bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of placental ischemia on cardiac structure and function during pregnancy and Post‐Partum
Authors: Bakrania Bhavisha , Granger Joey P.
Published: November 2023

Preeclampsia (PE) is a disorder prevalent in 5–7% of pregnancies. It is characterized by maternal hypertension and endothelial dysfunction. PE is thought to develop in response to impaired spiral artery remodelling of the placenta, which leads to placental ischemia and ultimately the release of anti‐angiogenic and pro‐inflammatory factors into the maternal circulation. More recently, it has been shown that cardiac function is impaired in women with PE, including hypertrophy and, systolic and diastolic dysfunction, with symptoms persisting one year post‐partum. In addition to the significant health risks imposed during pregnancy, multiple studies report increased risk of cardiovascular disease for mother and baby later in life. The placental factors, sFlt‐1 and TNF‐α, have been shown to play a role in cardiac dysfunction and the structural abnormalities seen during PE, however, the mechanisms remain unclear. Therefore, it is important to utilise an appropriate model of PE to assess cardiac function and investigate mechanisms of cardiac dysfunction, such as the Reduced Uterine Perfusion Pressure (RUPP) model, where silver clips are placed on the abdominal aorta and branches of the ovarian arteries to induce placental ischemia. We have previously reported that the RUPP model has many of the characteristics seen in women with PE. The aim of this study was to assess cardiac structure and function in RUPP rats during pregnancy and post‐partum to discern any similarities to the human condition. A normal pregnant (NP, n=8) and RUPP (n=8) group was included in this study. The RUPP group underwent surgery on gestational day (GD) 14 for placement of silver clips. Both the NP and RUPP rats had carotid catheters placed on GD 18, and all initial blood pressure and echocardiography measurements were made on GD 19. A second echocardiography analysis was performed 3 months post‐partum. The RUPP group had significantly increased mean arterial pressure compared to the NP group on GD 19 (123 ± 3 vs. 97 ± 2 mmHg, P<0.01). RUPP hearts also showed reductions in ejection fraction (60 ± 2 vs. 78 ± 2 %, P<0.01) and fractional shortening (46 ± 3 vs. 56 ± 1 %, P=0.05), in addition to cardiac hypertrophy (0.97 ± 0.04 vs.0.91 ± 0.02 g, P=0.02). Interestingly, systolic dysfunction was persistent post‐partum in RUPP hearts, where ejection fraction (67 ± 3 vs.76 ± 2 %, P<0.01) and fractional shortening (50 ± 5 vs. 56 ± 2 %, P=0.05) remained impaired. In conclusion, cardiac dysfunction is present in the RUPP model during pregnancy and post‐partum. Therefore, this model could be useful in understanding the mechanisms which underpin the cardiac structural and functional abnormalities seen in PE patients.

Support or Funding Information

This study was funded by the NIH (HL51971).
Endothelin receptor type B (ETB)‐deficient pregnant rats have exaggerated placental ischemia‐induced hypertension
Authors: Dent Edward Alexander , Spradley Frank Travis , Granger Joey P
Published: November 2023

Preeclampsia (PE) is a pregnancy‐specific disorder of new‐onset hypertension that threatens the lives of both mother and fetus. While the pathogenesis of PE is unclear, studies have implicated placental ischemia. Indeed, reduced uterine perfusion pressure (RUPP)‐induced placental ischemia in experimental animals stimulates the release of soluble factors into the maternal circulation, causing vascular dysfunction and hypertension. Pharmacological blockade of the vasoconstrictive endothelin type A receptor (ETA) abolishes RUPP‐induced hypertension. Although blockade of vasodilatory ETB receptors increases blood pressure during late pregnancy in rodents and placental ischemia has been shown to reduce ETB receptor expression, the importance of the ET/ETB receptor pathway in blood pressure regulation during pregnancy or in response to placental ischemia is unclear. Therefore, we tested the hypothesis that ETB deficiency results in exaggerated placental ischemia‐induced hypertension. At eighteen weeks old, ETB deficient (def) and transgenic control (Tg) timed‐pregnant rats were generated using Wistar Hannover males. Rats underwent RUPP or Sham surgeries at gestational day 14, with assessment of mean arterial blood pressure (MAP, carotid catheter) and pregnancy weights and plasma collection at day 19. This resulted in 4 groups: Sham Tg (N=13); RUPP TG (N=10); Sham ETB def (N=11); and RUPP ETB def (N=6). MAP was greater in Sham ETB def compared to Sham Tg (109±3 vs. 79±3 mmHg, P<0.05). MAP levels were increased by RUPP in both Tg (99±3 mmHg, P<0.05) and ETB def (139±6 mmHg, P<0.05), but the degree of this RUPP hypertension was exaggerated in ETB def rats (30 vs. 20 mmHg). Average fetal weights were lower in Sham ETB def than Sham Tg (1.79±0.11 vs. 2.24±0.06 g, P<0.05) and were reduced by RUPP in Tg (2.08±0.06 g, P<0.05) but not further reduced in RUPP ETB def (1.66±0.13 g). Average placental weights were similar in Sham ETB def and Sham Tg (0.52±0.01 vs. 0.50±0.01 g). RUPP reduced placental weights in Tg (0.43±0.03 g, P<0.05) but not RUPP ETB def (0.55±0.04 g). Placental sufficiency (fetal weight divided by placental weight, a marker of placental nurturing capacity) was lower in Sham ETB def compared to Sham Tg (4.05±0.15 vs. 4.58±0.20, P<0.05). This was not reduced by RUPP in Tg (4.41±0.36) but was further reduced in RUPP ETB def (3.11±0.38, P<0.05). Circulating levels of cGMP, a surrogate measure of bioavailability of the vasodilatory nitric oxide (NO), were reduced in Sham ETB def compared to Sham Tg (43±5 vs. 178±18 pg/mL, P<0.05), which were reduced by RUPP in Tg (18±3 pg/mL), but not further reduced in RUPP ETB def (35±11 pg/mL). In conclusion, these data not only suggest an important role for ETB in blood pressure regulation during normal pregnancy but also in buffering the response to placental ischemia‐induced hypertension.

Support or Funding Information

K99HL130577, HL51971, P20GM104357
Inhibition of Angiotensin II Type 1 Receptor Agonistic Autoantibody Binding Improves Maternal Outcomes in Rats with Placental Ischemia
Authors: Cunningham Mark Wayne , Castillo Javier , Vaka Venkata Ramana , Amaral Lorena , Cornelius Denise , Spradley Frank , Ibrahim Tarek , Campbell Nathan , Usry Nathan , Williams Jan , LaMarca Babbette
Published: November 2023

Women with preeclampsia (PE) have hypertension, placental ischemia, reduced glomerular filtration rate (GFR), nitric oxide bioavailability, elevated sFlt, preproendothelin (PPE), agonistic Angiotensin II type 1 receptor autoantibodies (AT1‐AA), oxidative stress, and activation of cytolytic natural killer (NK) cells during pregnancy. The reduce uterine perfusion pressure (RUPP) rat model of placental ischemia displays these same characteristics, indicating that it is a good model to examine factors contributing to the pathophysiology of PE. We hypothesis that administration of a specific 7 amino acid peptide sequence (7AA) to inhibit the AT1‐AA from binding to the AT1receptor, will improve maternal blood pressure and other PE features in RUPP rats.

Methods

Day 14 of gestation RUPP rats receive the AT1‐AA inhibitory peptide, 7AA, (2ug/μl saline) via mini‐osmotic pump. On day 19 of gestation, conscious blood pressure (MAP) and GFR were determined. The blood, kidneys, and placentas were collected for molecular analysis and flow cytometry quantification of NK cells.

Results

MAP was elevated in RUPP rats (n=28) vs. normal pregnant (NP) rats (n=10) (121±1 mmHg vs 98±3 mmHg, p<0.05) and this hypertension was attenuated with 7AA (n= 15; 104±3 mmHg, p<0.05). Circulating sFlt1 (176±37 vs. 77±15pg/ml, p<0.05) was elevated in RUPP rats vs. NP rats and was normalized in RUPP + 7AA rats (86±9 pg/ml, p<0.05). There was a 4 fold increase in PPE in RUPP (n=7) vs. NP (n=6) rats which was reduced 2 fold in RUPP + 7AA (n=7) (p<0.05). There was no difference in GFR or plasma creatinine among the groups. Activated cytolytic placental NK cells was increased in RUPP vs. NP (8.29±1.73% vs. 2.05±1.56%, p<0.05), and was normal in RUPP +7AA (2.78±0.95%, p<0.05). Nitrate and nitrite (NOx), was decreased in RUPP vs. NP (14.0± 1.5 vs. 20.0± 1.0 μM NO3, p<0.05) and was improved to 18.3± 1.6 μM NO3 in RUPP + 7AA (ns). Circulating isoprostanes were elevated for RUPP vs. NP (20.1 ± 6.3 vs. 2.8 ± 1.4 ng/mL, p< 0.05) and was normalized with RUPP + 7AA peptide treated rats (4.3 ± 2.2 ng/mL).

Conclusion

Inhibition of AT1‐AA from binding to the AT1R improves many PE characteristics observed in RUPP rats, thus indicating that this may be a potential therapy to further explore for PE patients.

Support or Funding Information

Research Supported by T32HL105324 and RO1HD067541
Obesity‐induced hypertension is associated with increases in adipose tissue‐derived angiotensinogen in female mice exposed to early life stress (ELS).
Authors: Herald Joseph , Murphy Margaret , Cohn Dianne , Loria Analia
Published: November 2023

ELS is an established independent risk factor for cardiovascular disease. We use Maternal separation and early weaning (MSEW) during postnatal life as a mouse model of ELS. Previously, we have shown that MSEW exacerbates the development of obesity‐induced hypertension (HT) in female mice. Given the well‐known role of renin‐angiotensin system (RAS) during the development of obesity‐induced HT, the goal of this study was to determine the RAS status in female MSEW mice. MSEW was performed in C57BL/6 mice from postnatal days 2–16. Undisturbed litters served as control group (C). Mice were placed on a low‐fat diet (LFD, 10% kcal from fat) or HFD (60% kcal from fat) for 16 weeks upon weaning. Plasma renin concentration (PRC) was reduced in female MSEW compared with C (p<0.05), suggesting increased circulating AngII levels. Angiotensinogen (AGT) protein level, the only known precursor for angiotensin peptides, showed similar levels between groups in plasma (6.2±0.3 vs. 5.5±0.1 ug/ml, p<0.05) and liver (1.6±0.2 vs. 1.7±0.3 ng/ml/g tissue); however, adipose tissue and media explants displayed increased AGT secretion in female MSEW mice compared with C (29±9 vs. 98±8 and 15±1 vs. 22±2 ng/ml/g tissue, respectively, p<0.05). AngII receptor subtypes 1 and 2 (AT1 and AT2) were not different between groups in liver, adipose tissue, brain and kidney. In additional studies, we found increased adipose tissue‐derived AGT in media explants from female MSEW after one week on HFD (27±2 vs. 46±3 ng/ml/g tissue, p<0.05). Finally, AGT mRNA expression in visceral fat was increased in MSEW pups at postnatal day 7 (p<0.05). Taken together, these data indicate that adipose tissue‐derived AGT is particularly susceptible to MSEW and may serve as a major source of circulating angiotensin II peptide in MSEW mice thereby contributing to obesity‐induced HT.

Support or Funding Information

HL111354
HNF1B as a New Transcriptional Activator of the Renal KIR4.1/KIR5.1 Potassium Channel
Authors: Kompatscher Andreas , de Baaij Jeroen H.F. , Aboudehen Karam , Hoefnagels Anke P.W.M. , Igarashi Peter , Bindels Rene J.M. , Veenstra Gertjan J.C. , Hoenderop Joost G.J.
Published: November 2023

Background

Hepatocyte nuclear factor 1 homeobox B (HNF1B) is an essential transcription factor for the development and functioning of the kidney. Mutations in HNF1b cause autosomal dominant tubulointerstitial kidney disease (ADTKD‐HNF1B), which is characterized by renal cysts and maturity‐onset diabetes of the young (MODY). Moreover, patients suffer from a severe electrolyte phenotype consisting of hypomagnesemia and hypokalemia. Until now, genes that are regulated by HNF1B are only partially known and do not fully explain the phenotype of the patients.

Methods

Therefore, a chromatin immunoprecipitation and sequencing (chIP‐seq) was executed in immortalized mouse kidney cells (mpkDCT) to identify HNF1B binding sites at a genome‐wide scale. Luciferase‐promoter assays, siRNA‐mediated knockdown of Hnf1b in DCT cells and RT‐qPCR on HNF1B mutant mouse kidneys were performed to assess the transcriptional regulation of Hnf1b on candidate genes in vitro and in vivo.

Results

In total 7,421 HNF1B binding sites were identified, including several genes involved in electrolyte transport and diabetes. A highly specific and conserved HNF1B site was identified in the promoter of Kcnj16, encoding the potassium channel Kir5.1. Luciferase‐promoter assays showed a 2.2 fold increase in Kcnj16 expression when HNF1B was present. Expression of the Hnf1b p.Lys156Glu mutant that was previously identified in a ADTKD‐HNF1B patient, did not activate Kcnj16 expression. Knockdown of Hnf1b in mpkDCT cells significantly reduced the expression of Kcnj16 (Kir5.1) and Kcnj10 (Kir4.1) by 38% and 37%, respectively. These results were confirmed in a HNF1B renal knockout mouse, exhibiting downregulation of Kcnj16, Kcnj10 and Slc12a3 transcripts in the kidney by 78%, 83% and 76%, respectively, compared to HNF1B wild‐type mice.

Conclusion

HNF1B has been identified as a transcriptional activator of Kcnj16. Consequently patients with HNF1B mutations may have a reduced Kir5.1 activity in the kidney, resulting in hypokalemia and hypomagnesemia.
Attenuated cardiovascular‐renal sensitivity of blood pressure and heart rate to infused insulin in obese TallyHo/Jng mice
Authors: Alunan Ashley , Li Lijun , Lee Hwal , Fluitt Maurice , Tiwari Swasti , Ecelbarger Carolyn M.
Published: November 2023

Obese mice are known to be relatively insulin resistant with regard to cellular glucose uptake; however, it is still uncertain whether the cardiovascular‐renal system develops insulin resistance and what this means in terms of blood pressure (BP) and heart rate (HR). To address this problem, we tested the impact of pre‐existing metabolic syndrome (MetS) on BP, HR (radiotelemetry), and blood chemistry responses to an insulin infusion using a newer inbred polygenic model of MetS. Male TallyHo/Jng (TH) mice were infused with 50 U/kg bw insulin by osmotic minipump for 2‐weeks. C57Bl6 (B6) mice were used as insulin‐sensitive controls (n = 6/strain). All mice were provided with 20% glucose, 0.5% NaCl, 0.5% K2HPO4 water to drink to combat hypoglycemia during the infusion. Mean arterial BP of TH mice was significantly (p < 0.05) higher than B6, e.g., 126±2 versus 112±5 mm Hg prior to infusion. Infusion led to an initial fall in BP in B6 (Day 3‐ 108±2) followed by a slow rise (Day 13‐ 122±8). In contrast BP remained fairly steady in TH (Day 3‐ 123±1; Day 13‐ 125±5). Similarly HR was elevated in TH in the basal period: 649±8 versus 527±20 beats per min, (bpm) in B6, and was reduced by insulin infusion to 614±21 (Day 13). In B6, HR rose to 563±30 (Day 13). Insulin infusion caused a number of changes in blood chemistry including, a reduction in pH and HCO3 levels. Most of these changes were independent of strain; however, insulin infusion caused a reduction in blood glucose levels only in the B6 mice. Overall, we found greater sensitivity of BP and HR to insulin in B6 versus TH. The rise in HR with insulin is likely the result of a rise in sympathetic activity. Insulin infusion produced somewhat “beneficial” changes in TH with a slight fall in HR and BP. In contrast, in B6, after the first few days, BP and HR rose, suggesting primarily detrimental effects likely as mice developed a resistance to the infusion. These findings demonstrate that insulin resistance extends to non‐traditional target tissues in MetS; and that insulin resistance, not insulin action per se, is correlated to rises in both BP and HR during MetS.

Support or Funding Information

Georgetown University internal funding and Georgetown/Howard University CTSA
Thick Ascending Limb‐derived TNF production regulates the blood pressure response to high NaCl intake
Authors: Ferreri Nicholas R , Hao Shoujin
Published: November 2023

We previously showed that the thick ascending limb (TAL) produces tumor necrosis factor‐alpha (TNF) in response to diverse stimuli and acts as an endogenous inhibitor of NKCC2 expression and activity. In this study we determined the effect of TNF derived from the TAL on the renal adaptation to high NaCl (HS) intake. TNF levels were higher in urine (40±4 vs 18±5 pg/24 h) of mice that ingested 1% NaCl in the drinking water for three days compared with tap water. The increase in urinary TNF levels was associated with a 4‐four fold increase (p<0.05) in TNF mRNA abundance in mTAL tubules isolated from mice given HS. In contrast, plasma levels of TNF remained unchanged in these mice (HS: 20±3 vs tap water: 18±3pg/ml). The increase in renal TNF mRNA levels induced by HS intake decreased by more than 80% (p<0.05) in mice given an intrarenal injection of a lentivirus construct that silences TNF in the kidney. Neither injection of a control lentivirus (U6) nor EGFP‐TNF‐ex4 altered baseline blood pressure in mice ingesting a normal salt diet and tap water. However, renal‐specific silencing of TNF rapidly increased blood pressure in mice drinking 1% NaCl (U6: 110±4 vs shTNF: 136±6mmHg; p<0.05), an effect that was sustained for 11 days. Baseline blood pressure was rapidly restored when mice were switched to tap water (days 12–16), and rapidly increased when they were switched back to HS (days 17–21). Renal outer medullary NKCC2A mRNA accumulation increased approximately 3‐fold (p<0.05) after injection of EGFP‐TNF‐ex4 followed by ingestion of HS for 3 days compared with U6 control groups. The Cre/loxP recombination system was then used to generate TAL‐specific TNF knockout mice (TAL‐TNF KO). Specific recombination in the TAL was validated by PCR of microdissected nephron segments. The recombined allele product (0.45kb) was present in TAL tubules but not PT, IMCD, or spleen. Baseline blood pressure was similar in TNF‐TAL KO mice and littermate controls but increased when TNF‐TAL KO mice were given HS for three days. Food, water intake, and body weight were not different between groups in either model of TNF deletion and the increases in blood pressure were not associated with damage to glomeruli, blood vessels, or tubules. The increase in renal TNF levels in response to HS intake without a concomitant increase in blood pressure or an inflammatory component, and the increase in blood pressure in response to HS when TNF is silenced in the TAL suggests deficiency of TAL‐derived TNF is sufficient to render normotensive healthy mice salt‐sensitive.
Forskolin (cAMP) Promotes Insertion of Large Conductance Potassium (KCa1.1) Channels into Apical Membranes of Rat Distal Colon
Authors: Nickerson Andrew , Rottgen Trey , Minor Emily , Rajendran Vazhaikkurichi M
Published: November 2023

Mammalian colon contributes significantly to overall K+ homeostasis. Under normal conditions, the proximal colon secretes and distal colon absorbs K+. Distal colon also has a secretory capacity, which becomes important to K+ homeostasis under pathological conditions, such as end‐stage renal failure. K+ secretion in the distal colon is primarily mediated through apical large conductance (BK) channels. We hypothesized that the BK opener BMS‐204352 (BMS) and the adenylate cyclase activator forskolin (FSK) induce K+ secretion through separate mechanisms. K+ flux studies were performed in distal colonic mucosal tissues obtained from male Sprague‐Dawley rats (200–225g). Unidirectional 86Rb fluxes (K+ surrogate), and short‐circuit current (ISC) were measured under voltage‐clamped conditions in an Ussing chamber setup. Fluxes were performed in the presence of amiloride (10 mM) to inhibit ENaC‐mediated ISC, as well as VO4 (1mM) to inhibit apical H+/K+ ATPase and unmask BK‐mediated K+ secretion. Results indicate that BMS (10mM) and FSK (10mM) induced a net K+ secretion of 0.083 ± 0.04 and 0.274 ± 0.05 mEq/cm2·hr, respectively (n=4, p<0.05). FSK, but not BMS, increased ISC compared to baseline (220.5 ± 30.5 vs 132.2 ± 18.7 uA/cm2). Immunofluorescence studies performed on the same tissues used in flux experiments showed markedly increased apical BK staining in tissues exposed to FSK, but not in those exposed to only BMS. Taken together, these data suggest that while BMS may only open BK channels already present on apical membranes, FSK may cause further K+ secretion by triggering the insertion of more BK channels into the apical membrane.

Support or Funding Information

• R01 DK104791/DK/NIDDK NIH HHS/United States
Proximal Tubule Glutamine Synthetase is Critical for the Response to Low Protein Diet
Authors: Weiner I. David , Lee Hyun‐Wook , Osis Gunars , Handlogten Mary E. , Chen Chao , Verlander Jill W.
Published: November 2023

The mechanisms through which the kidneys increase ammonia generation in response to increased endogenous acid loads have been extensively studied; the mechanisms underlying decreased ammonia metabolism are less well understood. Glutamine synthetase is expressed in the kidney, and mediates reaction of NH4+ with glutamate, generating glutamine and decreasing net ammonia generation. Our previous studies show that dietary protein restriction, which decreases endogenous acid production, increases proximal tubule glutamine synthetase expression. The purpose of the current studies was to determine the functional importance of glutamine synthetase in the response to dietary protein restriction. We used mice with proximal tubule‐specific glutamine synthetase deletion (PT‐GS‐KO), using floxed GS alleles and Cre‐recombinase under control of the PEPCK promoter. Control (C) mice was floxed, Cre‐negative littermates. Mice were studied in metabolic cages, and daily 24‐hour urine samples collected. Changing from normal, 20%, protein to low, 6%, protein diets for one week resulted in a rapid decrease in urinary ammonia excretion that was maximal at day 2 in C mice. In PT‐GS‐KO mice, the ability to decrease ammonia excretion was impaired significantly on day 2–7 as compared to C mice. This impaired ability to alter urinary ammonia excretion occurred despite no significant difference in urine pH on any day between C and PT‐GS‐KO mice. The difference in ammonia excretion was unique to this component of net acid excretion; titratable acid excretion did not differ significantly between C and PT‐GS‐KO mice. Other components of renal ammonia metabolism, specifically PDG, PEPCK, NHE3, NKCC2, Rhbg and Rhcg, were not detectably different between C and PT‐GS‐KO mice. We conclude that glutamine synthetase, whose expression increases in response to dietary protein restriction, is necessary for adaptive changes in renal ammonia excretion, thereby enabling maintenance of acid‐base homeostasis and nitrogen metabolism.

Support or Funding Information

The studies were supported by funds from the NIH (R01‐DK045788 and R01‐DK107798) and from the Department of Veterans Affairs (1I01BX000818).
Identification of a Stress‐Inhibitory Pathway from the Nucleus of the Solitary Tract (NTS) to the Bed Nucleus of the Stria Terminalis (BNST)
Authors: Scheuer Deborah , Hiller Helmut , McCowan Michael , Wang Lei , Krause Eric G
Published: November 2023

We and others have previously shown that the NTS influences physiological responses to both acute and chronic stress. The NTS sends a robust neuronal projection to the BNST consisting of both catecholaminergic and 11βhydroxysteroid dehydrogenase‐expressing neurons. The BNST contributes importantly to regulation of the stress response, however, the functional significance of the NTS to BNST pathway is not known. We conducted the present experiments to test the hypothesis that activating this pathway would influence stress‐associated physiological endpoints. The Cre‐inducible channelrhodopsin construct, rAAV2/EF1a‐DIO‐hChR2(H134R), was microinjected into the NTS (500 nl bilaterally and at the midline) and the retrograde Cre recombinase expressing virus, CAV2‐Cre, was injected into the BNST (1000 nl bilaterally) in six adult male Wistar‐Kyoto rats. After recovery, rats were implanted with chronic indwelling fiber optics targeting the BNST. At least 12 weeks later, tests were performed in conscious rats to measure the effect of stimulation of NTS neuron terminals in the BNST. Optogenetic activation of the NTS to BNST pathway reduced baseline plasma corticosterone concentration from 5.4±0.9 to 2.1±0.8 μg/dl (P<0.05, within subjects design) and increased the percent of time the rats spent in the open arms of the elevated plus maze from 20±7 to 87±6 % (P<0.05, between subjects design). Rats also underwent tests for sucrose and saline preference. Activation of the NTS to BNST pathway reduced 1.8% saline intake over a 30 min period from 1.40±0.32 to 0.55±0.16 ml (P<0.05, within subjects design). Sucrose (1% solution) intake and water intake were not affected by activation of the pathway. Sucrose intake was 2.01±0.64 ml without stimulation and 1.59±0.24 ml with stimulation. Average water intake was 0.94±0.37 ml without stimulation and 0.89±0.29 ml with stimulation. We conclude that in unstressed normal male rats activation of the neuronal pathway from the NTS to the BNST inhibits the hypothalamic‐pituitary‐adrenal axis, anxiety‐like behavior and saline intake. The pathway may act to restrain the stress response under these conditions. Future studies will determine the function of this pathway under conditions of chronic stress, its role in the control of cardiovascular function, and the role of mineralocorticoid receptors in both catecholaminergic and 11βhydroxysteroid dehydrogenase‐expressing NTS neurons.

Support or Funding Information

R01 HL076807, R01 HL‐122494
Differential Role of Nav Isoforms in the Induction vs Conduction of Action Potentials in Vagal Afferent Nerves
Authors: Sun Hui , Meeker Sonya , Herbstsomer Robert A. , Kollarik Marian , Undem Bradley J.
Published: November 2023

Based on compound action potential studies, it has long been known that tetrodotoxin (TTX) blocks action potential (AP) conduction in the vagus nerve. We recently reported that airways specific nodose and jugular neurons express mainly NaV 1.7, 1.8 and 1.9 channels; and selectively silencing NaV1.7 mRNA expression with an in vivo shRNA strategy mimics the blocking effect of TTX on AP conduction in vagal sensory nerves (J. Physiol, 2011). Here we evaluate the role of TTX‐sensitive Nav 1.7 and TTX‐resistant Nav 1.8 channels in the induction and conduction of APs in airway‐specific vagal afferents derived from nodose and jagular gaglia. Extracellular recordings from nodose or jugular ganglia were used to evaluate the activities of single afferent fibers arising from the isolated, innervated trachea and intrapulmonary airways. The trachea is innervated by jugular C‐fibers and nodose Aδ fibers. TTX 1 μM perfused selectively through the tracheal lumen abolished citric acid‐induced AP discharge in nodose Ad fibers (AP number: 21 ± 6 vs. 0 ± 0, n=7 each group), but did not inhibit citric acid induced AP discharge from jugular C‐fibers (50 ± 8 vs. 50 ± 9, n = 15 each group; the responses in jugular C‐fibers were blocked with lidocaine, n=4). When applied to a segment of the desheathed vagus, however, TTX abolished the conduction of APs induced by vagal electrical stimulation in all nodose Ad fibers and 70% of jugular C‐fibers (n=9). The intrapulmonary airways are innervated by both nodose and jugular C‐fibers. Again, TTX applied via intrapulmonary artery and trachea virtually abolished bradykinin‐induced AP discharge in nodose C‐fibers (AP number: 494 ± 80 vs. 2 ± 2, n=6 each group), but did not affect bradykinin‐induced responses in the jugular C‐fibers (285 ± 95 vs. 318 ± 122, n=6 each group). The NaV1.8 blocker, PF‐01247324, at 30 μM, a concentration we found inhibit the guinea pig NaV1.8 current by about 75%, inhibited the citric acid responses in the jugular C‐fibers inervating the trachea (50 ± 13 vs. 12 ± 6, n=6 each group, P<0.05). Consistent with these findings, patch clamp studies revealed that the Nav1.8‐mediated TTX resistant INa is larger in jugular than nodose neurons (Gmax=6.81±0.81 nS, n=12 vs. 2.38±0.32nS, n=13, p<0.001) while the amplitudes of TTX‐sensitive INa are similar. The voltage dependence of activation and inactivation of Nav1.8 currents was not different between nodose and jugular neurons. Furthermore, TTX increased the amount of current required to evoke an AP to a greater extent in nodose compared to jugular neurons (10‐ vs. 4‐fold increase). In summary, in nodose nociceptors conduction of APs along axons and induction at the nerve endings within the tissue depends on NaV 1.7; whereas in jugular nociceptors, conduction is largely NaV 1.7 dependent, but TTX‐resistant Nav1.8 channels are sufficient to induce APs in the terminals.

Support or Funding Information

NIH (5R01HL22228‐04)
Glutamatergic Neurons in the Dorsal Hypothalamic Area Dissociate Thermogenesis from Heat Conserving Pathways During Stress Responses
Authors: Machado Natalia Lima , Abbott Stephen , Fuller Patrick , Fontes Marco , Saper Clifford
Published: November 2023

Stress elicits a variety of autonomic responses, including thermal changes in humans and animals. In this present study we investigate the circuit basis for stress‐induced hyperthermia with focus on the hypothalamic‐medulary pathway in adaptive thermoregulatory responses.

We injected retrograde tracer (CTb) in the raphe pallidus (RPa) of Vgat and Vglut2‐IRES‐cre L10‐GFP reporter mice to identify the phenotype of neurons in the dorsal hypothalamic area (DHA) projecting to RPa.

In a separate series of experiment, we bilaterally injected AAV‐Flex‐hGlyR‐mCherry in the DHA of Vglut2‐IRES‐cre mice to promote inhibition of DHAVglut2 neurons. hGlyR is a mutated glycine receptor in which ivermectin (hGlyR ligand) gates a chloride channel. Using AAV8‐hSyn‐DIO‐hM3D(Gq)‐mCherry (DREADD) we induced activation of DHAVglut2 neurons and measured changes in brown adipose tissue (BAT) and tail temperature. An optogenetic approach in freely‐behaving mice was used to promote inhibition of the synaptic terminal of the DHAVglut2 neurons in the RPa.

We identified that glutamatergic neurons in the DHA (DHAVglut2) sending direct inputs to RPa are activated during cage switch stress (singly housed male was switched to a cage previously occupied by another male) causing 39 + 6% of Vglut2/CTb neurons to express cFos, whereas only 13 + 2% of Vglut2/CTb neurons expressed cFos in the control animals (mean + SEM, n=4,3 respectively, *p=0.016).

The long‐lasting inhibition induced by hGlyR‐IVM reduced cFOS expression in our stress protocol (after IVM 6 + 1% cFOS+/hGlyR Vglut2 labeled neurons vs vehicle 40 + 8% cFOS+/hGlyR Vglut2 cFos doubly labeled neurons in the DHA; n=3 each group, *p=0.013) and also decreased stress‐induced hyperthermia (2.8 + 0.1°C after vehicle vs. 2.0 +0.1°C after IVM, n=11, FTc(interaction) (164,1278)=1.556).

During the stress protocol we found a large increase in BAT thermogenesis (rise of 1.2 + 0.2°C), and tail artery vasoconstriction (fall in tail temperature by −2.3 + 0.4°C). Surprisingly, activation of DHAVglut2 neurons using hM3D excitatory receptor caused profound BAT thermogenesis (1.0 + 0.14°C), but it did not cause vasoconstriction, instead resulting in tail artery vasodilation (2.5 + 0.6°C) as a delayed reflex response to central hyperthermia.

Finally, optogenetic experiments identified that DHAVglut2—>RPa pathway is a mediator of stress‐induced hyperthermia, as photoinhibition of terminals in the RPa reduced the peak of stress‐induced hyperthermia by 0.4+0.06°C, (comparing same group before vs. during photoinhibitoin). We propose that the DHAVglut2—‐>RPa is a thermogenic pathway regulating BAT thermogenesis but not thermoregulatory vasoconstriction during stress.

Support or Funding Information

Grant support: NIH Grant NS085477 (C.B.S.), NS073613 (P.M.F.), CAPES and CNPq
Post‐traumatic stress sensitizes the angiotensin II‐elicited hypertensive response
Authors: Xue Baojian , Yu Yang , Wei Shun‐Guang , Beltz Terry G , Guo Fang , Felder Robert B , Johnson Alan Kim
Published: November 2023

Post‐traumatic stress disorder (PTSD) is associated with an activated renin‐angiotensin system (RAS) and elevated levels of proinflammatory cytokines (PICs) in the circulation and in the central nervous system (CNS). Using an Induction‐Delay‐Expression experimental paradigm, our previous studies demonstrated that activation of central RAS and inflammation by various challenges sensitizes angiotensin (ANG) II‐elicited hypertension. The present study investigated whether the ANG II‐elicited hypertensive response is also sensitized in a model of PTSD. The resident‐intruder paradigm was used to model PTSD. Each intruder rat (male Sprague‐Dawley) was exposed to a different resident (male Long‐Evans) for 2 hours on three days with each session separated by 1 day. Beginning on day 3 after the last exposure, the intruder (PTSD) rats and unstressed control rats received a subcutaneous infusion of ANG II (120 ng/kg/min) for 2 weeks. The PTSD rats had a significantly enhanced hypertensive response to the ANG II infusion when compared to control rats (Δ35.9±2.5 mmHg vs. Δ17.9±3.9 mmHg). In a separate group of rats, the brains of control and PTSD rats were collected on day 3 after the last exposure to determine mRNA expression of RAS and PIC components in the lamina terminalis (LT) and the hypothalamic paraventricular nucleus (PVN). The results showed that PTSD had upregulated expression of angiotensin type 1 receptor, tumor necrosis factor‐α, interleukin (IL)‐1β and IL‐6 in the LT, but not in the PVN. These results support the hypothesis that conditions upregulating the central RAS and PICs sensitize ANG II‐induced hypertension, and suggest that disorders such as PTSD may predispose individuals to exacerbation of renin‐angiotensin‐elicited hypertension.

Support or Funding Information

This work was supported by the NIH grants HL‐14388 (AKJ), HL‐98207 (AKJ), HL‐096671 (RBF) HL‐073986 (RBF), with partial support from the Department of Veterans Affairs (RBF).
Characteristic changeable shape of the otolith organs in Meniere's disease
Authors: Tanioka Hisaya
Published: November 2023

INTRODUCTION

Theprimary function of the otolith organs is the determination of bodily positionand movements. Meniere's disease is characterized by recurrent spontaneousattacks of vertigo, fluctuating hearing loss, tinnitus, and aural fullness. Theotolith organs are correlated with vertigo.

OBJECTIVE

To investigatethe shape of the otolith organs in Meniere's disease according with clinicalcourse and non‐affected ear. No one knows the shape of the otolith organs inthe living subject suffered from Meniere's disease.

METHOD

3Dvirtual histology of the otolith organs by a volume rendering was reconstructedby a conventional temporal HR‐CT. The analysis of the otolith organs was undergone by the bone algorithm of a volume rendering.

RESULTS

In Meniere's disease, the affected utricular otolith was thin, and the saccular otolith was large. In the clinical course of Meniere's disease, during the attack period, the utricular otolith was thinner, and the saccular otolith was larger than both pre and post status like the figure.

DISCUSSION

I provide 3D virtual image of the otolith organs in the inner ear by which the internal state of living subjects can be observed in detail using misinformation associated with calcium carbonate. During suffering from Meniere's disease, the utricular otolith was decreased in volume. Otherwise, the saccular otolith was increased in volume. After effective treatments, the utricular otolith was increased and the saccular otolith was decreased in volume. These quantitative methods for assessment of vestibular otopathology have defined the changeable otolith in the utricle and saccule.

Support or Funding Information

None
Role of Dlx5 in the development of the palatal‐pharyngeal region in higher vertebrates
Authors: Chai Yang , Sugii Hideki , Grimaldi Alexandre , Ho Thach‐Vu , Parada Carolina
Published: November 2023

Distal‐less (Dll) genes belong to an evolutionarily conserved family of homeobox transcriptional regulators, which are important for the early embryonic development of bone in the limbs and craniofacial region. Mutations in human Dll orthologs, Dlx5/6, cause split‐hand/split‐foot type 1 malformation (SHFM1) associated with sensorineural deafness, cleft palate, developmental delay, and micrognathia. Dlx5 mutant mice have craniofacial abnormalities including cleft soft palate and micrognathia. However, the role of Dlx5 in the palatal‐pharyngeal development, which is important for speech, remains unclear. This study examines the role of Dlx5 in the formation of the soft palate, the Eustachian tube (ET), and the pharyngeal region. Gene expression analysis demonstrates that Dlx5 is expressed in muscles and osteogenic progenitor cells. Homozygous Dlx5 mutant mice lack soft palatal muscles including the levator veli palatini (LVP) and palatopharyngeus (PLP). The posterior regions of the hard palate and pterygoid plate are smaller in Dlx5−/− mice than in controls. Morphometric analyses of the premaxilla, maxilla, palatine bone, and mandible show that these bones are smaller in height, width, and length in Dlx5−/− mice compared to controls. Taken together, our data reveal that Dlx5−/− mice recapitulate craniofacial phenotypes similar to those of SHFM patients. Further studies of Dlx5 expression in the proximal region of the mandible, posterior palate, and pharyngeal region may reveal the mechanism responsible for the pharyngeal morphology. These studies hold potential for improving our understanding of the evolution of speech in the human lineage.

Support or Funding Information

Supported by the NIDCR, NIH R37 DE012711
Functional specializations of hypoglossal motoneurons innervating intrinsic and extrinsic tongue muscles
Authors: Wealing Jesse Cole , Cholanian Marina , Fregosi Ralph
Published: November 2023

The mammalian tongue contains eight muscles that collaborate to ensure that breathing, swallowing, suckling and other important behaviors are robust and reliable. Seven of these muscles are innervated by hypoglossal motoneurons (XIIMNs). Most studies of XIIMNs have considered them to be a homogeneous population, however a given tongue movement requires precisely allocated contributions from all tongue muscles. Thus, XIIMNs may have unique functional properties that reflect the usage pattern of the muscle that they innervate. To better understand the functional specializations of muscle‐specific XIIMNs, intrinsic (superior longitudinalis) and extrinsic (genioglossus) muscles were injected with dextran‐rhodamine to illuminate the XIIMNs innervating those muscles. The labeled XIIMNs were anatomically and electrophysiologically differentiable. Intrinsic XIIMNs were located more medial and dorsal, and extrinsic muscles more lateral and ventral. Whole‐cell patch‐clamp experiments showed that resting membrane potential was on average 10 millivolts more depolarized in intrinsic compared to extrinsic XIIMNs, and that the firing rate response to current injection was higher in intrinsic compared to extrinsic XIIMNs. These observations show muscle‐specific functional specializations in XIIMNs, and this information lays the foundation for more detailed experiments, and the development of “neuron‐specific” therapeutic approaches for tongue‐related respiratory disorders.

Support or Funding Information

NIH (R01 HD071302).
Aging‐Related Deficits in Neuromuscular Transmission and Force Loss in the Diaphragm Muscle
Authors: Mantilla Carlos B. , Gransee Heather M. , Greising Sarah M. , Sieck Gary C.
Published: November 2023

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Aging‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in mice at 6, 18, and 24 months of age. At each age, DIAm‐phrenic nerve preparations were used to determine maximal force by ex vivo contractility of the DIAm as well as neuromuscular transmission failure by repetitive nerve and muscle stimulation. By 18 months, animals had reduced Pdi during maximal forces (e.g., during tracheal occlusion) and increased DIAm neuromuscular transmission failure compared to 6 months, but no change in DIAm maximum specific force. By 24 months, Pdi remained reduced during occlusion, DIAm neuromuscular transmission failure remained increased compared to 6 months, and DIAm maximum specific force was decreased compared to both 6 and 18 months. Using this global measure of neuromuscular transmission, it is evident that impaired transmission precedes muscle weakness.

Support or Funding Information

Supported by R01‐AG044615.
Functional evaluation of prorrenin/renin receptor in immune cells from rats with pregnancy‐induced hypertension
Authors: Galicia‐Galicia Erick , Anguiano‐Robledo Liliana , Lopez‐Sanchez Pedro , Hernandez‐Campos Maria Elena
Published: November 2023

Pregnancy‐induced hypertension (PIH), known before as preeclampsia, is a multisystemic disorder characterized by hypertension, proteinuria and low intrauterine development. Its etiology remains unknown. Several systems seem to participate in PIH genesis. The renin‐angiotensin system (RAS) has been implicated in PIH development. RAS is essential for blood pressure control and hydro‐saline balance. The prorrenin/renin receptor (PRR) is a component of this system. Recent studies have shown that PRR activates PLZF, MAPK pathways (ERK1, ERK2), as well as the Wnt pathway. V‐ATPase has also been related with PRR activation, lowering pH within cells. It is not described yet, however, if PRR activates these mechanisms in leukocytes during pregnancy. In the present work, we evaluated if PRR activates MAPK (ERK 1‐2, TGF‐beta), WNT (β‐catenin, DVL‐1, PKC alfa) pathways and V‐ATPase in leukocytes from normal pregnant and pregnancy‐induced hypertensive rats. We induced PIH in female Wistar rats using sub‐renal aortic coarctation (CASR). We measured proteinuria, systolic and diastolic blood pressure, pups body weight, length, and number. The expression of PRR, β‐catenin, PLZF, ERK1‐2, pERK 1‐2, PCK alfa and TGF‐b was determined by immunoblot in leukocytes from spleen. Our results show a significant increase in PRR and PLZF expression in the PIH model compared to normal pregnancy groups. β‐catenin and, ERK 1‐2 showed a moderate increase. However, DVL‐1 and PKC alfa increased significantly, indicating that PRR seems to activate WNT preferentially over MAPK pathways. Intracellular pH was not modified significantly. Our findings indicate that PRR is functional in immune cells during healthy and complicated pregnancy

Support or Funding Information

SIP 20150467 and SIP20160667
LRP‐1 Targeted Neuroprotection in a Mouse Model of Diabetic Retinopathy
Authors: El‐Desoky Dalia Hany , Bhattacharjee Partha , Hossain Ahamed
Published: November 2023

Human apolipoprotein E (apoE) is known to exert neuroprotective function in central nervous system disorders through low‐density lipoprotein receptor‐ related protein‐1 (LRP‐1). Retina being the same embryological origin like the brain, we rationale that retinal neurodegeneration can be inhibited by peptidomimetic blocking of LRP‐1. Thus, we predict a short peptide (apoEdp) mimicking the LRP‐1 binding region of apoE may have a role in the protection of retinal neuronal cells in the diabetic mouse eye model. Vascular changes in the retina are traditionally considered as classic diagnostic feature of Diabetic Retinopathy (DR). However, color blindness, lack of contrast sensitivity and abnormal electroretinogram characteristic to retinal neurodegeneration are documented in diabetic patients before clinically evident blood vessel changes. In fact, diabetes‐induced retinal neurodegeneration precedes vascular damage. Critical barriers to the development of adequate therapeutic strategies for the treatment of DR are related to the lack of information about the mechanism of early retinal neurodegeneration and its progression to late vascular abnormalities. Our studies with diabetic mouse retina suggest a signaling pathway involving low‐density lipoprotein receptor‐ related protein‐1 (LRP‐1) protects neurons from apoptosis by regulating the pro‐survival pathways of PI3k/Akt and MAPK/Erk pro‐survival pathway. We found apoEdp has anti‐PP2A activity in the diabetic retina and is effective in the treatment of retinal neurodegeneration in vivo. Our study suggests that intravitreal administration of apoEdp and its possible therapeutic neuroprotection will generate a proof of principle for treating diabetic neuronal complications.

Support or Funding Information

Support is provided in part by grant number 2G12MD007595‐06 from the National Institute on Minority Health and Health Disparities (NIMHD), National Institutes of Health (NIH), Department of Health and Human Services (DHHS) and its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIMHD or NIH.
Effect of PPAR Partial Agonist, GQ‐16, on Viability of Breast Cancer Cells in Culture
Authors: Ferreira Anna Paula B , Coelho Michella Soares , Amato Angélica Amorim , de Assis Rocha Neves Francisco , Rodrigues Izabel Cristina , Royer Carine
Published: November 2023

Breast cancer (BC) is the most frequent cancer in women around the world. Tumor cells have several mechanisms of resistance to chemotherapy treatment, so the relevance of searching for new drugs for the treatment of this pathology. Studies have shown that agonists of peroxisome proliferator‐activated receptors g (PPARg) are a promising target in the development of drugs for treatment of various cancers, including BC. Drugs in this class include thiazolidinediones (TZDs), total PPARg agonists with serious side‐effects. Recently, it was proposed that adverse effects of totals PPARg agonists could be avoided using partial agonists. Previous studies showed that GQ‐16 presents partial agonist activity and specificity to PPARg, as well as similar performance to TZDs with decreased adipogenic effect.

Objective

To investigate the antitumor activity of partial PPARg partial agonist, GQ‐16, in human breast tumor cell line (MCF‐7).

Methods

MTT (3 bromide (4, 5dimetiltiazol‐2il)‐diphenyl tetrazolium‐ ‐2.5) assay was used in human BC tumor cell lines (MCF‐7) with different doses of rosiglitazone and GQ‐16 (10−8 – 10−4 M) for 24 and 48 hours and DMSO 0.01% as negative control.

Results

Rosiglitazone decreased cell viability (49%) after 48 h of treatment on concentration of 10−4 M. On the other hand, GQ‐16 was able to decrease cell viability after 48h of treatment on several concentrations (36.92% at 10−6 M; 39.08% at 10−5 M; 75.81% at 10−4 M).

Conclusion

Partial PPARg agonist, GQ‐16, proved to be more potent than rosiglitazone on reducing the viability of breast cancer cells line MCF‐7. This effect was shown to be dose and time‐dependent. Therefore, it is suggested that this drug can be studied as an antitumor agent. Our results indicate that GQ‐16 is a promising target for antitumor activity research

Support or Funding Information

CNPq/CAPES/FAPDF