bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐Term Potentiation (LTP) in Syrian Hamster Hippocampal Slices Can Be Generated at Warm (35[deg]C[endash]20[deg]C) but Not Cool (15[deg]C) Slice Temperatures over a Hibernation Cycle
Authors: Horowitz John M , Hamilton Jock S , Chau Sat M , Horwitz Barbara A
Published: November 2023

Previous studies have shown that LTP, a form of synaptic plasticity supporting the formation of memories, can be generated in the Syrian hamster, a facultative hibernator. However, the full range of brain temperatures at which LTP can be generated at various times of the hibernation cycle has yet to be fully delineated. We hypothesized that LTP in hamsters prepared to enter hibernation could be generated at lower temperatures than in hamsters not prepared to hibernate. To test this hypothesis, we compared responses of slices from non‐hibernating hamsters housed in a summer‐like environment (SH slices), to responses of slices from hamsters housed in a winter‐like environment but had not yet entered torpor (WH slices), and to slices from hamsters housed in a winter‐like environment that had entered torpor (HH slices). For this, we measured tetanus‐induced LTP [a sustained enhancement of population spike (PS) amplitude] and the dispersion of a packet of action potentials over a network of CA1 pyramidal cells [the width of a PS]. In all three groups, we observed sustained enhancement of PS amplitude lasting at least 1 hour, the hallmark of LTP generation, at temperatures above 20°C. However, no enhancement was seen at or below 15°C in any group. Moreover, at 15°C, synchronization of pyramidal cell firing following single‐shock stimulation, necessary for LTP generation, was degraded – i.e., PS width at 15°C was 5 times that observed at 35°C. Overall, our data negate our hypothesis and suggest that new memories cannot be formed via LTP synaptic enhancement during the late stages of entry into hibernation, during hibernation itself, or during the early stages of arousal from hibernation. Additionally, our results from the SH and WH slices imply that LTP may be a major cellular plasticity mechanism underlying the finding that hamsters that master a hippocampal behavioral task prior to hibernation can execute the task after hibernation. That is, LTP changes occurring at postsynaptic sites before hibernation persist through a series of hibernation bouts (and changes in dendritic spine structure) to a degree sufficient to allow hamsters to perform the task upon arousal.
Do Trigeminal Nerves that Innervate the Nose and External Nares Project to Medullary Dorsal Horn Locations Activated during Voluntary Diving in Rats?
Authors: McCulloch Paul , DelPrete Benjamin , Lahrman Kenneth , Warren Erik , DiNovo Karyn
Published: November 2023

Underwater diving initiates the diving response, an autonomic cardiorespiratory reflex that includes parasympathetically‐mediated bradycardia, a sympathetically‐mediated increase in total peripheral resistance, and apnea. The initial afferent node of the reflex circuitry includes the ventral tip of the medullary dorsal horn (MDH), between the pyramidal decussation and obex. This region is also the location of the central terminations of trigeminal nerves that innervate the nose and external nares. Accordingly, the objective of this research was to compare the pattern of neuronal activation of secondary neurons activated by voluntary diving, as determined by neuronal Fos production, with the central terminations of specific trigeminal nerves that innervate the nose and external nares, as determined by the transganglionic tracer Wheat Germ Agglutinin (WGA). Sprague‐Dawley rats (N=12) were trained to voluntarily dive through a 5 m underwater maze. Six rats then received bilateral AEN sectioning, while 6 rats received sham surgery. Nine days post‐surgery all rats had 24 dive trials over 2 hours to activate brainstem neurons to produce Fos. Other rats had 1 μl of 100% WGA injected into the anterior ethmoidal nerve (AEN; N=4), the main orbital branch of the infraorbital nerve (ION; N=7), terminal branches of the ION that innervate the nasal region (N=9), or supratrochlear nerve (N=8). Results indicate Fos was primarily located within the ventral tip of the superficial MDH and adjacent paratrigeminal nucleus, between the pyramidal decussation caudally and the obex rostrally. The Fos labeling was heaviest when the area postrema was present, especially when Spinal Trigeminal Nucleus Interpolaris displaces the ventral MDH medially. This pattern of Fos labeling within the MDH was similar in rats both with and without intact AENs. Within the MDH WGA central termination sites showed varying degrees of overlap with the location of Fos‐positive neurons. The AEN WGA labeling had the best overlap with Fos‐positive neurons. The main orbital branch of the ION had extensive WGA labeling throughout the MDH, both ventrally and dorsally, and did partially overlap the MDH Fos labeling. The WGA labeling from both nasal branches of the ION and supratrochlear nerve were located more caudally within the MDH near the pyramidal decussation, thus showing poor overlap with the Fos labeling. We conclude that the AEN, and possibly the ION, provides the anatomical pathway that provides the afferent information necessary to initiate the mammalian diving response. However, in the absence of the AEN, it is not clear whether the ION can actually provide this afferent information.

Support or Funding Information

Supported by MWU Intramural funding (PFM), CCOM Suarez Summer Research Fellowships (BDP and KAL), and CHS MBS program (EAW).
Second hand smoke exposure depresses excitability and SK channel function of cardiac vagal neurons in the nucleus ambiguus
Authors: Sun Jun‐Qing , Karey Emma , Pan Shiyue , Chen Yi‐Je , Pinkerton Kent E , Chen Chao
Published: November 2023

BACKGROUND

Second hand smoke (SHS), a major indoor air pollutant, is a significant risk factor for cardiac dysfunction including arrhythmias and sudden cardiac death. Reduced heart rate variability (HRV), an index of autonomic function, has been implicated as one of the underlying mechanisms of these cardiac maladies. We have previously shown that exposure to air pollution in the form of iron‐soot significantly reduced HRV and spiking activity of cardiac vagal neurons in the nucleus ambiguus (NA), the final vagal cardiac output regulating HRV. Here, we sought to test the hypothesis that SHS exposure, at an environmentally relevant concentration that reduces HRV, depresses neuronal excitability of cardiac vagal neurons in the NA by altering potassium channel function.

METHODS

Adult male C57BL6/J mice underwent open chest surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. After two weeks of recovery from surgery, mice were randomly assigned to either filtered air (FA) or SHS (3 mg/m3, 6 hr/d, 5 d/wk) for 4‐, 8‐ or 12‐weeks. Whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the NA with fluorescent labeling.

RESULTS

Four weeks of SHS exposure was sufficient to increase action potential (AP) threshold and reduce spiking response to depolarizing current injections (p < 0.05) in cardiac vagal neurons. The minimal current required to evoke an AP is also significantly higher in SHS group. Perfusion with apamin (20 nM), an SK channel blocker, had no effect on AP threshold or on minimal current required to evoke an AP in either FA or SHS mice. However, apamin significantly increased neuronal spiking response to depolarization in both groups; this effect was significantly smaller in SHS animals (p < 0.05). These data suggest that SHS exposure reduces excitability of cardiac vagal neurons that is associated with attenuated SK channel modulation of spiking response. These data also suggest that SHS exposure may reduce dynamic spiking range of these cardiac vagal neurons, consistent with reduced cardiac vagal regulation of HRV. Resting membrane potential was not significantly changed until 12 weeks of SHS exposure (became more depolarized). These data suggest that there may be compensatory changes in potassium channel function to increase the neuronal excitability in response to extended SHS exposures.

CONCLUSIONS

We conclude that environmentally relevant SHS exposure can reduce neuronal excitability of cardiac vagal neurons and contribute to reduced vagal regulation of HRV resulting in reduced HRV.

Support or Funding Information

R01 ES025229; T32 HL086350
Effect of Sedentary Conditions on the Expression of GABAAα2 Receptor Subunits in Bulbospinal C1 and non‐C1 Neurons along the Rostrocaudal Extent of Rat Rostral Ventrolateral Medulla (RVLM)
Authors: Fyk‐Kolodziej Bozena E , Azar Toni A , Llewellyn‐Smith Ida J , Mueller Patrick J
Published: November 2023

Bulbospinal RVLM neurons play a crucial role in the regulation of sympathetic nerve activity and blood pressure, and can be characterized phenotypically as catecholaminergic (C1) or non‐catecholaminergic (non‐C1). Although excitatory in nature, the tonic activity of the RVLM is strongly influenced by the prevailing level of the inhibitory GABAergic tone. Following sedentary conditions, RVLM neurons also exhibit structural and functional plasticity in a region‐dependent manner, with enhanced dendritic branching and increased glutamate responsiveness in rostral versus caudal regions. Because withdrawal of GABAergic tone produces net excitation of RVLM neurons primarily via GABAA receptors, it is possible that sedentary conditions also alter GABA receptor expression in a region‐dependent manner. Thus, the purpose of the present study was to test the hypothesis that sedentary conditions reduce GABAA receptor expression in rostral regions of the RVLM. To this end, male Sprague‐Dawley rats were divided into sedentary (no wheels) and physically active (running wheels) conditions for 12 wks (n=4 ea). Spinal cord injections of cholera toxin B (CTB) labeled bulbospinal neurons. We examined coronal sections processed for triple‐labeling immunofluorescence using laser confocal microscopy, and quantified CTB‐positive C1 and non‐C1 neurons expressing the GABAAα2 receptor subunit. As expected, more bulbospinal C1 cells expressing GABAAα2 occurred in caudal versus rostral RVLM (p<0.05). Sedentary rats had fewer bulbospinal C1 cells that expressed GABA Aα2 only in the rostral most subregions of the RVLM when compared to active rats (p<0.05). The number of bulbospinal non‐C1 neurons also varied rostrocaudally. However, there were more non‐C1 neurons in rostral compared to caudal RVLM with no overt differences between sedentary versus active groups. Decreases in RVLM neurons expressing GABAA receptors in rostral RVLM are consistent with previous data demonstrating enhanced sympathetic outflow following sedentary conditions, which may in turn contribute to the increased incidence of cardiovascular disease in sedentary individuals.

Support or Funding Information

R01‐HL096787‐06; NHMRC 1025031; AHA25810010
Arcuate NPY/AgRP Neurons Decrease Arterial Pressure and Sympathetic Nerve Activity in part via a projection to the Dorsomedial Hypothalamus in Mice
Authors: Shi Zhigang , Madden Christopher J. , Brooks Virginia L.
Published: November 2023

Using designer receptors exclusively activated by designer drugs (DREADD) technology, we recently demonstrated that select activation of arcuate nucleus (ArcN) neurons that express Neuropeptide Y (NPY) and agouti‐related protein (AgRP) rapidly and profoundly decreases splanchnic sympathetic nerve activity (SSNA), mean arterial pressure (MAP), and heart rate (HR) in male mice. However, the downstream targets of ArcN NPY neurons are unknown. The present study tested the hypothesis that this response is mediated in part by a projection to the dorsomedial hypothalamus (DMH). The mutated excitatory muscarinic receptor (hM3Dq) was selectively expressed via bilateral injection of Cre‐recombinase‐dependent adeno‐associated virus (AAV)‐hM3Dq‐mCherry into the ArcN of AgRP‐Ires‐cre mice. After >2 wks recovery, mice were anesthetized with α‐chloralose and the otherwise inert ligand Clozapine‐N‐oxide (CNO; 0.3 mg/kg i.p.; n=5), to activate hM3Dq, or saline (sal; n=5), was injected i.p. After ~30 min, CNO decreased SSNA (−25±2 % control), MAP (−9±1 mmHg), and HR (−62±17 bpm) (P<0.01); however, sal had no effects (ΔSSNA: 3±2 % control; ΔMAP: 0±2 mmHg; and ΔHR: 11±9 bpm). Subsequent bilateral injections (30 nL) of the NPY Y1 receptor antagonist BIBO3304 (Y1x; 0.3 nmol) into the DMH of sal‐treated mice elevated (P<0.05) SSNA to 157±10 % control, MAP by 14±4 mmHg, and HR by 141±29 bpm, indicating that DMH NPY tonically inhibits SSNA. In mice given CNO, DMH Y1x increased (P<0.05) these variables (SSNA to 140±9 % control, and MAP by 14±4 mmHg and HR by 125±33 bpm above baseline) to levels similar as in sal‐treated mice given PVN Y1x, suggesting that in CNO‐treated mice Y1x is blocking the actions of both tonic and DREADDs‐induced NPY release in DMH. Next, because DREADDs can be expressed in terminal fields, in another group of AgRP‐Ires‐cre mice (n=5) that received Cre‐dependent hM3Dq into the ArcN, CNO (30 nL of 10 μM/L) was injected into the DMH. DMH CNO decreased SSNA by 23±3 % control, MAP by 11±2 mmHg, and HR by 40±9 bpm (P<0.05). Finally, a Cre‐dependent synaptically‐targeted mCherry AAV was injected into the ArcN of AgRP‐Ires‐cre mice (n=3) and the retrogradely transported indicator Cholera toxin B (CtB) was injected into the rostral ventrolateral medulla (RVLM). A modest number of CtB‐positive neurons were visible in the DMH. However, while mCherry‐labeled fibers were abundant in the DMH, these fibers only occasionally apposed CtB cell bodies, suggesting that NPY in DMH does not inhibit a population of neurons that directly innervates the RVLM. In conclusion, ArcN NPY/AgRP neurons decrease SSNA, MAP, and HR via a projection to the DMH. The downstream neuropathway remains to be determined.

Support or Funding Information

NIH HL128181, AHA 12GRNT11550018 and 15POST23040042, ADA 1‐13‐BS‐120
DREADD‐Induced cFos Expression in the Basal Forebrain of Male Rats
Authors: Marciante Alexandria Beverly , Cunningham J. Thomas
Published: November 2023

Designer Receptors Exclusively Activated by Designer Drugs, or DREADDs, are genetically modified G‐protein coupled receptors (GPCR) that are sensitive to an exogenous pharmacological agent, clozapine‐N‐oxide (CNO). DREADDs can be packaged in viral vectors with specific promoters or combined with CRE dependent platforms to express these receptors in specific neuronal phenotypes. This chemogenetic approach can be used to activate (Gq), inhibit (Gi), or stimulate cAMP (Gs) in neurons. In the present study, we tested the effects of a CRE independent Gq DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5‐CaMKIIa‐hM3D(Gq)‐mCherry) on Fos staining in the basal forebrain. Adult male Sprague‐Dawley rats (250–300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(Gq) or a control virus (rAAV5‐CaMKIIa‐mCherry) in either the diagonal band of broca (DBB) or the median preoptic nucleus (MnPO). Animals were allowed two weeks of recovery prior to administered CNO or vehicle. CNO was dissolved into dimethylsulfoxide (DMSO) and saline (ratio 20% to 80%) and given via intraperitoneal injection (IP) at a concentration of 10mg/kg. Rats were food and water deprived for 90 minutes following the injections. Each rat was anesthetized with inactin (100 mg/kg IP) and prepared for immunohistochemistry. Forebrains were sliced into 40 um coronal sections using cryostat. The sections were processed for Fos immunohistochemistry using DAB‐peroxidase and mCherry immunofluorescence to verify cells transfected with either the DREADD or control virus. Overall, rats transfected with the Gq DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either Gq DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the Gq DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either Gq DREADD and treated with vehicle (P<0.001) or the control virus and treated with CNO (P<0.001) or vehicle (P=0.002). The CNO did not appear to have a significant effect on Fos staining in rats injected with the control vector. Injection of Gq DREADD virus into the DBB also appears to have less of an effect on downstream targets, specifically the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), than did injection of Gq DREADD virus into the MnPO. These results indicate that Gq DREADD can be used to differentially activate neurons in either the DBB or the MnPO to influence activity in downstream regions that control autonomic and neuroendocrine function.

Support or Funding Information

Supported by P01 HL088052
Role of Alpha‐adrenergic Inputs to PVN in the Cardiovascular Responses to Acute Hypoxia
Authors: Beig Irfan , Mifflin Steve
Published: November 2023

Previous studies indicate that catecholaminergic neurons in nucleus of solitary tract (NTS) contribute to intermittent hypoxia hypertension and that the hypothalamic paraventricular nucleus (PVN) plays a role in the cardiovascular response to hypoxia. The present study was designed to examine the role of alpha‐adrenergic receptors in PVN in the cardiovascular responses to acute hypoxia. Rats were anesthetized with chloralose and urethane and mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA) and end tidal CO2 measured. The rat was placed in a stereotaxic frame for microinjections in PVN. After paralysis with gallamine, the rat was artificially ventilated with room air. Hypoxia was induced by ventilating the animal with 10% O2 for 30 seconds. When the parameters returned to the baseline, bilateral injections of vehicle or either α1 antagonist prazosin (500nmol) or α2 antagonist idazoxan (5nmol) were made in PVN and responses to hypoxia observed after 20 minutes. At the end of experiment brain was removed for localization of microinjection site. Hypoxia increased MAP with no change in HR or RSNA. The increase in MAP induced by systemic hypoxia was significantly blunted by bilateral administration of either prazosin (36 ± 3mmHg to 19 ± 4mmHg, n=11) or idazoxan (36 ± 2mmHg to 25 ± 5mmHg, n=10) in PVN. Changes in baseline blood pressure were not significant following prazosin (−2 ± 4mmHg) or idazoxan (0 ± 3mmHg). The results suggest that catecholaminergic inputs to PVN play a role in acute hypoxia induced increases in MAP.

Support or Funding Information

Supported by PO1 HL88052
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 Information

Supported by G. Harold and Leila Y. Mathers Charitable Foundation
For Patients with Chronic Musculoskeletal Pain, Does Therapeutic Neuroscience Education (TNE) Complement Exercise and/or Manual Treatment in Decreasing the Perception of Pain?
Authors: Gamper Jacob , Bryant Alexandria , McCallum Josh , Wilson Storm , Lehman David
Published: November 2023

Chronic pain treatment contains a cognitive and a musculoskeletal component, the musculoskeletal component more often addressed by a physical therapist with exercise and/or manual therapy. Meanwhile, the cognitive aspect of treatment is neglected or considered less of a significance in the physical therapy treatment. This evidence based review aims to determine whether Therapeutic Neuroscience Education (TNE), a cognitive approach, could be an effective adjunct with manual treatment and/or exercise (musculoskeletal approaches) in treating chronic musculoskeletal pain. According to the review of pain science and pain treatment literature, TNE can be equally or more effective as manual therapy and/or exercise in treating chronic musculoskeletal pain. Understanding the neurophysiology of pain can decrease the severity of how one perceives pain. Through multiple fMRI studies, TNE has also been shown to decrease activity in various sections of the brain associated with pain perception. As a goal of physical therapy is to address all variables of pain, a cognitive component in addition to a musculoskeletal component must be implemented to effectively decrease the perception of chronic pain.
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 Information

NIH 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 Information

This 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

Background

Bronchopulmonary 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.

Methods

Pregnant 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.

Results

Decreases 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.

Conclusion

Our 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 Information

NHLBI 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 Information

Supported 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 Information

This 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 Information

We 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 Information

Supported by a pilot grant from the University of Iowa Hospitals and Clinics Center for Hypertension Research.
The effects of high fat diet‐induced obesity and exercise training on hematopoietic stem and progenitor cells following sub‐lethal radiation
Authors: Emmons Russell , Hernández‐Saavedra Diego , Parks Zachary S , Chen Hong , De Lisio Michael
Published: November 2023

Of the 14 million cancer survivors in the United States, approximately 2/3 have received radiation therapy. Hematopoietic stem and progenitor cells (HSPCs) are among the most sensitive cells to radiation damage resulting in anemia and immunosuppression acutely following radiation exposure, and decreased hematopoiesis in the long‐term. Increased adiposity and physical inactivity are common among cancer survivors and both have negative effects on hematopoiesis. However, the impacts of obesity and exercise on the late effects of radiation exposure on HSPCs are unknown. The purpose of the present investigation was to evaluate the impact of obesity and exercise on HSPCs following sub‐lethal radiation exposure. A total of 40 CBA mice were divided into 4 groups (CON+SED n=10, CON+EX n=10, HF+SED n=10, HF+EX n=10). Mice were put onto a control (CON) or high fat diet (HFD) to induce obesity for 4 weeks before being placed into a progressive treadmill exercise (EX) program for 9 weeks or remained sedentary (SED). Four weeks into the training program, all mice were exposed to a 3 Gy dose of gamma radiation and completed their respective exercise and diet programs. At the conclusion of the 9 week intervention, bone marrow cells were collected and lineage negative cells were separated and analyzed for total HSPCs (LSK, SCA+cKit+), and HSPC subpopulations: long‐term (LT‐HSC, LSK CD150+CD48−), short‐term hematopoietic stem cell (ST‐HSC, LSK CD150+, CD48+), and multi‐potent progenitors (MPP, LSK CD150−,48+). HFD‐induced obesity reduced bone marrow cellularity compared to control mice (p<0.01). EX attenuated these changes to bone marrow cellularity (p<0.01). A decrease in the total number of LSK resulted from consuming HFD (p<0.01) that was attenuated with EX (p<0.01). Similar HFD‐induced decreases were seen in LT‐HSC (p<0.01), ST‐HSC (p<0.01), and MPPs (p<0.01) which were attenuated with EX (LT‐HSC, p<0.01, ST‐HSCs, p<0.05) and strongly trended in MPPs (p=0.067).

Conclusion

Here we show that HFD‐induce obesity decreases bone marrow cellularity and HSPC content following radiation exposure which is rescued by EX. As such, exercise training may serve as a promising therapy to preserve HSPCs following radiation therapy.

Support or Funding Information

Funded by the UIUC Research Board Grant, ACSM Student Research Grant, and ACSM Foundation Grant
CL‐K1, a Novel Lectin Pathway Component is a Leptomeningeal Protein
Authors: González‐Losada Cristóbal , Castillo‐González William , Jensenius Jens Christian , Zerr Inga , Schmitz Matthias , Dorta‐Contreras Alberto Juan
Published: November 2023

The universal validity of the molecular diffusion/cerebrospinal fluid (CSF) flow model is valid for the brain‐derived molecules, is the base for a systematic approach to determine the source of a CSF protein either derived from blood, from brain or from leptomeninges.

Objective

To determine if CL‐K1, a new component of the lectin pathway, should be consider a leptomeningeal protein.

Methods

36 serum and CSF paired samples were obtained from control persons without neuroinflammation process and without oligoclonal IgG bands. CL‐K1 and albumin were quantified by ELISA and by an immunoturbidimetric analysis. Q albumin= CSF albumin/serum albumin and QCL‐K1= CSF CL‐K1/serum CL‐K1 was calculated.

Results

Serum and CSF CL‐K1 follow a normal distribution according to Kolmogorov‐Smirnov test. Mean values of CL‐K1 in serum was higher than its mean content in CSF. CSF CL‐K1 concentration increased with the increase of Q albumin. Cl‐K1 values in serum are higher than in CSF. This fact notes that CL‐K1 is not a brain‐derived protein. In Figure 1 there is the relationship between CL‐K1 vs. Q albumin. Note that the regression line has a positive slope. It means that CL‐K1 increases with increment of Q albumin. It is a general rule that follows the proteins that was located in the leptomeninges.

Conclusions

These results means that the function is not solely diffusion dependent and allowed the finding that CL‐KI in CSF derives from the leptomeninges as well as mannan‐binding lectin.
Features of metabolic syndrome in male mice with deficiency of estrogen receptor alpha
Authors: Winn Nathan C , Jurrissen Thomas , Lin Gabriela S , Gastecki Michelle L , Welly Rebecca J , Lubahn Dennis B , Vieira‐Potter Victoria J , Padilla Jaume
Published: November 2023

Attenuation of estrogen action through estrogen receptor α (ERα) has been implicated in the development of metabolic syndrome in male mice; yet, some studies remain inconclusive which may be attributed to controversy regarding a commonly used mouse model that contains a splice variant of ERα. Herein we examined whether common features of the metabolic syndrome are present in male mice with deficiency of ERα (i.e., bred at our facility), including excess adiposity, adipose tissue inflammation, glucose intolerance, fatty liver, hyperlipidemia, and vascular dysfunction. After weening, C57BL/6J wild type (WT; n=6) and mutated/non‐functional ERα (ERα−/−; n=7) male mice were fed a standard chow diet for 16 months. Despite no significant differences in body weight or energy intake, ERα−/− mice demonstrated increases in fat mass, visceral adiposity, fatty liver, hyperlipidemia, and glucose and insulin intolerance (P<0.05). In visceral adipose tissue, both histologic and gene expression markers of immune cell infiltration [CD68, CD11c (P<0.05), and ITGAM (P=0.05] were elevated in ERα−/− mice along with increased markers of oxidative stress [p47phox and p22phox (P<0.05)]. Furthermore, ERα−/− mice exhibited impaired endothelium‐dependent vasorelaxation in the aorta (P<0.05), whereas aortic stiffness (in vivo or ex vivo, P>0.05) was unaffected. Collectively, our findings support that loss of ERα signaling promotes metabolic and vascular dysfunction in males.