bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effect of sodium depletion with or without concomitant dehydration on Fos immunoreactivity in the different subregions of the subfornical organ and organum vasculosum of the lamina terminalis of the rat
Authors: McKinley Michael J , Goga Melinda 
Published: November 2023

In rats, each of the two forebrain sensory circumventricular organs, the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) can be categorised anatomically and functionally into subregions. The main divisions of the SFO are its outer shell and its ventromedial core, while the OVLT has a lateral zone, dorsal cap and a central vascular plexus. The aim of these experiments was to determine the effect of Na depletion for 24 hours by furosemide (20 mg/kg s.c.) treatment and low Na diet on rats (4 per group) with or without water available to drink on Fos immunoreactivity (Fos‐IR) in the subregions of the SFO and OVLT. We also examined the effect after 2 hours on these responses of correcting the Na deficit by drinking either isotonic 0.15M or hypertonic 0.3M NaCl. After these treatments, rats were killed by Na pentobarbitone (100 mg/kg i.p.) and standard immunohistochemical methods used to identify Fos in cell nuclei of CVO neurons. Whereas control untreated rats on low Na diet for 24 h exhibited minimal Fos‐IR in any subregions of the SFO and OVLT, rats subjected to sodium depletion with water available to drink exhibited many Fos‐labelled cells in the SFO that were almost exclusively confined to its ventromedial core, while in the OVLT, Fos expression was observed predominantly in the lateral zone with little Fos‐IR in the dorsal cap. Fos‐IR was not observed in the supraoptic nucleus (SON), signifying that the rats were not dehydrated. Treatment with the angiotensin AT1 receptor antagonist valsartan (5 mg/kg s.c. × 2 doses) inhibited the Fos response in the ventromedial core of the SFO. If water was withheld from Na‐depleted rats, some Fos‐IR was now observed in the outer shell, although most Fos‐IR was still observed in the ventromedial core; in the OVLT, Fos‐IR was observed in both its dorsal cap and lateral zone and the SON expressed Fos‐IR. Correction of the Na‐deficit in water‐replete but Na‐depleted rats by drinking 0.15M NaCl reduced the intensity, but did not abolish the Fos‐IR in the ventromedial core of the SFO or the lateral zone of the OVLT by 2 h, whereas water‐replete, Na‐depleted rats that drank hypertonic 0.3M NaCl exhibited additional Fos‐IR in the outer shell region of the SFO and in the dorsal cap of the OVLT as well as the less intense Fos‐IR in the ventromedial core of SFO and lateral zone of OVLT. These data confirm that the ventromedial core of SFO and lateral zone of the OVLT are activated by loss of body Na, whereas neurons in the outer shell of the SFO and dorsal cap of OVLT are more attuned to loss of body water and hypertonicity.Support or Funding InformationSupported by G. Harold and Leila Y. Mathers Charitable Foundation
Role of TGFβ3 in periderm desquamation during palatal fusion
Authors: Nawshad Ali 
Published: November 2023

The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (−/−) TGFβ3, interferon regulatory factor 6 (IRF6) (−/−), and truncated p63 (ΔNp63) (−/−) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (−/−), ΔNp63 (−/−), and wild‐type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (−/−) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (−/−) palates because of premature fusion.Support or Funding InformationNIH NIDCR R01DE017986
Vimentin Directly Interacts with NLRP3 and Regulates Inflammation
Authors: Shumaker Dale , Ridge Karen 
Published: November 2023

Activation of the NLRP3 inflammasome and subsequent maturation of IL‐1β have been implicated in acute lung injury (ALI), which is characterized by inflammation, IL‐1β release, alveolar epithelial cell injury, hyperplasia of alveolar type 2 cells, accumulation of fibroblasts, deposition of collagen, and scar formation. We investigated the role of vimentin, a type III intermediate filament, in this process using three well characterized murine models of ALI which require NLRP3 for inflammasome activation. It was demonstrated that in these ALI models, the pathophysiologic changes are attenuated in Vim−/− mice. We hypothesized that vimentin directly interacts with NLRP3 for normal inflammasome activity. Therefore, we examined, by co‐immunoprecipitation, whether NLRP3 and vimentin interact in a human macrophage cell line, THP‐1, and mouse bone marrow derived macrophages (BMDM). Next, using bio‐layer interferometery we investigated whether NLRP3 and vimentin directly interact. Preliminary results indicated that full length vimentin directly interacts with NLRP3 with a KD around 240 nM. To investigate which region of vimentin interacts with NLRP3 we used peptide arrays. Our results indicate that the vimentin head domain has and a small region of the tail domain interact with NLRP3 while the central rod domain shows very little interaction. Since vimentin has a large number of amino acids which can be phosphorylated, the binding between phosphomimetic vimentin peptides and NLRP3 was also examined. Phosphomimetic peptides of the vimentin head domain inhibit binding to NLRP3 while increased binding was observed in the tail domain when T426D and S430D mutations were examined. To further demonstrate a functional interaction between vimentin and NLRP3, vimentin was deleted from BMDMs using crispr/Cas9 and vimentin domains were expressed in these Vim KO cells using lentiviral infection. These new cell lines were examined for IL‐1β release after activation with LPS and ATP. Additionally, localization of vimentin and vimentin domains near NLRP3 after activation was investigated using proximity ligation assays. Our data strongly indicate that NLRP3 and vimentin directly interact and that this interaction is important for NLRP3 inflammasome activity.Support or Funding InformationThis work was supported by National Heart, Lung, and Blood Institute (HL71643; HL128194)
AAV9‐miR‐29b Treatment Decreases Expression of Matrix Proteins and Improves Alveolarization in Mice Exposed to Maternal Inflammation and Neonatal Hyperoxia
Authors: Heyob Kathryn M , Pool Caylie A , Tipple Trent E , Rogers Lynette K 
Published: November 2023

BackgroundBronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication for preterm infants. Severe BPD is characterized by decreased alveolarization and obstructive and/or restrictive lung function due to increases in or dysregulation of extracellular matrix proteins. We have previously reported decreased pulmonary miR‐29b expression in our mouse model that mimics the pathophysiology observed in infants with severe BPD. The current studies tested the hypothesis that AAV9‐mediated restoration of miR‐29b in the developing lung will improve lung alveolarization and minimize the pulmonary fibrosis.MethodsPregnant C3H/HeN mice received an intraperitoneal LPS injection on E16 and newborn pups were exposed in 85% oxygen from birth to 14 days of life. On postnatal day 3, PBS containing adeno‐associated 9 (AAV9)‐mir‐29b or AAV9‐control was administered intranasally. On postnatal day 14 or 28, mouse lung tissues were analyzed for changes in miR‐29 expression, alveolarization, and matrix protein expression.ResultsDecreases in alveolarization and increases in septal thickness were observed at both 14 and 28 days after LPS/O2 exposure. Administration of AAV9‐miR29b modestly improved alveolarization at 28 days but completely prevented the increases in septal thickness observed at both 14 and 28 days. AAV9‐miR29b also prevented the increases in phospho‐SMAD2/3, aSMA, vimentin, and MMP‐9.ConclusionOur data suggest that miR‐29b restoration may represent a novel therapeutic strategy to treat or prevent severe BPD in prematurely born infants.
The Effect of in vivo High Molecular Weight Hyaluronan Treatment on Airway Reactivity in a Neonatal Mouse Model of Continuous Positive Airway Pressure (CPAP)
Authors: Mayer Catherine , MacFarlane Peter 
Published: November 2023

Although continuous positive airway pressure (CPAP) is a commonly used mode of respiratory support in the care of pre‐term infants, its long‐term effects on lung development and airway reactivity are largely unknown. Previously, we developed a neonatal mouse model of CPAP and showed that it caused a long‐term increase in airway reactivity to methacholine challenge. In the current study we tested the hypotheses that hyaluronan (HA), a major component of lung extracellular matrix, plays a role in the airway hyper‐reactivity (AHR) following neonatal CPAP and whether it can be prevented by exogenous administration of high molecular weight HA. Neonatal mice received CPAP (6cm H2O) for 3 hours per day for the first 7 postnatal days. Control animals experienced the same conditions as experimental animals only with no CPAP. On days 3 and 7, a subset of mice in each group received sub‐cutaneous injections of 1% high molecular weight HA (>1000kD, 20μl/g). Two weeks after CPAP ended (postnatal (P) age 21 days), mice were sacrificed and lungs prepared for measurements of airway reactivity to increasing doses of methacholine (0.25μM‐8μM) using the precision cut lung slice method. Airway reactivity to methacholine was increased in P21 mice that received CPAP during the first postnatal week compared to untreated control mice. Gel electrophoresis analysis of whole lung homogenates revealed CPAP also increased total lung HA levels. Immunohistochemical staining of HA with an antibody selective for the hyaluronan binding protein (HABP) showed increased HA expression around the airways within or in the vicinity of airway smooth muscle. However, mice that also received high molecular weight HA during CPAP displayed decreased reactivity to methacholine challenge when compared to airways from animals that received CPAP alone. High molecular weight HA administration also prevented the increased total HA expression caused by CPAP. We conclude that lung HA plays a role in the long‐term increase in AHR following neonatal CPAP, although exogenous administration of high molecular weight HA appears to be protective against such adverse effects of CPAP. These data may have important implications for the way CPAP may contribute to the pathophysiology of wheezing disorders commonly seen in preterm infants.Support or Funding InformationNHLBI PO1HL107147
Differential expression of serotonin 2A (5HT2A) receptors in the phrenic motor nucleus over disease progression in a rat model (SOD1G93A) of ALS
Authors: Borkowski Lauren Faith , Craig Taylor A , Nichols Nicole L. 
Published: November 2023

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and, ultimately, death by ventilatory failure. In a rat model of ALS (SOD1G93A), phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (a form of phrenic motor facilitation and respiratory plasticity) is NADPH oxidase dependent in pre‐symptomatic rats, but NADPH oxidase independent at disease end‐stage (Nichols et al., 2015). Naïve rats express pLTF via a 5HT2 and NADPH oxidase dependent mechanism. Furthermore, 5HT2A, not 5HT2B, receptor induced phrenic motor facilitation is NADPH oxidase independent in naïve rats (MacFarlane et al., 2014). Here, we tested the hypothesis that 5HT2A receptor expression in the phrenic motor nucleus is upregulated in end‐stage SOD1G93A rats compared to age‐matched, wild‐type littermates and pre‐symptomatic SOD1G93A rats using immunohistochemistry. However, our preliminary data suggest that end‐stage SOD1G93A rats have significantly less 5HT2A positive pixels in the phrenic motor nucleus and the non‐phrenic ventral horn (40±8 and 19±4 pixels, respectively; n=9) vs. age‐matched, wild‐type littermates (72±12 and 36±6 pixels, respectively; n=8; p<0.05), and pre‐symptomatic SOD1G93A rats (2773±474 and 1833±1556 pixels, respectively; n=3; p<0.05). 5HT2A positive pixels in the phrenic motor nucleus and the non‐phrenic ventral horn were unaffected when comparing pre‐symptomatic SOD1G93A rats to age‐matched, wild‐type littermates (2199±396 and 2678±1127 pixels, respectively; n=3; p>0.05). In summary, our preliminary data suggest that 5HT2A receptor expression is decreased with age and with disease progression. Although 5HT2A receptor expression is decreased in end‐stage SOD1G93A rats, this does not rule out the functional upregulation of 5HT2A receptors on surviving phrenic motor neurons or if they are required for pLTF. Future studies will be focused on: 1) completing 5HT2A histological analysis comparing pre‐symptomatic vs. end‐stage SOD1G93A rats; 2) determining 5HT2B expression in SOD1G93A rats; 3) determining if pLTF in end‐stage SOD1G93A rats is 5HT2A, not 5HT2B, dependent; and 4) determining if other reactive oxygen species (e.g. nitrogen oxygen species) that do not require NADPH oxidase compensate to exhibit pLTF in end‐stage SOD1G93A rats. This project increases our understanding of the expression of receptors associated with respiratory plasticity and its implications for breathing in motor neuron disease.Support or Funding InformationSupported by NIH K99/R00 HL119606
Perineuronal Net Expression in the Brain of an Obligate Hibernator
Authors: Schwartz Christine , Bonner Michael , Greife Augustus , Hammer Melissa , Johnson Reid , Marchand Anna , Morrow Bailey , Olson Lyn 
Published: November 2023

The brains of mammalian hibernators are naturally protected. Hibernating thirteen‐lined ground squirrels undergo rapid and extreme changes in body temperature and brain perfusion as they cycle between lengthy torpor bouts and brief periods of euthermia called interbout arousals (IBAs). Arousal from torpor to IBA occurs rapidly, but there is no evidence of brain injury accompanying this transition, despite the rapid and extreme change in physiology. Previous work investigating seasonal gene expression in the hypothalamus and cerebral cortex provided evidence for substantial regional differences in brain structural and functional regulation during hibernation. However, due to the extreme nature of the hibernation phenotype, protection must be a main priority for all brain regions. Interestingly, the hypothalamus showed a significant increase in aggrecan mRNA expression during hibernation, which is a main component of complex extracellular matrix structures called perineuronal nets (PNNs), which are known to be both protective. Hypothalamic function is important for hibernation and PNNs could help promote both protection and continued function during this time. Aggrecan expression was low in the cerebral cortex and did not show a seasonal change, suggesting that this change was specific to the hypothalamus. Here, we examined direct expression of PNNs in the thirteen‐lined ground squirrel brain using fluorescent Wisteria floribunda lectin histochemistry. Expression of PNNs was not found globally throughout the brain, but was found in discrete regions, including areas of the hypothalamus. However, PNN expression was also found throughout the cerebral cortex and other parts of the brain, including areas of the thalamus, epithalamus, and septum, indicating that PNNs are not specific to the hypothalamus. Additionally, PNNs were found in both hibernating (Torpor and IBA) and non‐hibernating (summer) animals, indicating that these extracellular matrix structures are present throughout the year. However, it is currently unknown if the structural components of the PNNs are the same in all brain regions or if these change seasonally. Overall, this work supports the idea that the extracellular matrix is contributing to neuroprotection during hibernation in the brain, but does not support the idea that the presence of PNNs is specific and unique to the hypothalamus during hibernation.Support or Funding InformationThis work is supported by UWL startup funding and a UWL Summer Faculty Research Grant to C.S.
IL‐10 improves cardiac remodeling post‐myocardial infarction by increasing M2 macrophage polarization to improve scar formation
Authors: Jung Mira , Ma Yonggang , Yabluchiansky Andriy , Iyer Rugmani Padmanabhan , DeLeon‐Pennell Kristine , Garrett Michael R , Lindsey Merry L 
Published: November 2023

Timely resolution of inflammation is important for better cardiac remodeling following MI and requires the coordinated actions of macrophages and fibroblasts. We hypothesized that interleukin‐10 (IL‐10), an anti‐inflammatory cytokine, contributes to post‐MI repair through actions on these cell types. C57BL/6J male mice (3–6 month old; n=13/group) were treated with saline or IL‐10 (50 μg/kg/day) starting at day 1 post‐MI by osmotic mini‐pump infusion and sacrificed at day 7 post‐MI. Tissue and cells (macrophages and fibroblasts) were isolated from the left ventricle infarct area (LVI). Despite similar infarct area and mortality rates, IL‐10 treatment significantly decreased LV dilation by 1.8 fold and improved ejection fraction by 1.5 fold (p<0.05 for both). IL‐10 treatment attenuated inflammation at day 7 post‐MI, evidenced by decreased numbers of Mac‐3+ macrophages in the infarct (p <0.05). LV macrophages isolated from day 7 mice treated with IL‐10 showed significantly elevated M2 markers (Arg1, Ym1 and TGF‐b1). We further performed transcriptomic analysis of isolated day 7 post‐MI cardiac macrophages and identified 410 significant different genes. IL‐10 treatment up‐regulated 155 genes and down‐regulated 225 genes compared to saline. These genes were grouped using functional network analysis; the majority of genes (120 out of 410) were part of the cellular assembly and repair functional group. Of these, hyaluronidase 3 (Hyal3) showed the biggest fold change between groups. IL‐10 treatment decreased Hyal3 by 28% (p<0.05), which reduced hyaluronan degradation and limited collagen deposition (p <0.05). IL‐10 treatment also significantly increased fibroblast activation, noted by elevated α‐SMA expression and increased proliferation and migration rates (all p <0.05). Combined, our results indicate that in vivo infusion of IL‐10 post‐MI improves the LV microenvironment to dampen inflammation and facilitate cardiac wound healing.Support or Funding InformationWe acknowledge support from AHA for 15SDG22930009, the National Institutes of Health HL075360, HL129823, GM114833, P01HL051971 and P20GM104357; and the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award 5I01BX000505.
Vasoprotective Actions of Chronic Vagal Nerve Stimulation in Hypertensive Rats – Role of inflammatory cytokines and vascular migration of inflammatory cells
Authors: Stauss Harald Martin , Tan Brianna K , Rotella Diane L , Ratcliff Jason A , Harwani Sailesh C , Chapleau Mark W 
Published: November 2023

In a previous study we demonstrated that four weeks of chronic vagal nerve stimulation (VNS) in stroke‐prone spontaneously hypertensive rats (SHR‐SP) on high‐salt diet prevents progression of endothelial dysfunction and aortic stiffening. This study was designed to test the hypothesis that anti‐inflammatory effects (e.g., reduced activation of inflammatory cells in the spleen, less vascular migration of inflammatory cells) of VNS contribute to these vasoprotective actions of chronic VNS. Aortic collagen I content, determined by immunohistochemistry, was less in SHR‐SP with VNS (n=8) than in sham‐stimulated SHR‐SP (Sham, n=8), confirming the effects of chronic VNS on aortic stiffening. Furthermore, aortic collagen I content correlated with serum levels of the pro‐inflammatory cytokines interleukin‐1 beta (IL‐1β, R=0.77, n=11, P<0.01) and tumor necrosis factor‐alpha (TNF‐α, R‐0.55, n=11, P=0.08). Protein content of the pro‐inflammatory cytokines interleukin‐5 (IL‐5), macrophage inflammatory protein‐1 alpha (MIP‐1α), and TNF‐α was reduced in spleens from SHR‐SP with VNS compared to Sham (IL‐5: 0.35±0.02 vs. 0.69±0.11 pg/mg; MIP‐1α: 0.91±0.04 vs. 1.44±0.13 pg/mg; TNF‐α: 0.18±0.02 vs. 0.28±0.05 pg/mg; n=3 for both groups). Finally, preliminary data based on aortic staining for CD3 (T‐cells), CD45 (leukocytes), and CD68‐positive (macrophages) cells suggest less migration of inflammatory cells into the aorta in VNS compared to Sham (CD3: 25±1% vs. 62±5%; CD45: 28±18% vs. 53±3%; CD68: 23±6% vs. 44±11%; n=2 for both groups; % values are positively stained cells relative to all cells identified by DAPI staining of the cell nuclei). Collectively, these data suggest that reduced splenic activation of inflammatory cells and less vascular migration of inflammatory cells contribute to the vasoprotective actions of chronic VNS in this rat model of severe hypertension.Support or Funding InformationSupported by a pilot grant from the University of Iowa Hospitals and Clinics Center for Hypertension Research.