Conscious Mice Research Articles

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1β3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

Evolving acidic microenvironments during colitis provide selective analgesic targets for a pH-sensitive opioid.

Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis.

0049 Orexin 2 receptor agonist activates pre-Bötzinger complex neurons, phrenic motoneurons and the diaphragm in rodents

Abstract Introduction Neurons in the pre-Bötzinger complex are critical for the generation of inspiratory rhythm and regulation of activity of phrenic motoneurons, which control the activity of the diaphragm. Dysregulation of this neuronal network and resulting dysfunction of the diaphragm is associated with central sleep apnea (CSA). Orexins by stimulating both orexin 1 receptor (OX1R) and/or orexin 2 receptor (OX2R) can activate these neurons and the diaphragm. In this study, we assessed effects of OX2R-selective agonists on the activity of neurons in the pre-Bötzinger complex, phrenic motoneurons, and the diaphragm. Methods OX2R-selective agonists danavorexton (TAK-925) and OX-201 (a tool compound with longer plasma half-life than danavorexton in rodents) were used. OX2R and OX1R agonistic activities were examined by calcium mobilization assay in hOX2R/CHO-K1 cells and hOX1R/CHO-K1 cells, respectively. Activity of neurons in the pre-Bötzinger complex and phrenic motoneurons was investigated by electrophysiological recording in rat tissues. Activity of the diaphragm was evaluated by electromyogram recording in anesthetized rats. Respiratory activity was evaluated by whole-body plethysmography recording in mice. Results Danavorexton and OX-201 showed potent OX2R agonistic activity with 50% effective concentration (EC50) values of 5.5 nM and 8.0 nM, respectively. Danavorexton and OX-201 had >5,000-fold and 1,000-fold OX2R selectivity over OX1R, respectively. In in vitro electrophysiological analyses, danavorexton and OX-201 increased the burst frequency of neurons in the pre-Bötzinger complex (EC50: 300 nM and 760 nM, respectively) and phrenic motoneurons (EC50: 1.6 µM and not determined, respectively). Intravenous administration of OX-201 at 1 and 3 mg/kg increased burst frequency of the diaphragm in anesthetized rats and oral administration at 3 mg/kg increased respiratory activity in conscious mice. Conclusion OX2R-selective agonists may enhance respiratory function via activation of the diaphragm through stimulation of neurons in the pre-Bötzinger complex and phrenic motoneurons. OX2R agonists may have therapeutic potential in patients with CSA if enhancement of respiratory function by OX2R agonists can be observed at non-arousal concentrations. Further preclinical and clinical studies are worth conducting in patients with CSA. Support (if any) This work was conducted by Takeda Pharmaceutical Company Limited.

Multichannel Extracellular Recording in Freely Moving Mice.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Echo2Pheno: a deep-learning application to uncover echocardiographic phenotypes in conscious mice

Echocardiography, a rapid and cost-effective imaging technique, assesses cardiac function and structure. Despite its popularity in cardiovascular medicine and clinical research, image-derived phenotypic measurements are manually performed, requiring expert knowledge and training. Notwithstanding great progress in deep-learning applications in small animal echocardiography, the focus has so far only been on images of anesthetized rodents. We present here a new algorithm specifically designed for echocardiograms acquired in conscious mice called Echo2Pheno, an automatic statistical learning workflow for analyzing and interpreting high-throughput non-anesthetized transthoracic murine echocardiographic images in the presence of genetic knockouts. Echo2Pheno comprises a neural network module for echocardiographic image analysis and phenotypic measurements, including a statistical hypothesis-testing framework for assessing phenotypic differences between populations. Using 2159 images of 16 different knockout mouse strains of the German Mouse Clinic, Echo2Pheno accurately confirms known cardiovascular genotype–phenotype relationships (e.g., Dystrophin) and discovers novel genes (e.g., CCR4-NOT transcription complex subunit 6-like, Cnot6l, and synaptotagmin-like protein 4, Sytl4), which cause altered cardiovascular phenotypes, as verified by H&E-stained histological images. Echo2Pheno provides an important step toward automatic end-to-end learning for linking echocardiographic readouts to cardiovascular phenotypes of interest in conscious mice.Graphical abstract

Effect of Sigma-1 Receptor Activation on Renal Injury and Hypertension in Female Mice with Lupus

Systemic lupus erythematosus (SLE) is a female dominant autoimmune disease that presents with prominent renal injury and hypertension contributing to the morbidity and mortality. While the causality and association of the renal injury and hypertension is not completely understood, novel therapies to reduce these detrimental outcomes could be beneficial to SLE patients. The sigma-1 receptor is a cytoprotective ligand-regulated chaperone protein that acts by decreasing protein aggregation, cellular stress and cell death, thus preventing tissue injury. Activation of the sigma-1 receptor with pharmacological ligands enhances cytoprotection in autoimmune diseases like multiple sclerosis and Huntington’s disease; however, the efficacy of sigma-1 receptor agonists in SLE is not known. We hypothesize that sigma-1 receptor activation with the agonist, LS-1-127, will reduce renal injury and halt the progression of hypertension in SLE mice.Female SLE ( NZBWF1) mice and control ( NZW) mice were weighed and urine collected via metabolic cages weekly starting at 30 weeks of age. Urinary albumin was measured via dipsticks. At 33 weeks of age, SLE and control mice were treated with the sigma-1 receptor ligand (LS-1-127; 10 mg/kg IP) or equal volume of vehicle (10% DMSO; IP) three times a week for two weeks. At 35 weeks, mean arterial pressure (MAP) was measured in conscious mice using indwelling carotid catheters for two consecutive days and then mice were euthanized. Markers of renal injury, including urinary neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and creatinine were measured by ELISA.Body weight (g) was not different in SLE vs. control mice (39.2 ± 2.9 vs. 35.4 ± 0.9; p=0.44) and LS-1-127 did not alter body weight in SLE mice (39.1 ± 2.2; p=0.97) or controls (34.1 ± 0.6; p=0.84). Albuminuria was present in 44.4% (4 out of 9) of SLE/Veh mice and none of the controls. LS-1-127 did not improve albuminuria in SLE mice (50%; 3 out of 6). MAP (mmHg) was higher in SLE mice compared to control mice (146 ± 4 vs. 123 ± 3; n=9-10, p <0.0001). LS-1-127 did not significantly alter MAP in SLE mice (150± 8; n=6) or controls (124 ± 2; n=10). The NGAL-to-creatinine ratio (ng/mg) was higher in the SLE mice compared to controls (327.3±119.8 vs 63.2±4.3, n=9-12, p<0.0007). LS-1-127 treatment did not significantly alter the NGAL-to-creatinine ratio in SLE mice (484.3±209.0, n= 6) or control mice (71.7 ± 5.2, n=10). The KIM1-to-creatinine ratio (ng/mg) was not significantly different between any of the groups.In conclusion, two weeks of sigma-1 receptor activation with LS-1-127 did not significantly alter blood pressure or renal injury in female SLE mice with advanced disease. Further inquiry into the effect of LS-1-127 on the expression of various cellular stress markers in the kidney will be conducted. Also sigma-1 receptor activation at different stages of SLE disease progression warrants investigation. NIH (R01HL153703, K01HL139859) and Department of Defense (LR210096) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Type 2 phosphodiesterase regulates cardiac pacemaker activity

Elevated heart rate (HR) and lower HR variability (HRV) predict cardiovascular morbi-mortality. HR is mainly controlled by the autonomic nervous system (ANS). During sympathetic stimulation, the activation of β-adrenergic receptors increases cAMP levels leading to positive chronotropic effect. In the heart, cAMP can be hydrolyzed by 6 different phosphodiesterases (PDEs). One of them is PDE2 which has recently been reported to be beneficial for the failing heart. However, whether this is due to a direct control sinoatrial node (SAN) function or an indirect effect via the ANS is unknown. To explore the role of PDE2 in regulating HR. The hydrolytic cAMP activity was measured with radioenzymatic assay. The in vitro pacemaker activity was assessed by measuring spontaneous Ca2+ transients in Fluo4-loaded-SAN tissue using confocal microscopy. ECG telemetry was recorded in WT and cardiac PDE2-overexpressing (PDE2TG) conscious mice, in basal conditions, after ANS inhibition with atropine (atro, 1 mg/kg, ip) + propranolol (propra, 2 mg/kg, ip), or after β-adrenergic stimulation with isoprenaline (Iso, 1.5 mg/kg, ip). If current was measured by patch-clamp (whole-cell). PDE2 is expressed in WT SAN and overexpressed by ∼4-fold in the PDE2TG SAN. Total cAMP-hydrolyzing activity is increased ∼3-fold in PDE2TG SAN. PDE2TG mice display lower basal HR, during day and night. This phenotype was maintained after atro + prop and after Iso. Basal beating rate of PDE2TG SAN tissue was lower. While no difference in Ca2+ transient amplitude time to peak and duration at half maximum were unchanged in PDE2TG SAN cells, the time constant of decay was increased in basal conditions. Ca2+ sparks analysis between WT and PDE2TG SAN did not show any modification on ryanodine receptor activity. In addition, If current density was decreased in PDE2TG SAN cells. PDE2 regulates HR in vivo by a mechanism independent from ANS which takes place at the level of SAN and involves the voltage clock.

Fecal microbiota transplantation attenuates hypertension in a female mouse model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a prototypic multisystem autoimmune disorder that disproportionately affects women. SLE is characterized by a breach of immunological self-tolerance and the expansion of autoreactive T and B lymphocytes, leading to the production of autoantibodies. Autoantibody production leads to downstream chronic inflammation resulting in high rates of hypertension, renal injury, and cardiovascular disease. Alterations in the gut microbiota, or gut dysbiosis, is associated with many autoimmune diseases, including SLE. In the present study, we hypothesized that gut dysbiosis in SLE may play a role in the development of immune system dysfunction and hypertension in an experimental model of SLE, and that fecal microbiota transplantation (FMT) with microbes from control mice may improve these parameters. To test this hypothesis, we treated 26-week-old female control (NZW, n=19) and SLE (NZBWF1, n=19) mice with broad spectrum antibiotics (0.4 g/L ampicillin, 0.4 g/L neomycin sulfate, 0.4 g/L metronidazole, and 0.2 g/L vancomycin) in drinking water for one week to deplete the existing microbiota. Subsequently, mice were given 0.1 mL of fecal microbes via oral gavage two times per week for four weeks in the following groups (n=9-10 mice/group): Control-vehicle, Control-SLE microbes, SLE-vehicle, SLE-Control microbes. Changes in the gut microbiome before and after FMT were assessed by 16S rRNA analysis of the microbiome. Mean arterial pressure (MAP, mmHg), measured in conscious mice by carotid artery catheter was higher in SLE mice that received SLE microbes (Control: 112±3 vs. SLE: 140 ±2 mmHg, p<0.0001). SLE mice that received FMT with control microbes had lower blood pressure (SLE-Control microbes: 126±4 mmHg, p=0.03). Renal injury, as assessed by urinary albumin excretion at the conclusion of the study, was not significantly different among the groups (p=0.3523). Circulating autoantibodies and immunoglobulins were also measured at the conclusion of the study. Anti-dsDNA IgG was elevated in SLE mice compared to control (Control: 149±38 vs. SLE: 527±114 kU/mL, p=0.02), and were significantly lower in SLE mice treated with control microbes (SLE-Control microbes: 126±4 mmHg, p=0.03). Circulating IgM was also elevated in SLE mice (Control: 0.053±0.008 vs. SLE: 0.32±0.04 mg/mL, p<0.0001), and FMT lowered IgM in SLE mice that received FMT (0.19±0.03 mg/mL, p=0.0069). These data suggest that gut microbiota present in NZBWF1 mice contribute to both autoantibody production and the development of hypertension, and that interventions to alter the microbiora could have a beneficial impact on disease activity in SLE patients. NIH NIGMS P20GM104357 to UMMC Department of Physiology and Biophysics, NIH NHLBI R00HL146888 to Erin Taylor This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of renal function by the peroxisome proliferator-activated receptor-alpha: A novel target for treating hypertension

Approximately 37 million people in the U.S. have chronic kidney disease, which is a major risk factor for cardiovascular and end stage renal diseases. We have identified a nuclear receptor, peroxisome proliferator-activated receptor – alpha (PPAR-α), that plays a major role in regulating renal glomerular filtration rate in an experimental model of hypertension. Targeting PPAR-α signaling provides novel therapeutic strategies for reducing hypertension associated chronic kidney disease. PPAR-α knockout (KO) mice exhibit increased renal inflammation and blood pressure response to Angiotensin II (Ang II). In this study, we investigated the role of PPAR-α in renal function in a mouse model of hypertension. Male 4-month-old wild type (WT) and PPAR-α KO mice were instrumented with radio transmitters by artery canulation (Data Science International). This method minimizes stress and artifacts by avoiding the use of tethering, restraining, or anesthetizing the mice during data sampling. After recovery from surgery, we continuously measured mean arterial pressure (MAP) via radio telemetry in conscious ambulatory mice. After baseline MAP was established, vehicle (Veh; saline) or Ang II were infused using an osmotic minipump at a slow pressor dose (400 ng/kg/min) for 12 days. On day 12, we injected an intravenous bolus of fluorescin-sinistrin (3.74 ml/g body weight) and collected 8 blood samples (20 μl/sample) during a 75-minute period to enable calculation of the glomerular filtration rate (GFR) using [GFR = I/(A/a + B/b]. Similar to our prior observations, no significant (ns) differences in baseline MAP were observed between WT and PPAR-α KO mice [(mmHg): WT (n=6), 111±20 vs. PPAR-α KO (n=6), 113 ± 10; ns] whereas after 12 days of the slow pressor effect of Ang II, MAP was increased in both strains [(mmHg): WT (n=8), 138 ± 11# vs. PPAR-α KO (n=8), 156 ± 16#; #p<0.05 vs. basal, same strain]; however, the increase in MAP was greater in the PPAR-α KO (43 mmHg) compared to WT (27 mmHg) mice. The absence of PPAR-α reduced GFR by 24% under basal conditions [(μl/min): WT (n=3), 346 ± 20 vs. PPAR-α KO (n=3), 275 ± 27*, *p<0.05 vs WT]. After 12 days of Ang II infusion, GFR was reduced in WT mice by 27% and did not further reduce GFR in PPAR-α KO mice [(μl/min): WT (n=6), 251 ± 11# vs. PPAR-α KO (n=6), 277 ± 61; #p<0.05 vs. basal, same strain]. PPAR-α protects mice from worsening hypertension and is critical to preserving GFR during normotensive conditions. Ongoing studies are further investigating how PPAR-α regulates renal function. These finding suggest therapeutics designed to increase PPAR-α activity could have clinical benefit in chronic kidney disease. Howard University Research Centers at Minority Institutions, Georgetown-Howard Universities Center for Clinical and Translational Science, Howard University Aging Grant, K01HL092593 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 525: Angiotensin Ii Type 1a Receptor Deletion In Vascular Smooth Muscle Cells Prevent Abdominal Aortic Aneurysm Formation Induced By Angiotensin Ii Plus β-aminopropionitrile

Abdominal aortic aneurysm (AAA) is multifactorial disease resulting from changes in the aortic wall structure, such as medial degradation and inflammation, causing life-threatening AAA rupture. Angiotensin II (AngII) type 1A receptor (AT 1A R) upon AngII binding mediates blood pressure elevation and several chronic cardiovascular diseases including AAA. Treatment with AngII plus β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, in normolipidemic C57BL6 mice similarly induces AAA. However, we have limited understanding whether smooth muscle AT 1A R affects AAA formation induced by AngII plus BAPN. Thus, the subject of this study is to test the contribution of smooth muscle AT 1A R to AngII-dependent AAA development in normocholesterolemic mice. At 8 to 12 week of age, smooth muscle AT 1A R silenced mice (knock-in Tagln-Cre +/- crossed with AT 1A R flox/flox mice) and control mice were infused with AngII (1 μg/kg/min) via osmotic mini-pump for 4 weeks and treated with 1 g/L BAPN containing water for the first 2 weeks or sham-operated for mini-pump implantation. After 4 weeks, significant increases in maximum external aortic diameter and structural alterations were observed in the control mice, which was attenuated in smooth muscle AT 1A R silenced mice (1.67 mm vs. 1.22 mm, p<0.05). AngII plus BAPN treatment increased perivascular collagen III positive cell ratio, which was normalized in smooth muscle AT 1A R silenced mice (p<0.01). Evaluation of blood pressure at week 4 in conscious mice showed significant reduction in AngII-induced hypertension in smooth muscle AT 1A R silenced mice (p<0.01). Furthermore, in contrast to control mice, no death duet to aortic rupture was observed in smooth muscle AT 1A R silenced mice. In conclusion, our study has demonstrated the significant contribution of smooth muscle expressing AT 1A R in mediating AAA development and rupture in response to AngII plus BAPN treatment. It is possible that attenuation of hypertension in the AT 1A R silenced mice may provide additional protection against AAA rupture.

Evidence of sex differences in cancer-related cardiac complications in mouse models of pancreatic and liver cancer.

Abnormal heart rate variability (HRV) is commonly observed in cancer patients who have undergone targeted therapy and/or surgery, yet the effects of cancer itself on cardiac function remain underexplored. Specifically, there is limited knowledge about sex-specific manifestations of HRV in cancer patients. Transgenic mouse models are widely used to study different types of cancer. Here, we aimed to investigate the sex-specific effects of cancer on cardiac function using transgenic mouse models of pancreatic and liver cancers. This study used male and female transgenic mice with cancer and wild-type controls. Cardiac function was assessed by recording electrocardiograms in conscious mice. RR intervals were detected to determine HRV using time and frequency domain analyses. Histological analysis with Masson's trichrome staining was performed to determine structural changes. In females, increased HRV was observed in both pancreatic and liver cancer-bearing mice. In contrast, in males, increased HRV was observed only in the liver cancer group. Male pancreatic cancer mice demonstrated autonomic balance shift showing an increase in parasympathetic to sympathetic tone. The heart rate (HR) was higher in control and liver cancer male mice groups than in females. Histological analysis did not show significant sex differences but suggested a higher degree of remodeling in liver cancer mice than in control, specifically in the right atrium and left ventricle. This study revealed sex differences in cancer's HR modulation. Specifically, female cancer mice had lower median HR and higher HRV. These findings indicate that sex must be considered when using HRV as a cancer biomarker.

378 Regulation of renal function by the peroxisome proliferator-activated receptor-alpha: A novel target for treating hypertension

OBJECTIVES/GOALS: Approximately 37 million people in the U.S. have chronic kidney disease, which is a major risk factor for cardiovascular and end stage renal diseases. PPAR-αknockout (KO) mice exhibit increased renal inflammation and blood pressure. In this study, we investigated the role of PPAR-αin renal function in a mouse model of hypertension. METHODS/STUDY POPULATION: Male 4-month-old wild type (WT) and PPAR-αKO mice were instrumented with radio transmitters by artery canulation (Data Science Intl). This method minimizes stress and artifacts by avoiding the use of tethering, restraining, or anesthetizing the mice during data sampling. After recovery from surgery, we continuously measured mean arterial pressure (MAP) via radio telemetry in conscious ambulatory mice. After baseline MAP was established, vehicle (Veh; saline) or angiotensin II (Ang II) were infused using an osmotic minipump at a slow pressor dose (400 ng/kg/min) for 12 days. On day 12, we injected an intravenous bolus of fluorescin-sinistrin (3.74µl/g body weight) and collected 8 blood samples (20µl/sample) over 75 minutes to enable calculation of the glomerular filtration rate (GFR) using [GFR = I/(A/α+ B/ß)]. RESULTS/ANTICIPATED RESULTS: Similar to our prior observations, no significant (ns) differences in baseline MAP were observed between WT and PPAR-αKO mice [(mmHg): WT (n=6), 111 ± 20 vs. PPAR-αKO (n=6), 113 ± 10; ns] whereas after 12 days of the slow pressor effect of Ang II, MAP was increased in both strains [(mmHg): WT (n=8), 138 ± 11# vs. PPAR-αKO (n=8) , 156 ± 16#; #p DISCUSSION/SIGNIFICANCE: PPAR-αprotects mice from worsening hypertension and is critical to preserving GFR during normotensive conditions. Ongoing studies are further investigating how PPAR-αregulates renal function. These finding suggest therapeutics designed to increase PPAR-αactivity could have clinical benefit in chronic kidney disease.

Autonomic Nervous System Adaptation and Circadian Rhythm Disturbances of the Cardiovascular System in a Ground-Based Murine Model of Spaceflight.

Whether in real or simulated microgravity, Humans or animals, the kinetics of cardiovascular adaptation and its regulation by the autonomic nervous system (ANS) remain controversial. In this study, we used hindlimb unloading (HU) in 10 conscious mice. Blood pressure (BP), heart rate (HR), temperature, and locomotor activity were continuously monitored with radio-telemetry, during 3 days of control, 5 days of HU, and 2 days of recovery. Six additional mice were used to assess core temperature. ANS activity was indirectly determined by analyzing both heart rate variability (HRV) and baroreflex sensitivity (BRS). Our study showed that HU induced an initial bradycardia, accompanied by an increase in vagal activity markers of HRV and BRS, together with a decrease in water intake, indicating the early adaptation to fluid redistribution. During HU, BRS was reduced; temperature and BP circadian rhythms were altered, showing a loss in day/night differences, a decrease in cycle amplitude, a drop in core body temperature, and an increase in day BP suggestive of a rise in sympathetic activity. Reloading induced resting tachycardia and a decrease in BP, vagal activity, and BRS. In addition to cardiovascular deconditioning, HU induces disruption in day/night rhythmicity of locomotor activity, temperature, and BP.

A277 A NOVEL PH-SENSITIVE Μ-OPIOID RECEPTOR AGONIST THAT DOES NOT INDUCE TOLERANCE IN A COLITIS MOUSE MODEL

Abstract Background Adequate pain control in inflammatory bowel disease (IBD) can require opioids due to their high analgesic potency. The long-term use of opioids, however, is limited by the development of tolerance. This leads to reduced analgesic efficacy over time, resulting in escalating opioid dosing and thus increased risk of serious side effects. We previously demonstrated the safety and effectiveness of a novel pH-sensitive µ-opioid receptor (MOR) agonist, NFEPP, in a murine colitis pain model, but its tolerance potential with chronic administration is unknown. Purpose To assess the tolerance potential of NFEPP compared to its parent compound fentanyl during acute colitis in a preclinical mouse model. Method Acute colitis in C57BL/6 mice was induced using 2.5% dextran sulphate sodium for 5 days. NFEPP or fentanyl were then administered s.c. every 4 hours between 7am and 11pm over 5 days in daily increasing concentrations (0.4-1.5 mg/kg/d). Analgesic tolerance to opioids was assessed in conscious mice by measuring visceromotor responses (VMRs) to noxious colorectal distensions. Tolerance to the MOR agonist DAMGO in NFEPP or fentanyl treated mice was evaluated using patch-clamp recordings from dorsal root ganglion (DRG) neurons and extracellular recordings from lumbar splanchnic nerves that innervate the colon. Inflammation was assessed by macroscopic analyses, histological scoring and tissue-pH measurements of the inflamed colon. Group differences were analyzed using two-way ANOVA with Bonferroni′s or Tukey′s post-test (p-value <0.05). Result(s) NFEPP significantly reduced VMRs before and after chronic NFEPP treatment (39% reduction, p<0.05 vs. 41% reduction, p<0.05, compared to baseline at 80 µl). No differences of NFEPP induced antinociceptive actions were observed comparing VMR measurements before and after chronic administration (p=0.44). However, the analgesic activity of fentanyl decreased over time with less VMR inhibition observed after chronic treatment compared to fentanyl naïve mice (p<0.05). Cross tolerance testing between these two opioids revealed a loss of NFEPP- induced VMR inhibition in fentanyl treated mice, but fentanyl effects in the NFEPP treatment group remained unchanged. In patch-clamp recordings, DAMGO (100nM) evoked antinociceptive actions in DRG neurons from NFEPP treated mice (rheobase increase: 50%, p<0.05, compared to vehicle) whereas no change in rheobase with DAMGO was observed in DRG neurons from fentanyl treated mice. Afferent nerve activity from lumbar splanchnic nerves in response to von Frey filament probing decreased after DAMGO (100nM) application in NFEPP treated mice (p<0.05, compared to control probing), but showed no change in fentanyl treated mice. Colonic length, diameter, histological damage and tissue-pH did not differ between the two opioid treatment groups. Conclusion(s) Chronic administration of NFEPP during acute murine colitis does not lead to analgesic tolerance within the visceral nociceptive system, in contrast to its parent compound fentanyl. Please acknowledge all funding agencies by checking the applicable boxes below CCC Disclosure of Interest None Declared

Sex and strain differences in renal hemodynamics in mice.

The present study was to examine sex and strain differences in glomerular filtration rate (GFR) and renal blood flow (RBF) in C57BL6, 129/Sv, and C57BLKS/J mice, three commonly used mouse strains in renal research. GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious mice. RBF was measured by a flow probe placed in the renal artery under an anesthetic state. In C57BL6 mice, there were no sex differences in both GFR and RBF. In 129/Sv mice, females had significantly greater GFR than males at age of 24 weeks, but not at 8 weeks. However, males had higher RBF and lower renal vascular resistance (RVR). Similar to 129/Sv, female C57BLKS/J had significantly greater GFR at both 8 and 24 weeks, lower RBF, and higher RVR than males. Across strains, male 129/Sv had lower GFR and higher RBF than male C57BL6, but no significant difference in GFR and greater RBF than male C57BLKS/J. No significant difference in GFR or RBF was observed between C57BL6 and C57BLKS/J mice. Deletion of eNOS in C57BLKS/J mice reduced GFR in both sexes, but decreased RBF in males. Furthermore, there were no sex differences in the severity of renal injury in eNOS-/- dbdb mice. Taken together, our study suggests that sex differences in renal hemodynamics in mice are strain and age dependent. eNOS was not involved in the sex differences in GFR, but in RBF. Furthermore, the sexual dimorphism did not impact the severity of renal injury in diabetic nephropathy.

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias

ARTICLES DISCUSSED: Tomsits, P., Schuttler, D., Kaab, S., Clauss, S., Voigt, N. Isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of Ca2+ transients and L-Type calcium current. Journal of Visualized Experiments. (165), 10.3791/61964 (2020). Seibertz, F., Reynolds, M., Voigt, N. Single-Cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (166), 10.3791/61890 (2020). Xia, R. et al. Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node. Journal of Visualized Experiments. (171), 10.3791/62236 (2021). Xia, R. et al. Whole-Mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. Journal of Visualized Experiments. (166), 10.3791/62058 (2020). Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice. Journal of Visualized Experiments. (173), 10.3791/62735 (2021). Scherschel, K. et al. Location, dissection, and analysis of the murine stellate ganglion. Journal of Visualized Experiments. (166), 10.3791/62026 (2020). Shimura, D., Hunter, J., Katsumata, M., Shaw, R. M. Removal of an internal translational start site from mRNA while retaining expression of the full-length protein. Journal of Visualized Experiments. (181), 10.3791/63405 (2022). Rotzer, R. D. et al. Implantation of combined telemetric ECG and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice. Journal of Visualized Experiments. (168), 10.3791/62101 (2021). Tomsits, P. et al. Analyzing long-term electrocardiography recordings to detect arrhythmias in mice. Journal of Visualized Experiments. (171), 10.3791/62386 (2021). Grab, M. et al. Development and evaluation of 3D-printed cardiovascular phantoms for interventional planning and training. Journal of Visualized Experiments. (167), 10.3791/62063 (2021).

Abstract WP216: Intraperitoneal Delivery Of Capsinoids (Non-pungent TRPV1 Agonists) Promotes Mild Hypothermia And Improved Long-term Outcome Following Stroke

Intro: Mild hypothermia has been shown to improve outcomes in cardiac arrest and experimental stroke models. Unfortunately, hypothermia protocols in awake subjects are hampered by inconsistent temperature management due to incomplete suppression of cold defense mechanisms (e.g. shiver response). To address a need for better strategies for cooling conscious subjects, we sought to promote mild hypothermia by pharmacological activation of heat-sensing nerve fibers in the thermoregulatory system. We hypothesize that capsinoids - non-pungent agonists of the transient receptor potential vanilloid 1 (TRPV1) channel - activate vagal afferents, triggering thermoregulatory regions of the brain to induce mechanisms of whole-body cooling, protecting the brain and preventing injury progression after a stroke. Methods: C57BL6 male mice of 11wks underwent permanent distal middle cerebral artery occlusion (pdMCAO) or sham. Mice recovered for 2hrs in warming cages to maintain normal body temperatures during post-stroke recovery before being administered capsinoids or vehicle (4 IP injections, 90 min interval) to produce 6 hours of mild hypothermia (or normothermic control). After 1 month, mice underwent functional tests and brains were collected for histological analysis. Results: Capsinoids provided a significant and consistent hypothermia whereas vehicle maintained a baseline temperature (two-way ANOVA, n=7, p=0.0036). Capsinoid-treated mice showed ~50% infarct volume reduction 1 month after stroke, measured by cresyl-violet staining (0.7% ± 0.1% SEM vs 1.5% ± 0.2% SEM, n=7, two-tailed unpaired t-test: p=0.0046). Additionally, a stroke-induced breathing dysfunction (decrease in tidal volume by plethysmography) was significantly restored toward normal in the capsinoid-treated group (7.7e-3 ± 5.6e-4 SEM vs 5.8e-3 ± 2.4e-4 SEM, n=8, one-way ANOVA: p=0.033). Conclusion: Capsinoids delivered to TRPV1-containing visceral afferents promotes reversible mild hypothermia in freely moving conscious mice. Application of this form of cooling in the pdMCAO model provides significant reduction of stroke injury volume and long-term improvement in functional outcome.

Repeated variate stress increased voiding frequency and altered TrpV1 and TrpV4 transcript expression in lower urinary tract (LUT) pathways in female mice.

Psychological stress is associated with urinary bladder dysfunction (e.g., increased voiding frequency, urgency and pelvic pain); however, the mechanisms underlying the effects of stress on urinary bladder function are unknown. Transient receptor potential (TRP) channels (vanilloid family) may be potential targets for intervention due to their distribution in the LUT and role in pain. Here, we examine a model of repeated variate stress (RVS) of 2 week (wk) or 4 wk duration in female mice and its effects on bladder function, anxiety-like behavior, and TRPV transcript expression in urinary bladder and lumbosacral spinal cord and associated dorsal root ganglia (DRG). Using continuous infusion, open-outlet cystometry in conscious mice, RVS significantly (p ≤ 0.05) decreased infused volume and intermicturition interval. Bladder pressures (threshold, average, minimum, and maximum pressures) were unchanged with RVS. Quantitative PCR demonstrated significant (p ≤ 0.05) changes in TrpV1 and TrpV4 mRNA expression between control and RVS cohorts in the urothelium, lumbosacral spinal cord, and DRG. Future directions will examine the contribution of TRP channels on bladder function, somatic sensation and anxiety-like behavior following RVS.

Transient Receptor Potential Ankyrin-1-expressing vagus nerve fibers mediate IL-1β induced hypothermia and reflex anti-inflammatory responses

BackgroundInflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines.MethodsTo monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant.ResultsHere, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis.ConclusionThus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity.

Determination of Catecholamines in a Small Volume (25μL) of Plasma from Conscious Mouse Tail Vein.

The determination of plasma catecholamine levels is commonly used as a measure of the sympathetic nervous system's response to stress and is highly important for diagnosis, therapy, and prognosis of cardiovascular diseases, catecholamine-secreting tumors arising from the chromaffin cells of the sympathoadrenal system, and affective disorders. Diseases in which catecholamines are significantly elevated include pheochromocytoma, Parkinson's disease, Alzheimer's disease, neuroblastoma, ganglioneuroblastoma, von Hippel-Lindau disease, baroreflex failure, chemodectina (nonchromaffin paraganglioma), and multiple endocrine neoplasia. Plasma norepinephrine levels provide a guide to prognosis in patients with stable, chronic, and congestive heart diseases. The method described here for the determination of plasma catecholamines is based on the principle that plasma catecholamines are selectively adsorbed on acid-washed alumina at pH 8.7 and then eluted at a pH between 1.0 and 2.0. Upon injection, catecholamines in elutes were separated by a reversed phase C-18 column. After separation, the catecholamines present within the mobile phase enter the electrochemical detector. Electrochemical detection occurs because electroactive compounds oxidize at a certain potential and thereby liberate electrons that create measurable current. Catecholamines readily form quinones under these conditions, get oxidized, release two electrons, and create current. The electrochemical detector detects this electrical current that linearly correlates to the catecholamine concentration loaded into the ultra-performance liquid chromatography instrument. A 15-min mixing time during the adsorption and desorption steps was found to be optimal. If the washing step was omitted, the catecholamines could not be eluted from the acid-washed alumina. To prevent dilution, the alumina had to be centrifuged and not aspirated to dryness after the washing step. We report here that by changing the range in the electrochemical detector, plasma catecholamines were measured with only 12.5μL of plasma and more reliably with 25μL of plasma. The detection limit was 1ng/mL. This assay method is very useful as blood can be collected from the tail vein in a conscious mouse and the same mouse can be used for time-dependent or age-dependent studies.