bluuebunny/crossref_metadata_2025 huggingface dataset:
Recruitment of RGS5 Protein to Mechanically Activated AT1R in Arteriolar VSMC
Authors: Hong Kwangseok , Li Min , Meininger Gerald , Hill Michael
Published: June 2021

Ligand‐independent activation of G protein‐coupled receptors (GPCR) contributes to pressure‐induced vasoconstriction. Thus, stretch/mechanical activation of angiotensin II (Ang II) type 1 receptors (AT1R) likely mobilizes second messengers (e.g. Ca2+ and diacylglycerol) leading to constriction through ion channel activation and/or PKC‐dependent Ca2+ sensitization or actin polymerization. As enhanced pressure‐induced constriction may contribute to vascular disorders, mechanoactivation of AT1R is likely regulated by negative feedback mechanisms. This study explored whether regulators of G‐protein signaling (RGS) proteins, key molecules for the negative regulation of GPCR signaling, are co‐localized with mechanically activated AT1R in vascular smooth muscle cells (VSMC). An in situ proximity ligation assay (PLA) was performed with or without candesartan, an AT1R blocker, in cremaster arteriolar VSMC. VSMC were treated with Ang II or hypotonic solution to determine if ligand dependent‐ or independent‐activation of AT1R leads to translocation of RGS proteins. qPCR data indicated that RGS5 protein was abundantly expressed in cremaster arterioles (n=6). In PLA using antibodies specific for AT1R and RGS5 protein there were significantly higher fluorescent labeling, indicative of co‐localization, per VSMC detected in the presence of Ang II (1 µM, 24.25 ± 2.19) or hypotonic solution (150 mOsm/L, 22.86 ± 2.83) than at baseline (7.23 ± 0.89). Labeling produced by hypotonic solution was significantly reduced by candesartan (10 µM) (24.00 ± 5.00 VS 11.46 ± 1.30). These findings indicate that mechanoactivation of AT1R causes recruitment of RGS5 protein, which may prevent exaggerated AT1R‐mediated myogenic vasoconstriction by terminating GPCR signaling.
Vascular Collateralization Investigated in the Chorio‐Allantoic Membrane (CAM)
Authors: Pries Axel , Reglin Bettina , Rong Wenwei , Nitzsche Bianca , Maibier Martin , Secomb Tim , Xiang Weiwei
Published: June 2021

Introduction

Collateralization upon obstruction of an arterial vessel is crucial to maintain tissue perfusion. The aim of this study is to quantify the dynamics of structural adaptation on the microvascular level in a model where vascular reactions can be observed continuously.

Method

White leghorn chicken eggs were transferred on incubation day E3 into petri dishes. On E12, a selected arteriole of the CAM was occluded by laser irradiation. At several time points before and after occlusion, the area perfused by the arteriole was scanned using video microscopy. Blood flow velocity in selected vessels in the recorded area was measured (spatial correlation) and hemoglobin oxygen saturation was determined (hyperspectral analysis).

Results and Conclusion

Before occlusion, the occluded arterioles were 64.86 ± 6.33 µm in diameter and microvasculature displayed an interdigitated pattern of arterioles and venules. Flow velocities exhibited a strong linear decline with decreasing vessel diameter. After the occlusion, functional collaterals were typically observed either between one daughter arteriole and an adjacent arteriole not fed by the obstructed vessel or between one daughter and the upstream sister arteriole. They developed from pre‐existing terminal arterioles (3 of 20) and/or by remodeling of capillary meshes (17 of 20). Collateralization resulted in reversal of flow in arterioles downstream of the occlusion. It is concluded that in the CAM model, collateralization maintains local microcirculatory function via pre‐existing arterial anastomoses vessels or via capillary meshes.

Supported by EU / ITN “REVAMMAD” # 316990
Hydrogen Sulfide (H2S) Donor Attenuates Systolic Blood Pressure and Enhances Placentas Weights in Preeclampsia
Authors: Possomato‐Vieira José Sérgio , Rizzi Victor Hugo , Silva Katiussia , Nascimento Regina , Mendes Gabryella , Pupo André , Dias‐Junior Carlos
Published: June 2021

Preeclampsia (PE) is a pregnancy disorder characterized by elevated blood pressure (>140/90 mmHg) and proteinuria. Several studies relate placenta as a key organ in preeclampsia. We developed PE in an animal model using nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME). Female rats (220‐250 g) (n=16) were mated and we started applying L‐NAME, (60mg/Kg, intraperitoneally – i.p.) from 15‐21th day of pregnancy in all dams. Another set of animals (n=8) were treated with sodium hydrogen sulfide (NaHS) (50µmol/Kg, twice daily, i.p.), a quick donor of H2S, a gasotransmitter with important cardiovascular responses. We measured pressure by tail cuff plethysmography at gestational days 14, 16, 18 and 20. Animals were killed on pregnancy day 21 (ethics committee CEUA IBB/UNESP protocol 619/2014). Injection of L‐NAME significantly elevated systolic blood pressure while treatment with NaHS reduced it on pregnancy days 16 and 18 (148.12±1.48 mmHg, 149.25±1.02 mmHg; PE vs. 132.00±1.30 mmHg, 129.37±0.95 mmHg; PE+NaHS on gestational days 16 and 18, respectively). Pups body weight did not change significantly. Placentas weight however (0.55±0.01 PE vs. 0.64±0.03 PE+NaHS) was meaningfully higher in treated animals. Thus, number of reabsorbed pups (0.62±0.26 PE vs. 1.75±0.31 PE+NaHS) was higher in PE+NaHS group. MTT assay, which verifies antioxidant status of plasma, showed no difference in PE and treated animals. Our data showed that systolic blood pressure was significantly reduced in animals with PE treated with NaHS and differences in placentas weights can be related to an enhancement in blood flow promoted by H2S.
The Spatial Distribution of Nitric Oxide‐evoked cGMP Can Produce Biphasic Effects on Na+/H+ Exchanger‐1 Activity in the Rat Ventricular Myocyte
Authors: Richards M , Paterson D , Vaughan‐Jones R , Swietach P
Published: June 2021

We investigated the effects of nitric oxide (NO)‐signalling on Na+/H+ Exchanger (NHE1), the principal H+‐extruding protein in ventricular cardiac myocytes.

H+‐extrusion by NHE1 was activated by ammonium pre‐pulse (HEPES‐buffered Tyrode) and measured by pH‐sensitive fluorescence (cSNARF1).

NHE1 was inhibited by high [NO] (20nM; released from NOC‐12), but activated by low [NO] (2nM; NOC‐12 plus NO‐scavenger CPTIO). These biphasic effects were blocked by the guanylate cyclase (GC) inhibitor ODQ, implicating the NO‐GC‐cGMP pathway. The stimulatory effect of low [NO] was mimicked by the phosphodiesterase (PDE)‐resistant cGMP analogue, 8pCPTcGMP. In contrast, the inhibitory effect of high [NO] may be attributed to a cGMP‐evoked rise in cAMP (NHE1 inhibitor) involving PDE3 (high cGMP inhibits cAMP degradation by PDE3). Indeed, the inhibitory effect of high [NO] was mimicked by the PDE3 inhibitor cilostamide. To explore the spatial aspects of NO signalling, dual microperfusion was used to restrict [NO] to the proximal region of the cell (confirmed with DAR4 fluorescence). High [NO] applied locally to one end of the cell stimulated NHE1 distally, beyond the compartmentalized [NO] zone, in an ODQ‐sensitive manner, arguing against direct S‐nitros(yl)ation. Results suggest that cGMP is a mobile messenger, reaching low concentrations distally, sufficient to stimulate NHE1 but below the threshold for evoking a local rise in cAMP.

The net effect of NO on NHE1 activity is set by the balance between activation by cGMP and inhibition via cGMP‐cAMP coupling. This balance depends on the spatial concentration profile of cGMP evoked by NO. NO can modulate intracellular pH with an unprecedented degree of plasticity.

Funded by BHF
In Vivo Effects of Platelet Apheresis on Oxygen Metabolism in Pigs
Authors: Martini Wenjun , Rodriguez Cassandra , Richardson Jonathan , Cap Andrew , Dubick Michael
Published: June 2021

Introduction

Platelet apheresis is a routine clinical practice. Although the efficacy of collected platelets has been studied, the impact of apheresis on the physiology of the donor is less clear. This study investigated oxygen metabolism in pigs during a 4 hour platelet apheresis as a prelude to initiating studies of hemorrhage and resuscitation in swine with reduced platelets.

Methods

Forty pigs (39.8±0.6 kg) were catheterized with a femoral vein catheter for platelet apheresis, using a Haemonetics MCS9000. Hemodynamics was recorded during the apheresis process. Arterial and venous samples were collected before and after apheresis for blood gases and CBC.

Results

During apheresis, blood‐withdrawing decreased MAP from 74±2 to 45±1 mmHg and increased heart rate from 104±3 to 129±4 bpm (both p<0.05); blood‐returning recovered MAP and HR to baseline values, with no changes observed in cardiac output. Platelet counts in pigs decreased to 40±3% of baseline values after 4 hours of apheresis (p<0.05). Pig blood glucose was increased by apheresis from 4.1±0.3mM to 5.2±0.2mM, Ca++ decreased from 1.34±0.01mM to 1.13±0.02mM, and lactate decreased from 2.2±0.1mM to 1.3±0.1mM (all p<0.05), with no changes in Hct, Hgb and RBCs. Oxygen delivery was not affected by apheresis, but oxygen extraction was decreased from 27±2% to 20±1%, and oxygen consumption was decreased from 4.0±0.2 O2/dL blood to 3.1±0.2 ml O2/dL blood (both p<0.05).

Conclusion

In addition to reducing platelet counts, platelet apheresis caused large fluctuations in MAP and compromised oxygen metabolism, which can impact the physiologic response to hemorrhage in these animals.
Distinct Mechanisms Underlie Cardiomyocyte Contractile Dysfunction in Male and Female Endotoxemic Mice
Authors: Hobai Ion , Aziz Kanwal , Siwik Deborah , Colucci Wilson
Published: June 2021

Sepsis induced cardiomyopathy results (at least partially) from the dysregulation of myocardial calcium (Ca2+) handling. In male (M) mice, this involves redox‐mediated dysfunction of cellular Ca2+ tranporters. No data is available about Ca2+ signaling in septic female (F) hearts.

We studied M and F C57Bl6 mice (WT), as well as mice deficient in soluble guanylyl cyclase (sGC) activity (sGCα1‐/‐). Lipopolysaccharide (LPS) administration (ip) induced an inflammatory shock syndrome and cardiomyopathy. A dose of 7 μg/g LPS induced 59% mortality in M WT mice, 44% in M sGCα1‐/‐, and no mortality in F WT or F sGCα1‐/‐ mice. 20 μg/g LPS induced 100% mortality in WT F and 50% mortality in F sGCα1‐/‐ mice.

We measured sarcomere shortening (SS) and Ca2+ transients (ΔCai) in isolated, externally paced cardiomyocytes. 12h after LPS (25 μg/g) challenge, WT M mice had decreased SS and ΔCai, to 60 ± 7 and 78 ± 4% of baseline (bl), respectively (n > 60 cells from 8 mice for all groups, 60/8; p<0.05 for this and following). In sGCα1‐/‐ M, the decrease in SS and ΔCai was more pronounced than in WT (to 26 ± 3 and 53 ± 3% of bl, respectively, n>60/8). In F WT, LPS induced a decrease in SS (to 39 ± 6% of bl, n>40/4), but not in ΔCai, consistent with myofilament dysfunction. SS decrease was less in sGCα1‐/‐ F mice (70 ± 8%, n>35/4) than in WT F.

In conclusion, in M mice, LPS induces a parallel, sGC‐independent decrease in SS and ΔCai. In contrast, in F, LPS does not decrease ΔCai, but induces a (partially) sGC‐dependent myofilament dysfunction. Different therapeutic approaches may thus be required in septic M and F patients.
Coronary Vascular Effects of Leptin and Calpastatin in Lean vs. Obese Hearts
Authors: Noblet Jillian , Goodwill Adam , Sassoon Daniel , Tune Johnathan
Published: June 2021

Recent evidence suggests a role for leptin and calpastatin in the pathogenesis of obesity‐induced coronary vascular dysfunction. However, how these factors affect coronary smooth muscle reactivity in lean vs. obese hearts is not well defined. Coronary arteries from lean and obese Ossabaw swine were isolated, cleaned of all surrounding adipose tissue, and incubated with or without leptin (3 – 30 ng/mL) or calpastatin (10 μM) for 30 min. Arteries were then pre‐constricted with the thromboxane mimetic U46619 (1μM). In untreated arteries, vasodilation to adenosine (0.01 – 30 μM) was decreased ~25% in obese compared to lean arteries. Administration of leptin dose‐dependently reduced vasodilation to adenosine, with the 30 ng/mL concentration attenuating dilation ~25% in both lean and obese arteries. In contrast, the presence of calpastatin impaired responses to adenosine (10 μM) in lean (~26%), but not obese arteries. In additional studies, coronary arteries from lean swine were cultured at 37oC in the presence/absence of leptin (30 ng/mL) or calpastatin (10 μM) for 3 days and subsequently subjected to functional isometric tension studies. Relative to untreated cultured arteries, incubation with leptin augmented active tension development to KCl‐induced depolarization (30 mM) and diminished vasodilation in response to adenosine (10 μM). Incubation with calpastatin modestly reduced vasodilation to adenosine but did not influence active tension development to KCl. These findings indicate that leptin and calpastatin differentially influence vascular function and suggest that these factors could contribute to the augmented contractile, impaired vasodilator phenotype of the obese coronary circulation.
Hydrogen Sulfide Is An Endogenous Cytoprotective Gaseous Mediator in Cerebral Circulation
Authors: Liu Jianxiong , Leffler Charles , Parfenova Helena
Published: June 2021

Hydrogen sulfide (H2S) is a gaseous mediator with vasodilator and antioxidant properties produced by cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS). In the brain, CSE and CBS are predominant H2S‐producing enzymes in vessels and astrocytes, respectively. We investigated cytoprotective properties of H2S in a model of cerebrovascular disease caused by neonatal seizures. In newborn piglets, bicuculline‐induced epileptic seizures produce long‐term cerebral vascular dysfunction. During seizures, brain CBS activity is increased as indicated by H2S elevation in periarachnoid cerebrospinal fluid (CSF) that was selectively inhibited by aminooxyacetate. We addressed the hypothesis that H2S improves the cerebral vascular outcome of seizures. An H2S donor NaHS (3 mg/kg) administered enterally or parenterally rapidly increased H2S in CSF. At this dose, NaHS had no effects on core body temperature, mean arterial blood pressure, or heart rate. To test the cytoprotective effects of NaHS, the drug was administered before or during seizures. Cerebral vascular reactivity to physiologically relevant vasodilators was tested by the cranial window technique 48 h after seizures. In the control group, we observed a great reduction in postictal dilator responses to endothelium‐ and/or astrocyte‐dependent stimuli bradykinin, glutamate, and hemin. NaHS administered enterally or parenterally before or during seizures improved all postictal vascular responses. We conclude that H2S, endogenously produced in the brain via CBS activation during seizures, or pharmacologically delivered to the brain during a therapeutic window, reduces the adverse cerebral vascular outcome of neonatal seizures thus leading to vascular neuroprotection.

The study was supported by NIH/NLBI.
Enhanced Serotonin (5‐HT)‐Mediated Carotid Vessel Constriction From The Fawn Hooded Hypertensive (FHH) Rat
Authors: Pabbidi Mallikarjuna , Kaur Gurjit
Published: June 2021

Impaired myogenic response associated with an increase in BK (large conductance potassium) channel function was suggested in cerebral resistance vessels of FHH rats. Introgression of BN (Brown Norway) 2.4 Mbp region of chromosome 1 into FHH rats restored the function of these vessels by normalizing BK channel function in an FHH.1BN congenic rat. However, conclusions derived from resistance vessels cannot be extrapolated to conduit vessels such as the carotid or aorta since they differ in structure, function, and expression of contractile proteins. Here we tested agonist‐mediated contraction and the regulatory role of PKC in mediating K channel function using isometric measurement of tension in carotid vessels isolated from FHH and FHH.1BN rats. Serotonin (5‐HT)‐mediated tension is enhanced in carotid rings isolated from FHH rats when compared to FHH.1BN rats. 5‐HT‐mediated constriction is enhanced further more by inhibition of Kv channel (4AP; 1mM) but not BK channel (Paxilline or Iberiotoxin; 100nM) in FHH rats. 5‐HT‐mediated constriction is not affected by PKC activation (PMA; 100nM) in FHH rats but significantly enhanced in FHH.1BN rats. While inhibition of PKC (GF 109203X, 3µM) diminished 5‐HT‐mediated contraction in both strains it has significantly less effect in FHH rats compared to FHH.1BN rats. Together, these results suggest that mutation in the genes located in 2.4 Mbp region of FHH rat may enhance PKC function that inhibit K channels contributing to augmented 5‐HT‐mediated carotid vessel constriction. This research has received partial funding support from the American Heart Association, Scientist Development Grant (13SDG14000006).
Severe Cerebrovascular Dysfunction in a Hypertensive Heart Failure Model
Authors: Brooks Steven , Stanley Shyla , Chantler Paul , Brock Robert , Frisbee Jefferson
Published: June 2021

The spontaneously hypertensive, heart failure‐prone rat (SHHF) is a preclinical model of heart failure, and is available in both lean/hypertensive (SHHF‐L) and obese/hypertensive (SHHF‐Ob) strains. While cardiac mechanics have been studied extensively in this model, vascular reactivity of conduit and small arteries has not been evaluated. The aim of this study was to assess vasoreactivity in aortic rings, isolated middle cerebral arteries (MCA), and gracilis arterioles (GA) of adult male SHHF‐L and SHHF‐Ob animals.

At 37‐40 weeks of age, vessels were harvested from SHHF animals for evaluation. Aortic rings were placed in a myobath, and MCA and GA into isolated microvessel chambers. Vascular reactivity to dilator/constrictor stimuli was measured by tension myograph or video micrometer. Vasodilation to ACh was significantly impaired in aortic rings and GA of SHHF‐L (70% compared to non‐hypertensive lean control) and SHHF‐Ob (60‐70% to control). However, dilation to ACh in MCAs was reduced more than 50 % to control in the SHHF‐L, and nearly ablated in SHHF‐Ob. Both GA and MCA of SHHF‐L and SHHF‐Ob were hyperconstrictive to adrenergic stimuli. Myogenic reactivity was significantly impaired in MCA of both SHHF‐L and SHHF‐Ob, even compared to GA from the same animals.

Our findings reveal severe cerebrovascular dysfunction in both lean and obese SHHFs. SHHF rats are severely hypertensive (MAP>175 mmHg), which impacts vascular tone; however, the difference in reactivity between GA and MCA indicates a more complex pathology. Further investigation is needed to determine the physiological mechanisms of vascular dysfunction in this preclinical animal model.
Cerebrovascular Abnormalities Supporting the Selfish Brain Hypothesis of Human Hypertension
Authors: Hart E , Rodrigues J , Ratcliffe L , Burchell A , Manghat N , Wise R , Nightingale A , Paton J
Published: June 2021

The selfish brain hypothesis of hypertension suggests that brainstem hypoperfusion triggers increased sympathetic activity (SNA) and blood pressure (BP) as a mechanism to maintain cerebral perfusion. We assessed: 1) prevalence of anatomical variations in the posterior cerebral circulation, which may cause brainstem hypoperfusion, 2) relationships of SNA to cerebral vascular resistance (CVR) in hypertensive (HTN) vs normotensive (NTN) humans. The vertebral arteries (VA) and Circle of Willis (CoW) were imaged in 121 HTN (MR angiography). VA hypoplasia (VAH), defined as <2mm diameter, together with CoW morphology were compared to known standards in >50 NTN. In a cohort of HTN (n=50) and NTN (n=40), muscle SNA (MSNA, microneurography), blood flow in the internal carotid arteries (ICA) and VA (MR phase contrast), and BP were recorded.

Prevalence of VAH and incomplete posterior CoW was higher in HTN vs NTN (51 vs 27% and 62 vs 38%, p<0.05). Of the posterior CoW variants, complete disconnection of anterior/posterior CoW was higher in HTN (31 vs 10%, p<0.05). Prevalence of having both VAH and incomplete posterior CoW variant was higher in HTN vs. NTN (38 vs 10%, p<0.05). Total CVR and CVR in VA was higher in HTN vs. NTN (2.6 ±0.1 vs 1.1±0.1 and 9.5±0.1 vs 6.7±0.1 mmHg/mL/dl.tissue/min). CVR was related to MSNA in both groups (HTN; r=0.40, NTN; r=0.38, P<0.05). CVR was more closely related to MSNA in the VA vs ICA in HTN (r=0.40 vs r=0.30, P=0.05).

The higher prevalence of VAH and incomplete posterior CoW may cause the observed increase in CVR in HTN. The higher CVR in the posterior circulation was linked to higher MSNA in HTN, supporting the hypothesis of brainstem hypoperfusion as a driver of neurogenic hypertension.

BHF.
Effects of Hyperandrogenemia on Cardiovascular and Metabolic Responses to Chronic Melanocortin‐4 Receptor Blockade in Female SHR
Authors: do Carmo Jussara , da Silva Alexandre , Hall John
Published: June 2021

Hyperandrogenemia in females has been associated with sympathetic nervous system (SNS) activation and increased blood pressure (BP). We demonstrated that activation of the central nervous system (CNS) melanocortin system contributes to increases in SNS activity and BP in male spontaneously hypertensive rats (SHRs). We examined if chronic antagonism of melanocortin‐4 receptors (MC4R) attenuates elevated BP in female SHRs treated with dihydrostestosterone (DHT, 7.5 mg/90 days). Cardiovascular and metabolic effects of MC4R antagonism were assessed in female DHT‐treated SHR (n=8) and control untreated SHRs (n=7). At 15 weeks of age, the rats were implanted with telemetry probes to measure BP and heart rate (HR), and an intracerebroventricular (ICV) cannula was placed into the lateral ventricle for infusions. After control measurements, the MC4R antagonist (SHU‐9119) was infused (1 nmol/h, ICV) for 10 days followed by a 5‐day recovery period. MC4R antagonism increased food intake and body weight in DHT‐treated SHR (14±1 to 35±1 g/day and 244±3 to 298±8 g) and controls (14±1 to 34±2 g/day and 207±4 to 269±8 g). No changes were observed in blood glucose. Compared to controls, DHT‐treated SHRs had slightly higher BP, but lower HR (146±3 vs. 142±4 mmHg and 316±2 vs. 363±4 bpm). Chronic SHU‐9119 infusion reduced BP and HR in DHT‐treated SHR (‐12±2 mmHg and ‐14±4 bpm) and control SHR (‐19±2 mmHg and ‐21±6 bpm). These results indicate that MC4R antagonism reduces BP and HR despite marked increases in food intake and body weight in hyperandrogenemic and control female SHR, although the reductions in BP and HR were slightly attenuated in hyperandrogenemic SHR. (NHLBI‐PO1HL51971, NIGMS‐ P20GM104357 and AHA SDG5680016)
The Effect of Multiple Sclerosis on Carotid Baroreflex Latencies of Arterial Blood Pressure and Heart Rate
Authors: Allen Dustin , Huang Mu , Davis Scott , Keller David
Published: June 2021

Multiple sclerosis (MS) is marked by conduction abnormalities within the central nervous system that lead to impaired blood pressure regulation. However, the impact of these abnormalities on dynamic blood pressure control, particularly the timing of the responsiveness (i.e., latency), has not been examined. We tested the hypothesis that carotid‐cardiac and carotid‐vasomotor latency would be delayed in individuals with MS (n=5) compared to healthy controls (CON, n=5). Carotid baroreflex function was assessed using 5‐sec trials of neck pressure (NP, +40 Torr, simulated hypotension) and neck suction (NS, ‐60 Torr, simulated hypertension). Latencies (i.e., time‐to‐peak responses) were determined for both carotid‐cardiac (peak HR responses) and carotid‐vasomotor (peak MAP responses) by summing the R‐R intervals from the onset of the respective stimuli to the R wave of the cardiac cycle associated with the respective peak responses. Carotid‐cardiac latency was not different between groups for either NP (MS: 3.7±1.3 sec, CON: 3.7±1.3 sec; p=0.97) or NS (MS: 1.6±1.1 sec, CON: 1.9±0.8 sec; p=0.62). In addition, the carotid‐vasomotor latency to NP (MS: 5.7±1.7 sec, CON: 5.6±1.4 sec; p=0.90), and NS (MS: 4.7±2.3 sec, CON: 6.8±0.6 sec; p=0.08) were not different between groups. It appears MS does not markedly alter the timing of peak carotid‐cardiac and carotid‐vasomotor responses.

Supported by NIH R15HL117224 and National MS Society RG4696A
SOD1 Overexpression Alters Aortic Depressor Nerve Function but Does not Protect Against Impairment of Baroreflex Heart Rate Control in Chronic Intermittent Hypoxia Exposed Mice
Authors: Hatcher Jeffrey , Gu He , Cheng Zixi
Published: June 2021

Mice overexpressing Cu/Zn Superoxide Dismutase (SOD1) have been used as a model for investigating effects of increased antioxidant activity in pathologies in which reactive oxygen species (ROS) play a role. Our objective in this study is to determine if human SOD1 overexpression can protect against dysfunction of aortic depressor nerve and baroreflex control of heart rate in chronic intermittent hypoxia (CIH) exposed mice. We used the transgenic mouse line C57B6SJL‐Tg (SOD1)2 Gur/J overexpressing human SOD1 and the non‐transgenic C57BL/6J mice (control). Mice (male, 4 months old, n=8‐9/group) were either maintained in room air (RA), or subjected to 6 minutes cycles of 21% and 5.7% O2 during 12‐h light cycles for 40‐50 days (CIH). Mice were anesthetized with isoflurane. Arterial blood pressure (ABP) and heart rate (HR) were measured from the femoral artery. Sodium nitroprusside (SNP) and phenylephrine (PE) were infused sequentially into the femoral vein to decrease and then increase ABP. Aortic depressor nerve activity (ADNA) was recorded. We found that: HR and mean arterial blood pressure (MABP) were similar between groups. Baroreflex control of HR in response to infusion of PE or SNP was not different in SOD1‐RA animals vs. C57‐RA, and it was decreased to a comparable extent in SOD1‐CIH and C57‐CIH. Recordings of ADNA in response to sequential SNP and PE injection‐induced blood pressure ramps revealed a significantly greater baroreceptor gain in SOD1‐RA compared to C57‐RA (p < 0.01), but CIH exposure significantly decreased ADN baroreceptor gain in SOD1‐CIH compared to SOD1‐RA (P<0.01). Our data suggests that SOD1 overexpression alters ADN function, but does not protect against CIH‐induced baroreflex impairment.
Augmentation of Functional Sympatholysis via Endothelial Signaling Is Independent of Nitric Oxide and Prostaglandins in Humans
Authors: Hearon Christopher , Kirby Brett , Crecelius Anne , Luckasen Gary , Larson Dennis , Dinenno Frank
Published: June 2021

Sympathetic vasoconstriction is blunted in contracting skeletal muscle in an intensity dependent manner, and increasing endothelium‐dependent vasodilatory signaling during mild exercise can augment this “sympatholysis” similar to that observed during moderate exercise. Here, we determine the role of nitric oxide (NO) and vasodilating prostaglandins (PGs) to the endothelium‐dependent augmentation of sympatholysis during mild exercise. In 10 young subjects we measured forearm blood flow (Doppler ultrasound) and calculated vascular conductance (FVC) responses to local intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during 1) infusion of the endothelium‐dependent dilator acetylcholine (ACH), 2) moderate (15% maximum voluntary contraction; MVC) handgrip exercise and 3) mild (5% MVC) exercise + ACH infusion. Trials were repeated after combined local blockade of NO/PGs (L‐NMMA/Ketorolac, respectively). Vasoconstriction to PE was blunted during 15% exercise compared to control ACH infusion (ΔFVC: ‐14±3% vs ‐30±3%, P<0.05). Augmenting endothelium‐dependent signaling during 5% exercise blunted vasoconstriction similar to 15% exercise (ΔFVC: ‐8±4%). After blockade of NO/PGs, vasoconstriction to PE was blunted in all conditions (ΔFVC: ACH: ‐20±5%; 15% MVC: ‐15±5%; 5%MVC + ACH: ‐7±4%; all P<0.05) relative to control ACH infusion. These data demonstrate that increasing endothelial vasodilatory signaling limits sympathetic vasoconstriction independent of NO/PGs, implicating a role for endothelial hyperpolarization in sympatholysis in humans. Support: HL102720
Acid Sensing Channel 1 in Astroglia Is Up‐regulated by Saporin
Authors: Lin Li‐Hsien , Jones Susan , Talman William
Published: June 2021

Acid‐sensing ion channels (ASICs) are activated by acidosis and may act as chemosensors in the CNS. RT‐PCR has revealed that ASIC type 1 (ASIC1) is the predominant ASIC transcript in the nucleus tractus solitarii (NTS), but anatomical studies of its expression in the NTS have been lacking. We performed confocal microscopic immunofluorescent histochemistry to examine the distribution of ASIC1 in rat NTS. Previously we showed that saporin causes gliosis in the NTS. Because astrocyte damage may lead to acidosis and result in ASIC1 activation, we tested the hypothesis that saporin treatment increases ASIC1 expression in the NTS. Confocal images showed cells and processes that contained ASIC1‐immunoreactivity (IR) scattered in the NTS, dorsal motor nucleus of vagus (DMV) and area postrema (AP). ASIC1‐IR predominantly colocalized with IR of the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42), in the NTS and DMV. The sup‐postremal NTS was the only area where neurons containing IR of a neuronal marker contained ASIC1‐IR. ASIC1‐IR as well as OX42‐IR increased markedly in the NTS 7 days after saporin injection. Saporin caused depletion of GFAP‐IR in the center of the injection, but GFAP‐IR increased in the surrounding area. The increase in ASIC1‐IR was found in cells and processes that contained GFAP‐IR or OX42‐IR. Our results confirm that ASIC1 is increased with saporin‐induced gliosis in the NTS. The increased ASIC1‐IR in microglia may be associated with saporin‐induced inflammation. Potentially increased ASIC1‐IR in astrocytes could play a role in cardiovascular reflex attenuation that occurs after administration of saporin into the NTS. Support: NIH RO1 HL 088090 and a VA Merit Review
Hypoxia activates neurons in the hypothalamic paraventricular nucleus (PVN) that project to the nucleus tractus solitarii (nTS) in the brainstem
Authors: Ruyle Brian , Coldren K. , Palmieri Elizabeth , Heesch Cheryl , Hasser Eileen
Published: June 2021

The PVN is critical to arterial chemoreflex function and projects to brainstem cardiorespiratory regions. Our group recently showed that efferent projections to the rostral ventrolateral medulla and the intermediolateral cell column are minimally activated by acute hypoxia (AH), suggesting other efferent pathways are involved. We hypothesized that PVN neurons with nTS projections are activated by AH. Rats were microinjected with a retrograde tracer in the nTS to label nTS‐projecting PVN neurons. Conscious rats were subjected to either two hours of normoxia (Norm; 21% O2) or hypoxia (AH; 10% O2). Brains were perfused and immunohistochemistry performed to identify activated neurons (Fos‐IR) and determine the phenotype (nNOS‐, AVP‐, CRH‐ and Oxy‐IR) of nTS‐projecting PVN neurons. AH significantly activated nTS‐projecting PVN neurons (Norm: 26±4; AH: 46±7 cells). Similar to our previous work, AH significantly activated nNOS‐IR and CRH‐IR neurons, but few AVP‐IR cells. OXY‐IR PVN neurons were significantly activated by AH but very few of the activated OXY‐IR cells projected to the nTS (1.6±0.9%). Subpopulations of nTS‐projecting cells contained nNOS (17±1%) or CRH (26±4%). Furthermore, these projecting cells were significantly activated by AH (nNOS, Norm: 16±3; AH: 24±3%; CRH, Norm: 13±0.05; AH: 29±2%). These results suggest the PVN contributes to chemoreflex output via efferent projections of CRH‐ and nNOS‐IR neurons to the nTS. (HL98602)
Early Spinal Cord Rewiring Revealed by Transcranial Magnetic Stimulation following C2 Partial Injury: Implication of the Crossed Phrenic Pathway
Authors: Vinit S , Petitjean M , Keomani E , Deramaudt T , Bonay M
Published: June 2021

High cervical spinal cord injuries lead to permanent respiratory deficits. The preclinical model of respiratory insufficiency in the adult rat is the C2 partial injury which conducts to unilateral diaphragm paralysis. This model allows the investigation of a particular population of bulbospinal axons which crossed the midline at C3‐C6 spinal segment, namely the crossed phrenic pathway. Transcranial magnetic stimulation (TMS) has recently proved to be a non‐invasive technique to study supraspinal descending respiratory pathways in the rat (Vinit et al., 2014). Interestingly, a C2 injury does not affect the amplitude and latency of the largest motor evoked potential recorded on the diaphragm (MEPdia) ipsilateral to the injury in response to a single TMS pulse compared to a sham animal. Although the rhythmic respiratory activity on the contralateral diaphragm remains 7 days post‐injury, no diaphragm activity can be recorded on the ipsilateral side. However, a profound reorganization of the MEPdia evoked by TMS is recorded. The MEPdia is reduced on the contralateral side compared to the one observed on the ipsilateral side. This suggests an increase in ipsilateral phrenic motoneurons excitability. Moreover, a correlation between MEPdia amplitude and spontaneous contralateral diaphragmatic activity is observed. The larger diaphragm activity is correlated with a larger MEPdia on the ipsilateral side, and a smaller MEPdia on the contralateral side. This suggests the occurrence of a neuroplasticity process involving motoneuron excitability balance changes between the ipsilateral and contralateral sides at a short post‐lesional delay.
Interdisciplinary approaches of transcranial magnetic stimulation applied to a respiratory neuronal circuitry model
Authors: Petitjean M , Vinit S , Keomani E , Deramaudt T , Spruance V , Bezdudnaya T , Lane M , Bonay M
Published: June 2021

Respiratory related diseases associated with the neuronal control of breathing represent life‐threatening issues and to date, no effective therapeutics are available to enhance the impaired function. The aim of this study was to determine whether a preclinical respiratory model could be used for further studies to develop a non‐invasive therapeutic tool applied to rat diaphragmatic neuronal circuitry. Transcranial magnetic stimulation (TMS) was performed on adult male rats using a human figure‐of‐eight coil. The largest diaphragmatic motor evoked potentials (MEPdia) were recorded when the center of the coil was positioned 6mm caudal from Bregma, involving a stimulation of respiratory supraspinal pathways. Magnetic shielding of the coil with mu metal reduced magnetic field intensities and improved focality with increased motor threshold and lower amplitude recruitment curve. Moreover, transynaptic neuroanatomical tracing with pseudorabies virus suggest that connections exist between the motor cortex, the periaqueductal grey cell regions, several brainstem neurons and spinal phrenic motoneurons (C3‐4 spinal cord). These results reveal the anatomical substrate through which supraspinal stimulation can convey descending action potential volleys to the spinal motoneurons. We conclude that MEPdia following a single pulse of TMS can be successfully recorded in the rat and may be used in the assessment of respiratory supraspinal plasticity. Non‐invasive stimulations aimed to neuromodulate respiratory circuitry will enable new avenues of research into neuroplasticity and the development of therapies for respiratory dysfunction associated with neural injury and disease.
Impact of prenatal nicotine and infection on postnatal development and forebrain gene expression
Authors: Martin Eric , Hernandez Morgan , Von Chamier Maria , Reyes Leticia , Brown Mary , Hayward Linda
Published: June 2021

Smoking and intrauterine infection are among the two greatest risk factors for adverse pregnancy outcome, but little information is available regarding the interaction of these two key risk factors on the developing brain. The central hypothesis tested in this study was that prenatal nicotine exposure promotes microbial infection in utero resulting in a more detrimental postnatal outcome than prenatal nicotine or prenatal infection alone. This hypothesis was tested in a rodent model of prenatal nicotine exposure (NIC) using an osmotic mini pump inserted on day 6 of pregnancy (3 mg/kg) and maternal infection (INF) presented via intravenous injection of an urogenital rat mycoplasma on gestational day 14. Preliminary data demonstrate that nicotine/infection (NIC/INF) significantly reduced fetal weight (gestational day 18). On postnatal day 15 (PD 15) all forms of prenatal stress significantly increased body weight compared to sham treated animals. Behavioral tests on PD 15 identified an impairment of both wire hang and cliff‐avoidance tests in NIC/INF offspring compared to SHAM, NIC alone and INF alone. Preliminary analysis of gene expression in female offspring demonstrated a dysregulation of CRH expression in the amygdala: CRH expression was unchanged by NIC and trended toward up‐regulation in INF but was down‐regulated in the NIC/INF offspring. These results indicate that prenatal stress associated with NIC/INF in utero significantly alters both physical development and gene expression in the forebrain differently than exposure to either prenatal stress alone. Funded by FL‐DOH James and Esther King Biomedical Research Grant (LFH&MB).
Renal Denervation Increases Renal Blood Flow Variability in Conscious Rabbits
Authors: Schiller Alicia , Pellegrino Peter , Zucker Irving
Published: June 2021

Activation of the renal nerves, as observed in multiple disease states, is known to have a profound impact on renal blood flow (RBF) and hemodynamics. However, little is known about their role in maintaining renal hemodynamics under normal resting conditions. We hypothesized that the renal nerves limited RBF variability. To test this, we instrumented 8 New Zealand White rabbits with radiotelemetry, and implanted a renal flow probe on the left kidney. Rabbits randomized to the denervation group underwent bilateral denervation by stripping the renal arteries of all visible fibers. In the innervated group a sham surgery was performed. Data were recorded in conscious rabbits resting quietly. Denervated rabbits had increased RBF (40.7 ± 4.2 mL/min vs. 62.0 ± 9.9 mL/min, p < 0.05) and greater beat‐to‐beat RBF variability quantified by the standard deviation of the beat‐to‐beat RBF (2.6 ± 0.2 mL/min vs 3.7 ± 0.3 mL/min p < 0.01). The increased variability persisted even after variance stabilization of the beat‐to‐beat RBF by square root transformation (0.20 ± 0.01√ (mL/min) vs 0.25 ± 0.01√(mL/min), p < 0.05). Similarly, greater dispersion of the beat‐to‐beat RBF was observed in the RBF histograms of denervated rabbits compared to innervated rabbits (p < 0.001). Frequency analysis revealed that the renal nerves decreased the proportion of high frequency variability (24.8 ± 3.8 % vs 38.5 ± 1.2 % of total power p < 0.01). Additionally, the transfer function gain between mean arterial pressure and RBF tended to be lower in innervated rabbits compared to denervated rabbits (1.00 ± 0.25 mL/min/mmHg vs. 1.89 ± 0.35 mL/min/mmHg, p = 0.08) in the high frequency range. These data indicate that the renal nerves serve to limit fluctuations in RBF. Supported by AHA PREDOC14700045.
Intermittent hypercapnia produces long‐term changes of phrenic and renal sympathetic nerve activities that are serotonin dependent
Authors: Valic Maja , Valic Zoran , Pecotic Renata , Pavlinac Dodig Ivana , Stipica Ivona , Dogas Zoran
Published: June 2021

This study was performed to investigate long‐term changes of peak phrenic (pPNA) and renal sympathetic nerve (RSNA) activities during and following acute intermittent hypercapnic exposures. Urethane anesthetized, vagotomized, paralyzed and mechanically ventilated male Sprague‐Dawley rats, were exposed to the acute intermittent hypercapnia protocol consisting of 5 hypercapnic episodes (15% CO2). Experimental groups were pretreated with either methysergide (3 mg/kg iv, MeHC) or WAY‐100635 (3 mg/kg iv, WAY) prior to the onset of the first hypercapnia. PPNA or RSNA and blood pressure were recorded throughout the protocol and analyzed before and during hypercapnic exposures, and 15 (T15), 30 (T30) and 60 (T60) minutes after the last hypercapnic episode. PPNA did not significantly change at T15, T30 and T60 compared to baseline. In the MeHC group, pPNA significantly decreased at T30 and T60 (by 41.3 ± 5.3%; and by 46.1 ± 6.9%, P<0.005), compared to baseline values. In the WAY group, there were no significant changes in the pPNA at T15, T30, and T60 compared to baseline values. RSNA increased during hypercapnia and remained elevated at T15, T30 and T60 compared to baseline. In the WAY group RSNA increased during hypercapnia but returned to the baseline level at T60 (95.4 ± 23.1%). Acute intermittent hypercapnia induced long‐term depression of the pPNA following acute intermittent hypercapnia exposure only after blockade of serotonin receptors in anesthetized rats. On the other hand, acute intermittent hypercapnia evoked long‐term facilitation of the RSNA that was attenuated after blockade of serotonin receptors. Support: Croatian Science Foundation 09/165.
Effects of A2a Receptors on the Hypoxic Ventilatory Response in Rats Exposed to Chronic Sustained Hypoxia
Authors: Basaran K , Moya Esteban , Fu Zengxing , Powell Frank
Published: June 2021

Recent studies show that long term facilitation of phrenic nerve activity (LTF) with acute intermittent hypoxia (IH) involves two signaling pathways. In addition to the well‐known serotonergic (Gq) pathway, a Gs pathway can be activated by adenosine receptor 2 (A2a) agonists and cause LTF without IH (J Neurosci 28:2033‐42, 2008). Gs can inhibit the Gq pathway as A2a antagonists increase LTF from IH by disinhibiting Gq (J Physiol 588:255‐66, 2010). We tested the hypothesis that Gs also contributes to plasticity in ventilatory control during chronic sustained hypoxia (CH) by measuring the effects of A2a antagonists and agonists on ventilatory responses to hypoxia and hypercapnia in adult male rats before and after one week of CH (PO2 = 80 Torr in a hypobaric chamber). Ventilation measured in awake unrestrained rats with barometric pressure plethysmography increased after CH; acute ventilatory responses to hypoxia or hypercapnia increased also. Acute administration of A2a antagonist (MSX‐3, 1 mg/Kg i.p.) after CH did not change ventilation or acute ventilatory responses. A2a agonists (CGS 21680, 100 µg/Kg i.p.) before CH had no effect on ventilation or ventilatory responses, in contrast to stimulation reported for sleeping rats (J Neurosci 28:2033‐42, 2008). The results do not support a role for Gs in the increased ventilatory drive and hypoxic responses with CH, in contrast to their role in LTF in IH.

Supported by NIH RO1 HL‐081823.
Assessment of Diaphragm EMG Activity Recovery Following Upper Cervical Spinal Cord Injury
Authors: Khurram Obaid , Vasdev Ranveer , Sieck Gary , Mantilla Carlos
Published: June 2021

Motor units are normally recruited in an orderly manner in order to accomplish ventilatory behaviors. The duration of non‐stationarity in electromyographic (EMG) bursts provides an estimate of the period of motor unit recruitment. Motor unit recruitment also reflects central drive, which can be estimated by the root‐mean‐squared (RMS) EMG value 75 ms after the EMG burst onset (RMS75). Following unilateral diaphragm muscle (DIAm) paralysis induced by upper cervical spinal cord hemisection at C2 (SH), there is gradual spontaneous recovery of ipsilateral EMG activity. Increased activity of the contralateral (uninjured) DIAm is sufficient to maintain ventilation following SH. We hypothesized that changes in central drive can be estimated by the period of non‐stationarity and RMS75 from the contralateral DIAm EMG following ipsilateral paralysis and spontaneous recovery of DIAm EMG activity. We found that the duration of non‐stationarity increased to a greater extent in rats that did not recover ipsilateral EMG activity by 14 days post‐SH (D14) compared to those that did. In addition, we found that RMS75 values following SH were reduced in rats displaying recovery of ipsilateral DIAm EMG activity compared to rats that did not. Collectively, these results suggest that the duration of non‐stationarity and RMS75 can be used to evaluate recovery of ipsilateral DIAm function following spinal cord injury.
Tauopathy in the Ponto‐Medullary Brainstem Nuclei of Aged Tau‐P301L Mice
Authors: Stanic Davor , Bautista Tara , Jones Sarah , Dutschmann Mathias
Published: June 2021

We recently identified key brainstem areas critically involved in coordinating swallowing and breathing: 1) Nucleus of the solitary tract (NTS), which generates a phasic or rhythmic ‘command’ to produce sequential swallowing in response to sensory stimuli; and 2) Kölliker‐Fuse nucleus (KF), which provides tonic drive for the laryngeal adductors and completely seals the trachea during, and between, swallows. Our aim was to identify whether an underlying pathology exists in the NTS and KF of an established model of neurodegeneration, the Tau‐P301 mouse, and humans that suffered from dementia, which may link dementia and swallowing dysfunction.

Tauopathy and neurofibrillary tangle‐related neuropathological morphology was identified by immunohistochemistry using antisera raised against phosphorylated tau. Double‐fluorescence immunohistochemistry was performed to characterise the identity and functionality of cell bodies expressing phosphorylated tau.

Cell bodies immunopositive for phosphorylated tau were distributed within the KF, and to a lesser degree, NTS, of Tau‐P301 mice, and the KF of dementia patients with a history of swallowing disorder. This coincided with an upregulated density/intensity of GABA and GAD67 immunoreactivity in fibers/terminals within the KF. Tau positive cell bodies in the KF coexpressed the transcription factor FOXP2.

Respiratory‐related neurons in the brainstem are susceptible to neurodegeneration in Tau‐P301 mice and humans suffering dementia, which may disrupt the KF's control of laryngeal adductor motor neurons in the NA and lead to miscoordinated swallowing/breathing patterns.
The Diaphragm Challenged by Acid‐Base Imbalance
Authors: Fitzgerald Robert , Howell Sandra , Roussos Charis
Published: June 2021

Respiratory and Metabolic Acidosis can result from a variety of diseases and be sensed by receptors in the respiratory control system. But acid/base imbalance also directly affects the performance of the principal muscle of respiration, the diaphragm (D). In this presentation we provide an overview of the in vitro (rat) and in vivo (dog) D responses to four challenges: Uncompensated/Compensated respiratory acidosis (HC/CHC) and Uncompensated/Compensated Metabolic Acidosis (MA/CMA). Our findings revealed that HC reduced the force of tetanic contraction (FC) in both rat and dog Ds. This phenomenon is often seen in human subjects as well. CHC also reduced the FC of the rat D but less that HC. Both MA and CMA reduced the FC in both rat and dog Ds. We found decreases in FC are due to decreases in peak twitch tensions and increases in ½ relaxation time (1/2RT). We found that in the rat D neither HC, MA, nor CMA reduced ATP or PCr. Further, we found that methylxanthines, neostigmine, isoproterenol acted to attenuate the decreases in FC, each by different mechanisms. But first a glance at how decreased pHi per se can affect striated muscle: (1)Increased binding of Ca++ by the sarcoplasmic reticulum (SR) and decreased rate of Ca++ uptake and release per impulse. The first would prolong twitch ½ RT. (2)ATPase activity is decreased and Ca++ concentration requirement for myofibril activation is increased. (3) K+ leaks from the cell, hyperpolarizing the cell and reducing membrane excitability.

This work was performed when S. Howell and Ch. Roussos were at Meakins‐Christie Laboratories, McGill University.

Support: NIH (HL RO1‐50712‐14; MRC, Canada, Quebec Thoracic Society.