adenosine, or ATP (p=NS). Thus, there was no attenuation of α1‐adrenergic vasoconstriction in response to intraluminal flow mediated dilation, adenosine, or ATP in 1A arterioles. These results suggest that endothelium mediated dilation does not blunt α1‐adrenergic vasoconstriction in first order skeletal muscle arterioles.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Remote Ischemic Conditioning in Rodents: Tourniquet vs. Cuff Occlusion
Authors: El Dabagh Yasmin Sanaya Hauge , Mohanakumar Sheyanth , Hagensen Mette K. , Pedersen Michael
Published: June 2021

Purpose

Remote ischemic conditioning (RIC) implies that transient cycles of ischemia and reperfusion in a limb can activate innate protective responses against ischemia. RIC is usually accomplished in rodents by tightening either a tourniquet or an inflatable cuff on the hind limb. However, the optimal method is not established. Therefore, the purpose of the present study was to perform a comparison of the effects of RIC performed with a tourniquet and an inflatable cuff.

Methods

Eleven male C57/Bl6 mice were randomized into three groups: sham occlusion (n=3), tourniquet occlusion (n=4) and cuff occlusion at 200 mm Hg (n=4). Animals receiving occlusions were subjected to two cycles of 5 minutes unilateral hind limb occlusion and 5 minutes of reperfusion. Occlusion of blood flow to the hind limb was verified with Near Infrared Fluorescence (NIRF) imaging (figure 1). Motor function of the hind limbs was evaluated using a Rotarod, muscle tissue was harvested for histological analysis, and wet‐to‐dry muscle weight ratio was measured.

Results

NIRF imaging showed that complete occlusion of blood flow to the hind limb was obtained with both tourniquet and cuff occlusion. The tourniquet group scored significantly lower in the motor function tests (p<0.05) compared with the sham and cuff group which did not differ significantly (figure 2). There were no significant histological signs of muscle damage and wet‐to‐dry muscle weight ratio did not imply edema in any groups.

Conclusion

Complete occlusion of blood flow to the hind limb can be obtained with both a tourniquet and an inflatable blood pressure cuff. Thus, both methods can be used to induce RIC. However, motor function of the hind limb was reduced in animals exposed to tourniquet occlusion indicating that the inflatable cuff should be the preferred method in order to reduce stress on the animals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Intermittent Hypoxia Enhances Respiratory Muscle Weakness in Dystrophindeficient mdx Mice
Authors: Burns David P. , Edge Deirdre , Murphy Kevin , O'Halloran Ken D.
Published: June 2021

Sleep‐disordered breathing (SDB) is a prominent feature of neuromuscular disease, likely due to pharyngeal dilator muscle weakness. Boys with Duchenne muscular dystrophy (DMD) experience nocturnal desaturation during sleep as a result of obstructive and central apnoea. We have previously described maladaptive changes in respiratory muscle structure and function in rodent models of chronic intermittent hypoxia (CIH), a hallmark feature of SDB driving many pathologies. We hypothesized that CIH exacerbates respiratory muscle dysfunction in muscular dystrophy. We sought to explore the effects of CIH on respiratory muscle structure and function in wild‐type (WT) and dystrophin‐deficient mdx mice.

Six week old male WT and mdx mice were exposed to CIH, consisting of alternating bouts of hypoxia (90s; 5–6.5% O2 at the nadir) and normoxia (210s, 21% O2) for 12 cycles per hour, 8 hours per day for 14 days. Sham animals were exposed to normoxia in parallel. The diaphragm and sternohyoid (representative pharyngeal dilator) muscles were excised and functional parameters were examined ex vivo.

Substantial muscle weakness was observed in dystrophic diaphragm and sternohyoid muscle compared with WT muscles. Exposure to CIH elicited significant muscle weakness in the diaphragm and sternohyoid of WT mice. In mdx mice, exposure to CIH resulted in further weakness in the compromised diaphragm. CIH‐induced potentiation of muscle weakness was less robust in the sternohyoid. We have recently established the essential role of NADPH oxidase in CIH‐induced respiratory muscle weakness. We are currently exploring the molecular mechanisms of CIH‐induced respiratory muscle weakness in WT and mdx mice.

Our findings demonstrate that exposure to CIH exacerbates respiratory muscle weakness in the mdx model of muscular dystrophy. Since SDB is a feature of DMD, we reason that SDB may accelerate respiratory insufficiency in DMD. An understanding of the pathogenic factors driving CIH‐induced muscle weakness in mdx may help inform therapeutic strategies that may have relevance to DMD boys.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α2δ‐1 Is Essential for Angiotensin II‐Induced Sympathoexcitation and NMDA Receptor Hyperactivity in the Hypothalamus
Authors: Ma Huijie , Chen Shao‐Rui , Chen Hong , Zhou Jing‐Jing , Li De‐Pei , Pan Hui‐Lin
Published: June 2021

Both the sympathetic nervous system and renin‐angiotensin system are critically involved in hypertension development. Although angiotensin II (Ang II) stimulates presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) to increase sympathetic vasomotor tone, the molecular mechanism responsible for this action remains unclear. The glutamate N‐methyl‐D‐aspartate receptor (NMDAR) in the PVN controls sympathetic outflow in hypertension. In this study, we determined how Ang II affects synaptic NMDARs in spinally projecting PVN neurons. Coimmunoprecipitation assays showed that α2δ‐1, commonly known as a voltage‐gated calcium channel subunit, interacted with the NMDAR in the hypothalamus of rats and humans. Ang II increased the synaptic expression level of α2δ‐1–NMDAR complexes. Also, Ang II increased pre‐ and postsynaptic NMDAR activity via AT1 receptors, and such effects were abolished either by treatment with pregabalin, an inhibitory α2δ‐1 ligand, or by interrupting the α2δ‐1–NMDAR interaction with α2δ‐1Tat peptide. In α2δ‐1 knockout mice, Ang II failed to affect the pre‐ and postsynaptic NMDAR activity of PVN neurons. In addition, the α2δ‐1Tat peptide blocked the sympathoexcitatory response to the microinjection of Ang II into the PVN. Our findings indicate that by promoting the synaptic trafficking of α2δ‐1–bound NMDARs, Ang II augments sympathetic vasomotor tone and excitatory glutamatergic input to PVN presympathetic neurons.

Support or Funding Information

This work was supported by the National Institutes of Health (Grant R01 HL131161) and the N.G. and Helen T. Hawkins Endowment (to H.‐L.P.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CCL2‐CCR2 Signalling in the Hypothalamic Paraventricular Nucleus Elicits Sympathetic‐Mediated Blood Pressure Elevations Through Monocyte and Lymphocyte Recruitment
Authors: Elsaafien Khalid , Korim Willian Seji , May Clive Newton , Yao Song Tieng
Published: June 2021

Chemokine C‐C motif ligand 2 (CCL2) is a chemotactic cytokine, which is elevated in the plasma of hypertensive patients. In experimental models of hypertension, CCL2 levels are increased by 3‐fold in the paraventricular nucleus of the hypothalamus (PVN). However, the underlying neural and molecular mechanisms leading to the development of hypertension still remain unknown. Here, we show for the first time that in Sprague‐Dawley rats activation of receptors for CCL2 (i.e. CCR2) in the PVN elicited biphasic blood pressure (BP) responses and changes in renal sympathetic nerve activity (RSNA), consisting of early reductions followed by long‐lasting increases in RSNA and mean arterial pressure (MAP). These responses were abolished by prior CCR2 blockade with the antagonist RS‐102895. Purinergic transmission between astrocytes and neurons mediated the initial sympathoinhibition, whereas monocyte and lymphocyte infiltration into the PVN were involved in the subsequent sympathoexcitation. Our anatomical results support these findings by showing that CCR2 are predominantly expressed in the membrane of astrocytes, which make close appositions with PVN neurons projecting to the rostral ventrolateral medulla (RVLM) ‐ where cardiovascular sympathetic premotor neurons are found. Finally, in renovascular hypertensive rats (2 kidney ‐ 1 clip) plasma and cerebrospinal fluid levels of CCL2 were elevated with an onset occurring on the first week ‐ prior to the development of hypertension. Microglia activation and lymphocyte infiltration in the PVN were also evident by the second week, when animals were still pre‐hypertensive. The results here suggest that CCL2 upregulation, microglia activation, and lymphocyte infiltration in the PVN precede the sustained increases in sympathetic activity and blood pressure observed in hypertension.

Support or Funding Information

Department of Health, Australian Government | National Health and Medical Research Council (NHMRC): Song T Yao, GNT1079680. Rebecca L Cooper Medical Foundation: Willian S Korim. Australian Government Research Training Program Scholarships: Khalid Elsaafien. Department of Industry, Innovation, Science, Research and Tertiary Education, Australian Government | Australian Research Council (ARC): Song T Yao, FT170100363.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Centrally administered Ang‐(1‐7) via Mas receptors reverses psychosocial stress‐induced cardiovascular responses in mice with muscular dystrophy
Authors: Yang Liping , Pang Hong Weng , Speth Robert C. , Narayanan Nandakumar , Weiss Robert M. , Sabharwal Rasna
Published: June 2021

Muscular dystrophy is a devastating disease of muscle weakness, atrophy and dilated cardiomyopathy (DCM). We have demonstrated that angiotensin II (Ang II)‐dependent autonomic dysfunction precedes and predicts development of DCM in sarcoglycan delta deficient (Sgcd−/−) mice, an established model of muscular dystrophy [Exp Physiol 2015]. More recently, we found that Sgcd−/− mice exhibit depression‐like behaviors and are susceptible to psychosocial stress‐induced bradycardia, hypotension and arrhythmias at older ages (>45 weeks of age) [FASEB J 2017]. Thus, we hypothesized that central administration of angiotensin‐(1‐7) in older Sgcd−/− mice protects against Ang II‐mediated cardiovascular abnormalities during acute psychosocial stress challenge. Mean blood pressure (MAP) and heart rate (HR) were measured by telemetry in control C57BL6 (n=3–5) and Sgcd−/− (n=3–8) mice during social defeat (~15 min exposure to intruder mouse). At 40±3 weeks of age, mice were randomly assigned to one of the four experimental groups – (i) vehicle (saline); (ii) losartan (Ang II‐type 1 receptor antagonist, 1 mcg/kg/hr); (iii) Ang‐(1‐7) (100 ng/kg/hr); and (iv) Ang‐(1‐7)+Mas antagonist A779 (100 ng/kg/hr and 0.5 mcg/kg/hr, respectively). Drugs were administered ICV (catheter connected to osmotic minipump) for 8 weeks. Stressed Sgcd−/− mice exhibit an increase in AT1R‐binding (receptor autoradiography) in subfornical organ, organum vasculosum laminae terminalis, and paraventricular nucleus of hypothalamus. Social defeat produced profound bradycardia and hypotension in Sgcd−/− mice (see Table). Interestingly, while these drastic effects on MAP and HR in Sgcd−/− mice were modestly attenuated by losartan, the effects were completely abolished by Ang‐(1‐7) (P<0.05 vs. vehicle) and remained unaltered by Ang‐(1‐7)+A779 (NS vs. vehicle). Moreover, the increased plasma corticosterone (+86%) and norepinephrine (+62%) in stressed Sgcd−/− mice were normalized by Ang‐(1‐7) (P<0.05 vs. control).

We conclude that Ang‐(1‐7) acting centrally via Mas receptors abrogates cardiovascular response to psychosocial stress and is likely a more effective treatment strategy than Ang II‐type 1 receptor antagonists in muscular dystrophy.

Support or Funding Information

AHA GIA17GRNT33670004

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of microRNA‐195 in age‐dependent impairment of cognitive function
Authors: Chan Julie YH. , Chao Yung‐Mei , Juo Suh‐Hang
Published: June 2021

MicroRNA‐195 (miR‐195) is a member of the miR‐16/15/195/424/497 family. It is abundantly expressed in normal tissue, including the brain. Recently, an anti‐senescence role of miR‐195 was reported. However, its significance in age‐associated change in cognitive function has not been properly characterized. Using a tamoxifen‐inducible miR‐195 knock out (miR‐195+/− KO) mice based on the Cre‐loxP system, we compared in this study temporal profiling of cognitive functions in the mice at age of 3, 6, 9, 12 and 15 months to their wild type (WT) control. The Morris water maze was performed to evaluate cognitive function, sequential magnetic resonance imaging (MRI), coupled with diffusion tensor imaging (DTI) acquisition was carried out to evaluate the brain connectivity. In both WT and miR‐195+/− KO mice, the slope of time latency to reach the hidden platform in the water maze during the 5 consecutive training days became gentle, but not different between the two groups, suggesting there is no difference in the age dependency of learning in both groups. However, at 12 and 15 months of age, both preference and time spending in the target quadrant were notably shorter in miR‐195+/− KO mice, compared to the WT mice. There is no difference in total distance traveled and velocity between WT and miR‐195+/− KO mice during the tests, suggesting the locomotor ability was not affected in the KO mice. Next generation sequence analysis further revealed that among the target genes of miR‐195, expression of both Plexin‐A1 and its ligand, Sema3A, were significantly increased in the hippocampus of miR‐195+/− KO mice. Sema3A binds with Plexin‐A1 to induce neuronal apoptosis. Tractographic analysis based on MRI/DTI of forebrain image revealed no apparent change in connectivity between the hippocampus and cortical areas of both WT and miR‐195+/− KO mice at age of 12 or 15 months. Taken together, these data indicate that while miR‐195 may not play a permissive role in spatial learning, it is crucial for memory retrieving during aging process. This may mediate via suppression of neuronal apoptosis in the hippocampus when changes in hippocampal‐cortical neural connectivity are not yet bechanced.

Support or Funding Information

MOST106‐2314‐B182A‐003 from the Ministry of Science and Technology, Taiwan; OMRPG8G0011 from Chang Gung Medical Foundation, Taiwan

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influences of Ethanol on Inflammation and Apoptosis Following Transient Focal Cerebral Ischemia
Authors: Li Chun , Xu Guodong , Parsiola Anne L. , Li Jiyu , McCarter Kimberly D. , Sun Hong
Published: June 2021

Background

Inflammation and apoptosis are two key elements involved in cerebral ischemia/reperfusion (I/R) injury. Ethanol is one of the most commonly and regularly used chemical substances. We determined the influences of chronic ethanol consumption on inflammation and apoptosis following transient focal cerebral ischemia.

Methods

Male C57/BL6J mice were divided into 6 groups and gavage fed with 0.175, 0.35, 0.7, 1.4, 2.8 g/kg/d ethanol or volume‐matched water once a day for eight weeks. Cerebral I/R injury, adhesion molecules, microglial activation, neutrophil infiltration, pro‐ and anti‐inflammatory cytokines/chemokines, MMPs, cleaved caspase‐3‐positive neurons, and TUNEL‐positive neurons were evaluated at 24 hours after a 90‐minute unilateral middle cerebral artery occlusion (MCAO).

Results

Cerebral I/R injury was only significantly reduced in 0.7 g/kg/d ethanol group (peak blood ethanol concentration: 9 mM) and increased in 2.8 g/kg/d ethanol group (peak blood ethanol concentration: 37 mM). Baseline and post‐ischemic upregulation of ICAM‐1 and E‐selectin were suppressed in all ethanol groups. Post‐ischemic microglial activation and neutrophil infiltration were significantly alleviated in 0.175–1.4 g/kg/d ethanol groups but dramatically exacerbated in 2.8 g/kg/d ethanol group. Ethanol tends to dose‐dependently increase both baseline pro‐ and anti‐inflammatory cytokines/chemokines, but dose‐dependently suppressed post‐ischemic increase in pro‐inflammatory cytokines/chemokines. Surprisingly, 2.8 g/kg/d ethanol strengthened post‐ischemic increase in anti‐inflammatory cytokines/chemokines. Furthermore, although baseline MMP‐9 activity was reduced in 0.7 g/kg/d ethanol group, the magnitude of post‐ischemic increase in MMP‐9 activity was significantly less in 2.8 g/kg/d ethanol group. Both cleaved caspase‐3‐positive neurons and TUNEL‐positive neurons were significantly less in 0.7 g/kg/d ethanol group.

Conclusions

Light alcohol consumption protects against cerebral I/R injury by suppressing post‐ischemic inflammation and apoptosis, whereas heavy alcohol consumption may exacerbate cerebral I/R injury via an inflammation‐ and apoptosis‐independent mechanism.

Support or Funding Information

This study was supported by a National Institutes of Health Grant (AA023610) and funds from Louisiana State University Health Sciences Center‐Shreveport to HS, a postdoctoral fellowship to CL and a predoctoral fellowship to KDM from the Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Science Center‐Shreveport.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C1q Intrathecal synthesis in patients without inflammatory diseases with blood‐brain dysfunction
Authors: Lumpuy‐Castillo Jairo , González‐Losada Cristobal , Schmitz Matthias , Hansen Anette Gudmann , Jensenius Jens Christian , Dorta‐Contreras Alberto Juan
Published: June 2021

The role of complement system in the central nervous system is not enough clarified. For this reason it requires to know what happen in extreme situations. The objective is to know if under blood‐cerebrospinal fluid dysfunction how the classical complement pathway by analyzing C1q is possible to be intrathecally synthesized. It should consider three topics: the molecular size dependent concentration between CSF and blood. The transfer variation between blood and CSF and, and the CSF C1q concentration correlation with the albumin CSF/serum quotient (QAlb). C1q was assayed in samples of CSF and serum by ELISA. Albumin, immunoglobulin‐CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the control groups. Group comprised 20 patients without inflammatory diseases but with increased QAlb. C1q concentration in CSF was at least five‐fold higher than expected for a molecular‐size‐dependent passage from blood. In a QC1q/QAlb quotient diagram 7/20 cases showed an intrathecal fraction in some cases over 80% of total CSF C1q concentration. The absolute C1q concentration in CSF increases with increasing Q Alb. C1q is possible to be intrathecally synthetized in patients with blood brain barrier dysfunction

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
M Ficolin: Diffusion dynamics from Blood to Cerebrospinal Fluid
Authors: Castillo‐González William , González‐Losada Cristobal , Lumpuy‐Castillo Jairo , Rodriguez‐Pérez Jose Alejandro , Jensenius Jens Christian , Zerr Inga , Schmitz Matthias , Dorta‐Contreras Alberto Juan
Published: June 2021

INTRODUCTION

Ficolins belong to the lectins family. They activate the complement system as an effect in defence of the carrier, and play a very important role in the innate immunity.

Three kinds of human Ficolins were earlier described: L, H and M ones. The theory of molecular dissemination is the basis of the behavior's approach from these proteins that can be found in cerebrospinal fluid (CSF).

OBJECTIVE

To demonstrate through the dynamic of the dissemination from blood to CSF and its concentrations the different ways of molecular adding towards CSF and its action in this biological liquid.

METHODS

80 serum and CSF paired samples were obtained from control persons without neuroinflammation process and without oligoclonal IgG bands. M ficolin and albumin were quantified by ELISA and by an immunoturbidimetric analysis. Q albumin=CSF albumin/serum albumin and Q, M Ficolin=CSF M Ficolin/serum M ficolin was calculated.

To calculate the different aggregation forms were determinate the inflection points of the frequency distribution of M ficolin vs. Q M Ficolin taking into account that the more complex structure should diffuse more than a most simple one.

RESULTS

Serum and CSF M ficolin did not follow a normal distribution according to Kolmogorov‐Smirnov test. Mean values of M ficolin in serum was higher than its mean content in CSF. CSF M ficolin concentration increased with the increase of Q albumin. M ficolin is not a brain‐derived protein. Three inflection points was determined. (Table 1 and figure 1). G‐1, G‐2, G‐3 described the subpopulation of samples diffusion limited by their molecular weight and its aggregation structure.

CONCLUSION

M ficolin is a predominant blood‐derived protein and the diffusion from blood to CSF indicates three aggregation forms.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Arginine Vasopressin (AVP) Deficiency, Conivaptan and Desmopressin on Clinical Symptoms and Blood Cytokine Levels in Rats with Experimental Autoimmune Encephalomyelitis
Authors: Quintanar‐Stephano Andres , Viñuela‐Berni Veronica , Macías‐Segura Noé , Valdez‐Urias Fernando , Kovacs Kalman Thomas
Published: June 2021

Introduction

The experimental autoimmune encephalomyelitis (EAE), the rat paradigm for the human multiple sclerosis (MS), are autoimmune diseases mediated by Th1 and Th17 cells and responsible of the nervous system demyelination and progressive paralysis. It is know that lymphocytes possess AVP receptors and that EAE increases AVP serum levels along the disease. Neurointermediate pituitary lobectomy (NIL) in rats causes AVP and oxytocin permanent low serum levels and decreased humoral and cell‐mediated immune responses. Nevertheless the role of AVP as a direct immune regulatory factor has not been well clarified.

Objective

To investigate the effects of AVP deficiency (by NIL), and the treatment with desmopressin (DP, a synthetic analog of AVP) and conivaptan (CNV, an AVP V1a‐V2 receptor antagonist) on the clinical symptoms of EAE, and to determine the serum levels of Interleukine‐2 (IL‐2), IL‐4, IL‐6, IL‐10, IL‐17A, INF‐γ and TNF‐α in the experimental groups.

Methods

Groups of female Lewis rats were divided in: (1) Intact control (Control) (2) Control+EAE, (3) Sham‐operated (SHAM), (4) Control+CNV, (5) NIL and (6) NIL+DP. Except the Control the remaining groups were immunized for EAE three weeks post‐NIL. DP and CNV administrations started 3 days before EAE immunizations. Serum cytokines were analyzed by flow cytometry and EAE clinical symptoms were evaluated by a conventional numerical scale.

Results

EAE clinical symptoms, INF‐γ, TNF‐α, and the IL‐2, IL‐6 and IL‐17A were significantly decreased in both NIL and CNV groups, whereas the IL‐4 and IL‐10 increased in the CNV group. In NIL+DP group we found a significant increase in TNF‐α, whereas the IL‐4 was significantly decreased.

Conclusions

1) The present clinical and molecular findings demonstrate that AVP deficiency and the block of the AVP receptors decreased immune response, and that DP restores the susceptibility of the NIL animals to develop EAE. 2) The results strongly support that acting directly on the AVP receptors, AVP plays an important immuneregulatory role in the generation and maintenance of the immune‐competence, 3) AVP receptors may be therapeutic targets in the treatment of the human MS.

Support or Funding Information

Supported by UAA‐PIFF14‐1 and CONACYT‐221262 (AQS). México.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Basolateral FFA2 of enterochromaffin cells contributes to 5‐HT release in rat and mouse duodenum
Authors: Akiba Yasutada , Takajo Takeshi , Kaunitz Jonathan D.
Published: June 2021

Enteroendocrine cells (EEC) express nutrient receptors that when activated release hormones such as serotonin (5‐HT). Although nutrient receptors are thought to be localized on the apical membrane of EEC, where they are well placed to sense luminal nutrients, immunohistochemistry mostly localizes the receptors to the cytosol or to either membrane pole. Since villous cells transport luminal nutrients via nutrient transporters or by passive diffusion to the submucosal space, it is possible that absorbed nutrients activate basolaterally‐expressed nutrient receptors rather than detect luminal nutrients. One of the short‐chain fatty acid receptors, FFA2, is expressed on enterochromaffin (EC) cells of rat duodenum; its activation increases HCO3− secretion in vivo, accompanied by 5‐HT release into the portal vein and mediated by 5‐HT4 receptor activation. We thus used muscle‐stripped mucosa‐submucosa preparations of rat and mouse duodenum mounted in Ussing chambers to pharmacologically localize FFA2 to the apical or basolateral membranes of EC cells.

The selective and allosteric FFA2 agonist phenylacetamide‐1 (PA1) was applied to the mucosal or serosal bath of the Ussing chamber, where short‐circuit current (Isc) was measured in the presence of serosal indomethacin (10 μM).

PA1 (1 – 100 μM) in the mucosal bath had no effect or slightly decreased Isc in both rat and mouse duodenum. In contrast, PA1 in the serosal bath dose‐dependently increased Isc that was greater in mouse than in rat duodenum; in both, the PA1‐induce Isc increase was reduced by the 5‐HT3 receptor antagonist granisetron (Gran, 10 μM) and abolished by the 5‐HT4 receptor antagonist GR113801 (GR; 10 μM). The effect of serosal PA1 was inhibited by the selective FFA2 antagonist GLPG0974 (0.1 mM). Furthermore, the native FFA2 agonists sodium acetate and sodium propionate (pH adjusted to pH 7.4) dose‐dependently increased Isc inhibited by GR, Gran, the selective Nav1.8 antagonist A803467, and the selective neurokinin receptor‐1 (NK‐1) antagonist CP99994, but not by TTX or atropine. Serosal PA1 increased serosal 5‐HT concentrations, which were not affected by TTX+A803467. FFA2 immunoreactivity was limited to EC cells, suggesting that EC cells rather than myenteric neurons are the primary source of 5‐HT.

These results suggest that FFA2 is localized at least to the basolateral membrane of EC cells and that its activation increases electrogenic anion secretion. The basolateral location of FFA2 is important for informing the design of experimental and clinical studies aimed at altering 5‐HT release from EC cells, which is related to mucosal defenses and dyspeptic symptoms.

Support or Funding Information

VA Merit Review and NIDDK R01 DK54221

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal epithelial‐specific NHE3 knockout causes metabolic acidosis
Authors: Valdez Alexandria , Dominguez Rieg Jessica A. , Fenton Robert A. , Rieg Timo
Published: June 2021

The Na+/H+ exchanger isoform 3 (NHE3) has an important role in intestinal and renal Na+ uptake and acid‐base regulation. While renal‐specific NHE3 knockout models have been successfully generated, no intestinal‐specific model has been described. Of note, renal‐specific NHE3 knockout does not result in altered blood pH or bicarbonate levels (Kidney International; 2017, 92:397–414) and conventional intestinal epithelial cell‐specific NHE3 knockout mice die shortly after birth. To determine the role of NHE3 selectively in the intestine, we generated tamoxifen (Tam)‐inducible epithelial‐specific NHE3 knockout mice (NHE3IEC‐KO) by intercrossing NHE3loxlox (control, Con) and VillinCreERT2 mice. Semiquantitative fluorescent labeling determined that application of Tam (5 days, 200 mg/kg via oral gavage) deleted NHE3 from the small intestine and colon of NHE3IEC‐KO mice, whereas labeling in the proximal tubule and thick ascending limb of the kidney was unaffected. Before Tam, Con and NHE3IEC‐KO (n=8 and 4, respectively) mice had comparable blood pH (7.4±0.01 vs 7.4±0.01), HCO3− (24.7±0.7 vs 24.6±0.6 mM), base excess (0.5±0.2 vs 0.6±0.1 mM) and Na+ concentrations (147±0.4 vs 148±0.8 mM). Two weeks after Tam, compared to Con mice, NHE3IEC‐KO mice show significant metabolic acidosis (Δ change: 0±0.01 vs −0.15±0.03, P<0.05), lower HCO3− (Δ change: 0±0.4 vs −7±1, P<0.05) and negative base excess (Δ change: 0±0.7 vs −9±2, P<0.05), whereas plasma Na+ was unaffected (Δ change: 0.5±0.6 vs −0.3±1, NS). Indirect evidence for impaired intestinal HCO3− uptake using intestinal flushes showed a significantly higher flush pH in NHE3IEC‐KO compared to Con mice (7.9±0.1 vs 7.4±0.1, P<0.05). In conclusion, we successfully generated intestinal epithelial cell‐specific NHE3 knockout mice. The mouse model will be useful to study different aspects of NHE3 in the intestine and colon. The data indicate a prominent unrecognized role of the intestine for acid‐base regulation.

Support or Funding Information

Novo Nordisk Foundation, the Lundbeck Foundation, Danish Medical Research Council, NIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Concurrent Airway Surface Liquid Absorption and Secretion in Human Bronchioles
Authors: Shamsuddin A. K. M. , Quinton Paul M.
Published: June 2021

Characterization of epithelial fluid and electrolyte transport in native small airway epithelia has been largely neglected due to the complex tissue architecture as well as by its inaccessibility and lack of applicable assays, especially in human tissues. To better understand how airway surface liquid (ASL) is maintained as a balance between absorbing and secreting functions, we measure ion transport properties of bronchioles with diameters of 1–2 mm isolated from freshly resected specimens of human lungs with a “mini”‐Ussing chamber (area≈1 mm2). We determined the effects of inhibiting absorption by applying amiloride to inhibit Na+ conductance through ENaC and secretion by applying bumetanide to inhibit the Na+‐K+‐2Cl− cotransporter to block Cldependent secretion demonstrated spontaneous absorptive as well as secretory short circuit currents in bilateral 150 mM NaCl‐Ringer solution. We also tested for the presence of the Clchannels, CFTR stimulating with Fsk/IBMX and CaCC with UTP. Both agonists independently stimulated additive equivalent short circuit current in the same specimen of small airway tissue, but evoked distinctly opposite effects on the transepithelial potential (Vt). While Fsk/IBMX (cAMP) activation depolarized Vt, UTP (Ca2+) stimulation hyperpolarized the Vt. The depolarization of Vt induced by cAMP suggests that activating CFTR may stimulate fluid absorption, while hyperpolarization of Vt by Ca2+ (UTP) suggest that activating CaCC may stimulate fluid secretion. Independent regulation of these fluid movements may be critical to regulation of ASL volume during conditions of stress. Human small airways constitutively and concurrently secrete and absorb fluid, structurally separated and independent epithelial cells for liquid secretion and absorption. Our results present new evidence for how the delicate layer of fluid lining the small airways is physiologically maintained in a steady state and appropriate airway hydration.

Support or Funding Information

The Nancy Olmsted Trust and the Cystic Fibrosis Research Inc. (CFRI).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of GPR40/120 omega‐3 fatty acid receptors on barrier function of airway epithelial cells
Authors: Wattanaphichet Panisara , Moonwiriyakit Aekkacha , Muanprasat Chatchai
Published: June 2021

The airway epithelium serves as a physical barrier, whose integrity is maintained by tight junctions. Disruption of epithelial barrier function leads to increased permeability of harmful particles and pathogens, causing the development of various airway diseases. G protein‐coupled receptor 40 (GPR40) and G protein‐coupled receptor 120 (GPR120) have been discovered as receptors of omega‐3 fatty acids, and activation of these receptors induces AMP‐activated protein kinase (AMPK) activation, which is known to promote tight junction assembly. This study aimed to investigate the effect of omega‐3 fatty acid receptors, namely GPR40 and GPR120, on tight junction integrity of airway epithelial cells (Calu‐3 cells). Cell junctional assembly was measured by calcium switch assays and transepithelial electrical resistance (TER) analyses. We found that GW9508, an agonist of GPR40/120, promoted tight junction assembly of airway epithelial cells. This effect was abolished by specific antagonists of GPR40 and GPR120. In addition, the enhancement of airway barrier function by GPR40/120 agonist was mediated via a phospholipase C‐ calcium/calmodulin‐dependent protein kinase kinase (CaMKK)‐AMPK pathway. Taken together, these results indicate that activation of GPR40/120 has a beneficial effect in promoting airway epithelial barrier integrity and may be of therapeutic potential in airway diseases resulting from airway epithelial disruption including chronic obstructive pulmonary disease.

Support or Funding Information

This work was supported by Thailand Research Fund and Mahidol University (Grant BRG5980008), Faculty of Science, Mahidol University, and Faculty of Medicine Ramathibodi Hospital, Mahidol University (Tonkla Ramathibodi Project).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.