bluuebunny/crossref_metadata_2025 huggingface dataset:
Subconcussive Head Impact Results in a Unique Circulating Exosomal MicroRNA Signature
Authors: Caban Carolina T , Jackson Kelli , Pinson Catherine L , Dos Santos Fernando V , Jeka John J , Tierney Ryan T , Hudson Matthew B
Published: November 2023

A large segment of the population participates in sports (e.g. football and soccer) and endure hundreds to thousands of low level head impacts over their lifetime. While a continuously growing body of evidence indicates concussive head injury can lead to long term medical problems, little is known about the effects of subconcussive head impact, that is mild head impact that does not result in a clinical indication of concussion. Recently our group has shown that subconcussive head impact results in acute vestibular dysfunction. Additional recent evidence indicates subconcussive head impact results in memory loss for up to 24 hours. Thus, the possibility exists that both high magnitude head impact like concussions as well as repetitive low levels of head impact lead to pathological changes. Recent research has shown that small circulating molecules termed exosomes contain cargo including microRNAs that may be indicative of pathological events and signaling occurring in the tissue from which they were released. Thus, the purpose of this study was to identify a unique circulating exosomal microRNA profile indicative of subconcussive head impact. We used a common soccer heading task as a controlled head impact model to elicit subconcussive head impact in college males (n=6), in which subjects headed a soccer ball 10 times at a set velocity from ~30 meters away. Pre, immediately post, and 24 hours post blood samples were collected. Exosomes were isolated from plasma, and total RNA, including microRNA, was isolated from exosomes. Small RNA Next generation sequencing (NGS) was performed on microRNAs isolated from exosomes in pre and 24 hour post plasma to unbiasedly identify alterations in levels of exosomal microRNAs. A unique microRNA signature in circulating exosomes was identified 24 hours following subconcussive head impact. Specifically, based on abundance and fold change a small unique panel was identified including three microRNAs that were increased four fold or more (miR‐92b‐3p, miR‐423‐5p, and miR‐24‐3p), and four microRNAs that were decreased three fold or more (miR‐7844‐5p, miR144‐5p, miR‐221‐5p, and miR‐22‐3p). To verify NGS results, quantitative real time polymerase chain reaction (qPCR) was performed on microRNAs at all time points. Further, to verify specificity of head impact exosomal microRNAs in the panel were measured via qPCR from a group the received soccer ball impact to the gastrocnemius muscle. Interestingly, several of the identified microRNAs are known to be involved in or associated with neurological dysfunction and disorders. In conclusion, subconcussive head impact leads to a unique circulating exosomal microRNAs signature that could potentially be indicative of head injury following repetitive subconcussive head impacts.

Support or Funding Information

Supported by NIH GM087239
miR429: Potential regulator of PAK2 in Inflammatory Monocytes
Authors: Stull Lauren Christine , Amin M Asif , Rabquer Bradley James
Published: November 2023

Purpose

Rheumatoid Arthritis is an inflammatory disease that leads to the eventual deterioration of joints. Monocytes are key players in its pathogenesis and migrate to the sites of inflammation. Micro (mi)RNAs regulate mRNAs and the synthesis of proteins and have the ability to silence specific mRNAs by preventing translation or aiding their degradation. Previous results from our lab have shown that specific microRNAs are elevated in monocytes from patients with Rheumatoid Arthritis. Using in silico analysis, we have also determined potential gene targets for these miRNAs in monocytes. miR429 is a differentially expressed miRNA in monocytes in response to inflammatory stimuli and has PAK2 as a theoretical gene target. miR429 has been shown to inhibit cancer cell migration by reducing the expression of its target genes. PAK2, which is highly expressed in leukocytes, including monocytes, is known to regulate inflammation. Therefore, we hypothesized that miR429 regulates inflammation by binding to PAK2 and mediating its degradation.

Methods & Results

In order to assess the binding between miR429 and PAK2 we cloned the 3′ untranslated regions (UTR) of PAK2 into the pmirGLO dual luciferase vector. The vector was propagated, purified, and treated with the restriction enzymes SacI and SaclI, and cleavage was confirmed by PCR analysis. THP‐1 monocytes were grown and used as a source of mRNA. cDNA was prepared and primers specific to the miR429 predicted binding site on the 3′ UTR of PAK2 were used to produce a PCR product of 238 base pairs. Another double digest involving the same enzymes was performed on the PCR product, and the product was ligated into the vector and confirmed by gel electrophoresis. Sanger sequencing was used to determine the sequence of the resulting DNA, ensuring that the plasmid contains the 3′ UTR of PAK2 with the predicted miR429 binding site.

Conclusion

The 3′ UTR of PAK2 was cloned into the pmirGLO vector for the assessment of miR429 binding.

Support or Funding Information

This work was supported by the Albion College Biology Department, Foundation for Undergraduate Research, Scholarship, and Creative Activity (FURSCA), Hewlett‐Mellon Fund for Faculty Development at Albion College, and the University of Michigan Division of Rheumatology.
Kidney‐specific Conditional Knockout of Klotho Gene Impairs Natriuresis and causes hypertension by upregulating Epithelial Sodium Channel alpha
Authors: Ali Quaisar , Lin Yi , Wang Shirley , Chen Kai , Chen Jianglei , Sun Zhongjie
Published: November 2023

Background and Purpose

Excessive reabsorption of sodium by the kidney tubules contributes to renal dysfunction and high blood pressure. Deletion of Klotho leads to hypertension. The purpose of this study is to investigate if renal epithelial sodium channel (ENaC) contributes to klotho deficiency‐induced hypertension.

Methods and Results

We generated kidney‐specific conditional klotho null (KspKL−/−) mice and found out that renal alpha subunit of ENaC is significantly upregulated compared to wild type (WT) mice. Blood pressure measured by telemetry demonstrated systolic hypertension and elevated pulse pressure suggesting vascular dysfunction in KspKL−/−. Pulse wave velocity, a marker of arterial stiffening was significantly increased in KspKL−/− mice. Amiloride, an ENaC inhibitor, completely prevented systolic hypertension and arterial stiffening in KspKL−/− mice. Ex vivo vascular relaxing responses to acetylcholine were diminished in mesenteric arteries in KspKL−/− group, indicating that klotho deficiency causes vascular endothelial dysfunction. Interestingly, treatment with amiloride abolished Klotho deficiency‐induced vascular endothelial dysfunction. We found that urinary sodium excretion (UNaV) was significantly impaired in KspKL−/− group. Na retention was also associated with a decrease in glomerular filtration rate (GFR), suggesting impaired renal function in KspKL−/− mice. Treatment with amiloride prevented sodium retention and improved GFR in KspKL−/− mice. Total renal nitric oxide bioavailabililty and eNOS activity were significantly decreased in KspKl−/− mice which was ameliorated by amiloride treatment. Histological and morphometric analysis showed glomerular and tubular damage in KspKL−/−, which was improved by amiloride treatment. Western blotting analysis demonstrated a significant decrease in the αγ subunits of ENaC in the kidney cortex following treatment with amiloride.

Conclusion

This study demonstrates for the first time that depletion of klotho causes impairment in Na‐excretion and hypertension by affecting ENaCα levels. ENaC may be a promising therapeutic target for klotho deficiency‐induced renal and vascular dysfunction and hypertension.
Preterm birth is associated with a decrease in cervicovaginal Liners during second trimester in Mexican women.
Authors: Rodriguez‐Martin Pedro , Palacios‐Gonzalez Berenice , Vadillo‐Ortega Felipe
Published: November 2023

The vaginal microbiota plays an important rol during gestation. The presence of cervicovaginal infections during pregnancy has been associated to preterm labor. Prematurity is associated to high neonatal morbidity and 60–80% of all neonatal deaths. The aim for the present study was to characterize changes throughout pregnancy in Lactobacillus iners abundance of women who developed preterm birth. Women were selected according outcome and two groups were analyzed: 1. Term outcome (n=30) (T) and 2. Preterm outcome (n=30) (P). Cervicovaginal swabs were collected and whole genomic DNA was extracted and Lactobacillus ssp and L. iners were analyzed by RT‐PCR. Women who delivered at preterm had a significant decrease in L. ssp y L. inners in the second trimester. Our data revealed that the composition of the vaginal microbiota is a dynamic process, which means that the microbiota is modified in several points across the pregnancy.
Feed Restriction and IGF‐1 Affect the Oocyte Maturation in Matrinxã Brycon amazonicus
Authors: Montrezor Luís H. , Urbinati Elisabeth C.
Published: November 2023

The feeding and nutrition of breeders are crucial aspects in the reproductive process. During the maturation period, metabolic changes occur aiming at mobilizing energy for growth and follicular development. The involvement of IGF‐1 in metabolic and reproductive events is important. The aim of this work was to evaluate if alternate feed restriction and re‐feeding have permissive effects on in vitro actions of IGF‐1 on oocytes development of matrinxã. In vivo experiments were performed during vitellogenesis period. Females (n = 60) were fed with a commercial feed (2% of biomass) and they were divided into two treatments: fish receiving food daily (control ‐ fed), and fish submitted to cycles of three days of feed restriction and two days of re‐feeding (no fed group). The ovaries removed at the end of in vivo experiment were maintained in phosphate‐buffered saline (PBS) and the oocytes were mechanically dissected. The collected oocytes (n = 20) were cultured in 24 well plates (20 oocytes/well/mL) in Leibowitz medium supplemented with 20nM HEPES, antibiotics (1000U/mL penicillin and 100 μg/mL streptomycin) and 0,1% bovine serum albumin (BSA), in a final volume of 1,0mL, pH 7,5. The oocytes were divided into four treatments: a) fed ‐ IGF‐1; b) fed + IGF‐1 (0; 1; 10 and 50nM); c) no fed ‐ IGF‐1; d) no fed + IGF‐1 (0; 1; 10 and 50nM). The assays were performed in triplicate and the plates were incubated at 26° C for 3 hours. Fish under restriction had lower body weight, decreased plasma glucose, increased triglycerides levels, and their final maturation and mature oocyte were reduced and the atresic ones were in higher number. Moreover, the IGF‐1, in vitro, increased the percentage of mature oocytes in fed females and decreased the atresic ones. In no fed females, IGF‐1 increased the final maturation and mature oocytes and reduced the atresic ones. This study demonstrates the importance of the feeding management of female breeders of matrinxã during the vitellogenesis period. The feeding management affects oocyte development, food restriction impairs and IGF‐1 restores the negative effects on oocyte development induced by feeding management.

Support or Funding Information

CNPq
Reversible Azoospermia after Long Term Sustained Delivery of DHT
Authors: Benghuzzi Hamed A
Published: November 2023

The major objective of this study was to assess whether male rats whose endogenous testosterone and gonadotropin (LH/FSH) levels were suppressed to undetectable levels (<0.2 ng/mL) for a 1‐year period by the sustained delivery of dihydrotestosterone (DHT) would return to fertility status after serum testosterone returned to control levels. A total of 105 adult Male rats (280–320 gm) were randomly divided into three equal groups. Group 1 animals were implanted with Tricalcium Phosphate Ceramic Delivery Devices (TCPLD) loaded with 80 mg DHT each following standard lab protocols. Groups 2 and 3 animals served as a sham group (empty devices), and a control group of proven male breeders were used. For the treatment and sham groups, serum testosterone, LH and FSH levels were monitored at treatment periodic intervals of 1, 3, 6, 9 and 12 months and post treatment periods at 1, 3 months. After serum testosterone levels returned to vehicle control levels each of the animals was housed with adult female, proven breeders. All the breeder females produced offspring. The mean size and weight of the litters from each group were not statistically different. Further evaluation of all animals (male and female) offspring were shown to be fertile. The results suggest that induced azoospermia upon long term sustained delivery of DHT can be reversed. Overall conclusion obtained from this study indicated that TCPL delivery devices can be used to regulate fertility without any untoward side effects.
A Cellular Mechanism of Leptin Mediated Responses in Mouse NTS Neurons
Authors: Do Jeehaeh , Kowal Anthony , Floreani Lavinia , McCrimmon Donald R , Martina Marco
Published: November 2023

The nucleus of the solitary tract (NTS) in the medulla is the site of first synapse for sensory afferents of the vagal and glosspharyngeal nerves. A dense termination of sensory input reflects a key function of the NTS as an integrative center for a number of vitally important homeostatic reflexes such as cardiovascular, respiratory and visceral reflexes. Little is known concerning the cellular rules of information integration in the NTS, especially how the metabolic state influences respiratory output. Leptin is an anorexigenic cytokine acting through activation of specific G‐protein‐coupled receptors (GPCRs). Leptin receptors are expressed in the NTS, but the mechanism of action following their activation remains unclear. Moreover, the precise identity of the cells expressing the leptin receptor in the NTS is also elusive.

Our study has two main goals: 1‐ to uncover the expression of individual genes that identify neuronal subpopulations of cells expressing the leptin receptor; and 2‐ to identify the ion channel(s) mediating the action of leptin in these different cellular populations. To pursue these goals we combine genetic tagging of leptin receptor expressing neurons, electrophysiological recordings and single cell RT‐PCR to test the expression of candidate channels and neuropeptides. Our data show that NTS leptin receptor expressing neurons belong to two distinct populations characterized by different firing patterns (bursting vs. regular firing). Further, we found that leptin receptor activation leads to membrane depolarization that is mediated by a cationic conductance. This mechanism appears to be the same in both the cell populations. The PCR data suggest that the conductance activated by leptin is, at least in part, mediated by NALCN channels.

Support or Funding Information

This study was supported by NIH grant HL122921
The Vagus Nerve in Development of Lung Tumor
Authors: Liu Jun , Song Nana , Guardiola Juan , Yu Jerry
Published: November 2023

Objective

Lung cancer is the leading cause of cancer death worldwide. Its treatment remains largely unsatisfactory, because our knowledge of its underlying mechanisms is limited. In addition to genetic factors, the initiation, development and progression of lung tumors result from a network of interaction among mediators, cytokines, and growth factors derived from various cells. The vagus nerve is closely related to each of these network components, thus must influence the outcome. Here we tested the hypothesis that the vagus nerve protectes the lung from tumor formation and explored its underlying mechanism.

Methods

Tumors were assessed microscopically by HE staining and macroscopically by directly counting tumor numbers and measuring their sizes in the two lungs in unilaterally vagotomized AJ mice following urethane treatment. Tumors were also dissected from the lung tissue and grinded for FACS analysis to assess myeloid‐derived suppressor cells.

Results

Morphologically, hyperplasia lesions and nodules were similar in the two lungs. However, tumor numbers and sizes (normalized with lung surface area) 20 weeks after urethane treatment were greater in vagotomized (0.068 /mm2 and 21.6 area%) than intact lungs (0.052 /mm2 and 16.0 area%) (n = 13 and n = 8; p < 0.05 and p < 0.01). Similar results were observed at 10 weeks after urethane treatment. In addition, CD11b+Ly6− C++Ly6G− suppressor cells were more in the vagotomized lung than in the intact lung.

Conclusion

Our data support that the vagus nerve may protect the lung from tumor formation by modulating immune cell expression.

Support or Funding Information

Funded by the Pulmonary Fibrosis Foundation
Multipotent Myoepithelial Progenitor Cells Are Born Early During Airway Submucosal Gland Development
Authors: Anderson Preston , Lynch Tom , Engelhardt John
Published: November 2023

Airway submucosal glands (SMGs) are facultative stem cell niches for the surface epithelium, but the phenotype of the SMG‐derived progenitor cells remains unclear. In other organs, glandular myoepithelial cells (MECs) have been proposed to be multipotent progenitors for luminal cells. We sought to determine the developmental phase during which mouse tracheal glandular MECs are born and whether these primordial MECs are progenitors for other cell phenotypes during SMG morphogenesis. To approach this question, we localized two MEC protein markers (α‐smooth muscle actin [αSMA/ACTA2] and smooth muscle myosin heavy chain 11 [SMMHC]) during various stages of SMG development (placode, elongation, branching, and differentiation) and used ACTA2‐CreERT2 transgenic mice to fate map MEC‐derived lineages during SMG morphogenesis. Protein localization ontogeny studies demonstrated that both αSMA‐ and SMMHC‐expressing cells emerged early following placode formation and during the elongation phase of SMG development. Tamoxifeninduction of newborn ACTA2‐CreERT2 mice led to lineage‐tracing of ~50% of αSMA+ MECs at the tips of invading tubules during the elongation phase and these cells contributed to ~25% of αSMA+ MECs in the fully formed gland by 21 days. During the differentiation phase of gland development, ~33% MEC derived cells that were lineage‐marked at birth gave rise to αSMA‐negative glandular cell types by 21 days, including serous, mucous, and duct cells. By contrast, lineage‐traced MECs did not contribute to cell types in the surface airway epithelium. These findings demonstrate that primordial MECs born early during SMG morphogenesis are multipotent progenitors with the capacity to differentiate into other glandular cell types.

Support or Funding Information

This work was supported by NIH grants DK047967 (to J.F.E), the University of Iowa Center for Gene Therapy (DK54759) (to J.F.E), and the Carver Chair in Molecular Medicine (to J.F.E).
Modulation of the lung inflammatory response to ozone by the estrous cycle
Authors: Silveyra Patricia , Cabello Noe , DiAngelo Susan
Published: November 2023

Background

Studies have shown that women are at increased risk of adverse health outcomes from air pollution (e.g. ozone) than men. Emerging data indicate that hormones could play a role in the regulation of lung innate immunity and inflammation in response to environmental challenges, but the molecular mechanisms involved in these responses remain unknown. The goal of this project was to investigate whether fluctuations in circulating hormone levels during the estrous cycle could modulate the inflammatory response to ambient ozone.

Methods

Adult female C57BL/6 mice, in different stages of the estrous cycle, were exposed to ozone (2ppm) or filtered air (FA) for 3 hours (n=6/group). The estrous cycle stage was determined by vaginal smear, and cyclicity was confirmed for at least 3 cycles. Four hours after exposure, lungs were harvested and RNA was extracted using Trizol. The mRNA expression of inflammatory cytokines and chemokines was analyzed by Real Time PCR (mouse genes: Ccl2, Ccl3, Cxcl2, Ccl20, Il1b, Il6, Tnf, and RANTES) using TaqMan probes, and normalized to 18S. Serum levels of LH, estradiol, and progesterone were also measured by ELISA to confirm the estrous cycle stage.

Results

Exposure to ozone significantly increased expression of Cxcl2, Ccl20, and Il6 genes in female mice exposed in all estrous cycle stages, with higher expression levels in proestrus vs. other stages (p<0.05). In contrast, the expression of both Ccl2 and Ccl3 was only increased in mice exposed to ozone at the proestrus stage (p<0.05), but not in animals exposed in diestrus/estrus. Ozone exposure did not affect the expression of Il1a, Tnf or RANTES, when compared to females exposed to FA. Serum hormone levels confirmed proper estrus cycle stage in all animals.

Conclusions

Our results confirmed differences in lung inflammatory gene expression in response to ozone in female mice exposed at different stages of the estrous cycle. Our observations suggest that the negative effects of air pollution in women's lung health may be affected by their hormonal status. Future studies investigating adverse health effects of ozone in women should consider their menstrual cycle phase, and/or circulating hormone levels.

Support or Funding Information

Supported by NIH HL‐133520
Activation of Bitter Taste Receptors Has Distinct Modulatory Effects on Different Lung Afferent Signaling in Rats
Authors: Gu Qihai (David) , Gilbert Carolyn , McLaurin Hugh
Published: November 2023

We have reported recently that activation of bitter taste receptors (T2Rs) in lung nociceptors significantly augments capsaicin‐evoked TRPV1 responses, likely through PLC and PKC intracellular signaling pathway. Using whole‐cell patch‐clamp recordings, the present study was carried out to investigate how T2Rs activation may affect lung sensory neurons responding to different airway stimulations. Three airway irritants that are known to stimulate different pulmonary sensory receptors are selected: zinc, an agonist for TRPA1; low pH or acid, an activator for both acid‐sensing ion channels (ASICs) and TRPV1; and ATP, an agonist for P2X purinoceptors. Our results showed that pretreatment with T2Rs activator chloroquine (0.01, 0.1 and 1 mM, 90 s) concentration‐dependently potentiated zinc (30 μM, 3–16 s)‐evoked TRPA1 currents, and markedly inhibited ATP (0.3 or 1 μM, 4–6 s)‐evoked P2X currents, whereas affected acid (pH 5.5 and pH 6.5, 4–16 s)‐evoked inward currents differently: an inhibition for the slow inactivation ASIC‐like current, and a potentiation for the TRPV1‐like current as well as the fast‐activation and fast‐inactivation ASIC current. Surprisingly, our results also showed that none of the above modulations was completely abolished by the inhibition of PLC, PKC or PKA transduction pathway. In summary, our results demonstrated that T2Rs activation in lung afferents has distinct regulatory effects on various ion channels that are sensitive to different airway irritants.

Support or Funding Information

Supported by funds from Navicent Health Foundation and MUSM
Impact of Gestational Chronic Intermittent Hypoxia and Hypertension on the Serotonergic System and Autoresuscitation in the Developmental Rat
Authors: Harvey Drayton , Leal Angel Carrillo , Li Aihua
Published: November 2023

Sudden infant death syndrome (SIDS) is characterized by the sudden death of a seemingly healthy infant during a sleep period under the age of 12 months. Abnormalities in the medullary serotonin (5HT) system, e.g. decreased tissue levels of 5HT and 5HTT, and increased immature 5HT neurons, have been identified in approximately 70% of infants that died of SIDS. Failed autoresuscitation (inability to recover breathing and heart rate following hypoxic apnea) is considered a possible mechanism of SIDS deaths, and animal studies have shown that 5HT neurons are critical for neonatal mice and rats to survive repeated episodes of severe hypoxia/asphyxia. Gestational chronic intermittent hypoxia (CIH) and preeclampsia both are risk factors for SIDS. In this study we asked whether the stressors, e.g. gestational/prenatal chronic intermittent hypoxia (CIH) and/or hypertension, would increase stress hormone levels, impair the maturity and integrity of medullary 5HT neurons and the ability to autoresuscitate from repetitive asphyxia in developing rat pups. The pregnant dams of normotensive Sprague Dawley (SD) rats and spontaneously hypertensive rats (SHR) were exposed to CIH or room air (RA) from gestation E4‐21, and then the serum corticosterone level (ELISA), the number and integrity of the medullary serotonin neurons (immunostainings), and in vivo autoresuscitation were assessed in the offspring pups at Postnatal (P) days 5–15.

We found 1) SHR‐CIH pups have significantly higher serum corticosterone levels than SHR‐RA and SD‐RA pups at P7 (11,485 vs. 8078 & 8970 pg/mL respectively, P<0.05) and P10 (11,708 vs. 9040 & 6,529 pg/mL respectively, P<0.05). At P15 SD‐CIH, SHR‐CIH and SHR‐RA pups all have significantly higher corticosterone levels than SD‐RA pups; 2) Relative to the SD‐RA pups, SD‐CIH pups have a significantly higher total number of 5HT neurons and primarily due to increased number of Ki67/5HT‐ir positive neurons in the medulla. When compared to SD rats, we noticed that SHR pups appear to have more Ki67/5HT‐ir neurons which contribute to an increase in total number of 5HT neurons in the medulla than SD‐CIH & SD‐RA (P<0.01); and 3) the offspring pups of SHRs have the highest mortality rate during repetitive exposure of anoxia at all ages tested. Our data suggest that both gestational CIH and uncontrolled hypertension may lead to an impaired 5HT system in the offspring pups. The uncontrolled high blood pressure during pregnancy may cause placental insufficiency and contribute to failed autoresuscitation in response to repetitive anoxia in early postnatal life.

Support or Funding Information

Dartmouth UGAR, NIH HD036379, NIH HL28066
Intermittent Hypoxia during Gestation Disrupts Respiratory Rhythm and Neuroplasticity in Newborn Rat Brainstem‐spinal cord Preparations In Vitro
Authors: Johnson Stephen Mark , Watters Jyoti , Randhawa Karanbir
Published: November 2023

Pregnant women with sleep‐disordered breathing experience intermittent hypoxia, especially during late pregnancy. To test whether intermittent hypoxia exposures during pregnancy (G‐IH; gestational intermittent hypoxia) alter breathing in newborn rats, pregnant rats were exposed to intermittent hypoxia (2 min 10.5% oxygen / 2 min normoxia for 8 h) from gestational day 10 to 21 (exposures completed prior to birth). Newborn rats (P0–P3) were anesthetized and their brainstem‐spinal cords isolated under in vitro conditions. Spontaneous respiratory motor bursts were recorded on cervical spinal roots (C4–C5). G‐IH preparations from P0–P1 rats had a lower baseline burst frequency (8.2 +/− 0.6 bursts/min; n=10) compared to normoxic controls (11.0 +/− 0.3 bursts/min; n=21). After 90 min, burst frequency decreased with time nearly 2× times greater in G‐IH preparations compared to controls. In most G‐IH preparations, there were frequent “pauses” in the rhythm, which resembled spontaneous apneas. With respect to respiratory neuroplasticity, dihydroxyflavone (DHF) application (5 micromolar, 9 min; TrkB receptor agonist) induced long‐lasting 19% increase in respiratory motor burst amplitude in control preparations (n=5) after 90 min. However, only 2/4 G‐IH preparations expressed a DHF‐dependent increase in motor burst amplitude. Also, episodic substance‐P application (100 nanomolar, 3 min on / 7 min wash ×3) induced a long‐lasting (60‐min) increase in respiratory burst frequency in control preparations. However, there was no substance‐P induced frequency plasticity in any G‐IH preparations (n=4). These data suggest that gestational intermittent hypoxia disrupts respiratory rhythm generation and reduces the capacity for expressing respiratory neuroplasticity in newborn rats.

Support or Funding Information

NIH Grant: NS085226
Changes in Physiological Variables During Slow‐ and Rapid‐Onset Airway Obstruction in Swine
Authors: Blackburn Megan Bardgett , Nawn Corinne
Published: November 2023

Airway compromise is a significant cause of death in emergency and battlefield settings. While novel airway management devices are available, there is no reproducible animal model of airway obstruction for testing these new product solutions. The goals of this study were to develop a slow‐ and rapid‐onset model of airway obstruction and compare changes in blood gas and cardiovascular parameters. Female swine were anesthetized, intubated, and instrumented for blood sampling and arterial blood pressure and heart rate recordings. Pigs were allowed to breathe spontaneously and a hemostatic clamp on the endotracheal tube was either slowly ratcheted closed over 30 minutes (slow‐onset) or fully clamped immediately (rapid‐onset) until a real‐time pulse oximeter reading of 70% or below was attained. Maximal decreases in blood oxygen saturation (Slow: −66±10% vs Fast: −49±5%) and oxygen partial pressure (Slow: −70±14 mmHg vs Fast: −96±12 mmHg) did not differ (P>0.05) between slow and rapid‐onset obstructions. In contrast, the increase in CO2 partial pressure was significantly greater during slow (23±1 mmHg) vs rapid (13±2 mmHg, P<0.001) obstruction. Surprisingly, mean arterial pressure did not change significantly from baseline during either slow‐(Baseline: 109±8 mmHg vs Obstruction: 112±12 mmHg) or fast‐onset (Baseline: 105±7 mmHg vs Obstruction: 104±8 mmHg) obstruction. Heart rate significantly increased during slow‐onset obstruction (Baseline: 70 ± 8 bpm vs Obstruction: 88±11 bpm, P<0.05) but was unchanged during fast‐onset obstruction (Baseline: 72±7 bpm vs Obstruction: 80±5 bpm). The changes in mean arterial pressure (Slow: 3±9 mmHg vs Fast: −1±5 mmHg) and heart rate (Slow: 19±5 vs Fast: 8±6 mmHg, p=0.09) were not significantly different between groups. In conclusion, our model of slow‐ and fast‐onset airway obstruction induced significant hypoxemia and hypercapnia similar to what is expected during airway compromise and shows promise for use in testing components of airway management devices. Further studies are needed to determine whether obstruction for longer time periods (to induce a greater reduction in real‐time pulse oximetry blood oxygen levels) will cause greater cardiovascular changes.
Effects of cervical spinal cord injury on cough
Authors: Pitts Teresa , Huff Alyssa , Greene Clint , Cheffer Kimberly A , O'Steen Wilbur A , Howland Dena R
Published: November 2023

Action of the diaphragm is necessary for negative force production during breathing and cough. However, the effect of cervical spinal cord injury on action of the diaphragm during cough is not known. It was hypothesized that C2 hemisection would result in a loss of cough excitability and depression of the diaphragm during breathing and cough. Electromyograms (EMGs) of the crural diaphragm, abdominal oblique complex, thyroarytenoid, posterior cricoarytenoid, thyropharyngeus, cricopharyngeus and thyrohyoid muscles were measured along with esophageal and stomach pressures were recorded in spontaneously breathing (non‐paralyzed) anesthetized (sodium pentobarbital) cats. Cough was elicited by mechanical stimulation of the lumen of the intrathoracic trachea. Following acute C2 hemisection there was an ~50% reduction in cough number from 1:1 sec to 1:2 sec; additionally there was a bilateral reduction in crural diaphragm recruitment with a significant increase in upper airway EMG activity. Temporal progression of changes in cough characteristics was captured for up to 2 hours following acute hemisection. Lesion levels were verified post‐mortem and histology was used to assess lesion extent in each animal. Of note the blood pressure and pCO2 remained relatively consistent, and the animals were not placed on a ventilator. We conclude that there is a balance of excitation and inhibition from the spinal cord to the brainstem regulating the excitability of airway protective behaviors and cranial/spinal motor drive during coughing.

Support or Funding Information

Supported by R00‐HL 111215, The Kentucky Spinal Cord and Head injury Trust, The Commonwealth of Kentucky Challenge for Excellence, the Rebecca F Hammond Trust and RCS‐VA RR&D B9249S. The contents of this abstract do not represent the views of the DVA or US government.
In vivo Studies of Brainstem Neurons with Respiratory Rhythm Activity in a Rat Model of Rett Syndrome
Authors: Wu Yang , Cui Ningren , Xing Hao , Zhong Weiwei , Johnson Christopher M. , Jiang Chun
Published: November 2023

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations of the MECP2 gene, show breathing abnormalities that are attributable to the high incidence of sudden death. To understand the underlying mechanisms for the breathing disorders, direct in vivo recordings from brainstem neurons with respiratory rhythm are needed, which is a challenge in mouse RTT models. The newly available rat model may allow such a study. Therefore, we performed electrophysiological recording from individual neurons of the medullary ventral respiratory group (VRG) in decerebrate Mecp2‐null and wild‐type (WT) rats. In both groups of rats, neurons with expiratory rhythm were recorded from the rostral and caudal VRG, and cells with inspiratory rhythm were found in between. Firing frequency of the inspiratory neurons was significantly lower in null rats than in WT rats, while expiratory neurons in both rostral and caudal VRG did not show such a difference. Firing durations of both expiratory and inspiratory neurons were elongated in null rats compared with the WT. The ratio of inspiratory to expiratory firing duration (Ti/Te) was lower in null rats. A large number of cells showed E‐I phase‐spanning in null rats. The null rats had a frequent breathing pattern of apneusis followed by apnea, during which inspiratory neurons showed increased firing frequency and caudal expiratory neurons showed decreased firing frequency, whereas no significant change was observed in rostral expiratory neurons. Although this breathing pattern of apneusis followed by apnea was also seen in WT rats, the firing frequency of either respiratory neurons did not seem to change. These findings suggest that Mecp2 disruption appears to cause a large scale of changes in firing activity and pattern of medullary respiratory neurons consistent with the broad range of breathing abnormalities seen in people with RTT and animal models.

Support or Funding Information

This work was supported by NIH grant R01‐NS‐073875 and International Rett Syndrome Foundation.
The differential effect of systemic inflammation and chronic intermittent hypoxia on the respiratory networks generating Inspiration and Postinspiration.
Authors: Agosto Ibis M , Ramirez Sanja , Garcia Alfredo J , Ramirez Jan‐Marino
Published: November 2023

Novel evidence suggests that the neural control of breathing is controlled by three distinct, yet interacting oscillators. These rhythm‐generating networks have been identified as the Pre‐Bötzinger complex (preBötC), the Post‐inspiratory complex (PiCo) and the parafacial respiratory group (pFRG). Each rhythmic network has a specify function and together they are thought to control the three distinct phases of breathing, inspiration, post‐insipration and active expiration, respectively. The “three‐oscillator hypothesis” is a novel concept, and emerging evidence suggests that each oscillator differentially responds to endogenous and exogenously applied neuromodulators. Yet, little is known whether these networks also respond in a differential manner to clinically relevant conditions such as inflammation or chronic intermittent hypoxia (CIH). In this study we explore how CIH and systemic inflammation elicited by lipopolysaccharide (LPS, 2μg/g) affects two of the oscillators, the PreBötC and PiCo, in young mice (P5‐8). Systemic inflammation elicited by LPS induces a neuroinflammation by indirect pathways resulting in an upregulation of proinflammatory cytokines (IL‐β, IL‐6, TNF‐α), and oxidative stress. IL‐6 and TNF‐a have been shown to alter the CNS by decreasing inhibition/excitation ratio that can lead to a hyper‐excitable state. We extracellularly recorded spontaneous rhythmic population activity in horizontal slices containing preBötC and PiCo 24hrs after an LPS insult. Our data suggest that PiCo becomes hyper excited, while the PreBötC burst frequency remains unchanged to control (saline). This data complements our CIH data, in which young mice that receive CIH from P0‐8, also revealed increased excitability in PiCo, while the preBötC characterized in transverse slices becomes less active and more desynchronized. The decreased activity in the PreBötC, after prolonged CIH, results in irregular bursting patterns and failed transmission to the hypoglossal nucleus, which can contribute to apneas at the level of the hypoglossus. By contrast, the increased excitability in PiCo may contribute to an increased activation of postinspiratory activity following CIH and inflammation. Thus, we hypothesize that inflammation and CIH differentially affects the two phases of breathing, inspiration and postinspiration, by differentially acting on the rhythm generating networks that provide excitatory drive for inspiration and postinspiration, the preBötC and PiCo.

Support or Funding Information

NIH: R01HL126523, P01 HL090554
Modulation of KCNQ Channels is a Potential Mechanism for Controlling Inspiratory Burst Duration
Authors: Revill Ann L , Katzell Alexis , Funk Gregory D
Published: November 2023

The preBötzinger Complex (preBötC) is a key site of inspiratory rhythm generation in mammals and significant progress has been made toward defining the mechanisms underlying inspiratory burst generation. In contrast, the mechanisms that control burst duration have received relatively little attention. Here we test the hypotheses that KCNQ channels help determine inspiratory burst duration and contribute to burst termination. KCNQ channels mediate a slow, voltage‐gated outward K+ current that is active at resting membrane potential and becomes larger with membrane depolarization. This current helps determine resting membrane potential and also acts to stabilize membrane potential. Using rhythmically‐active brainstem spinal cord (BSSC) and rhythmic medullary slice preparations from neonatal rats (postnatal day 0–4), we examined the role of KCNQ in controlling inspiratory behavior by assessing the effects of KCNQ inhibitors and activators on inspiratory burst duration and inspiratory frequency recorded from the C4 ventral nerve root in BSSC preparations and the XII nerve in slices. Bath application of the KCNQ channel blockers XE991 (0.1 μM and 1 μM) and linopirdine (6 μM, BSSC; 3 μM, slice) caused a time‐ and concentration‐dependent increase in burst duration (XE991, 1 μM: BSSC: 1.35 ± 0.05 fold increase, n = 6, p < 0.05; slice: 3.59 ± 0.20 fold increase, n = 5, p < 0.05; linopirdine: BSSC: 1.22 ± 0.04 fold increase, n = 8, p < 0.05 slice: 1.94 ± 0.13 fold increase, n = 6, p < 0.05), but only XE991 caused a decrease in burst frequency (1 μM: 0.52 ± 0.03 of control levels, p < 0.05). Local microinjection of XE991 (10 μM) directly into the preBötC produced very similar effects on burst duration (3.65 ± 0.86 fold increase, n = 3, p < 0.05) and burst frequency (0.59 ± 0.04 of control, p < 0.05). In contrast, potentiation of the KCNQ current via bath application of retigabine (2 μM) caused a significant decrease in inspiratory burst duration (BSSC: 0.62 ± 0.05 times control, n = 5, p < 0.05; slice: 0.72 ± 0.06 times control, n = 8, p < 0.05) and a decrease in burst frequency. Inspiratory burst generation ceased when retigabine concentration was increased to 3 μM. These data suggest that activation of KCNQ channels specifically within the preBötC contributes to inspiratory burst termination and that their modulation may provide a mechanism for controlling inspiratory burst duration.

Funding: CIHR, NSERC, WCHRI, AIHS, CFI.

Support or Funding Information

CIHR, NSERC, WCHRI, AIHS, CFI
Cigarette Smoke Induced Alterations in Bone Microarchitecture: Regional Differences in a Mouse Model
Authors: Calendo Gennaro , Stapor Daniel T , Baker Sandy T , Amin Mamta , Barbe Mary F. , Wolfson Marla R.
Published: November 2023

Background

Oxidative stress from prolonged exposure to tobacco smoke plays a major role in the pathogenic process leading to COPD. An increased respiratory load due to altered lung and airway mechanics increases the demand on respiratory muscles and contributes to difficulties in maintaining body weight. Theoretically, the increased muscle demand, by imposing additional stress on the muscular‐skeletal insertion, may alter bone characteristics of the rib cage and contribute to rib fracture in patients with respiratory disease. There is growing evidence that smoking delays or inhibits vertebral and long bone healing after surgery or trauma. Finally, the incidence of osteoporosis is increased in humans with poor nutrition as well as a history of current smoking, with female gender an additional risk factor in developing osteoporosis. However, direct linkage between CS exposure and bone health has proven elusive due to co‐morbidities and/or therapeutics. Here, we sought to test the hypothesis that CS smoke‐exposure alone, in the absence of glucocorticoid use, will cause alterations in bone structure.

Methods

Female C57BL/6J mice (17–21 g;12–14 weeks) were randomly assigned to either whole body exposure to filtered room air (n=15) or cigarette smoke (CS; n =15, SciReq; In‐Expose; 3R4F University of Kentucky reference cigarettes; ISO 1991 standard exposure; 1 hr/2× daily, 5 days/week; total particulate matter was 100 mg/m3 for the first 3 days then 300 mg/m3 for a total of 6 weeks). Mouse carcasses were imaged with microCT (Skyscan 1176; 9 um). Reconstruction with volume rendering and 3D model creation was performed. Morphometric analyses were performed on cortical bone of the 1st, 6th, and 10th ribs and sternum, and on trabecular bone of the sternum, femur and 6th lumbar vertebrae (LV6).

Results

Cortical bone thickness was significantly (p < 0.05) reduced in the 1st and 10th rib in CS‐exposed relative to RA‐exposed mice, but was unchanged in the sternum and 6th rib. Percent trabecular bone/tissue volume, a reflection of bone mass, was significantly reduced in femurs of CS‐exposed animals (p< 0.05) but comparable in sternum and LV6 between exposure groups.

Conclusions

These data demonstrate in a mouse model, that chronic CS exposure alone results in thinning of the ribs that experience the greatest torque, and reduced trabecular bone mass in the femur. This profile is likely to predispose to fracture and impaired bone healing, consistent with observations in actively smoking humans.

Support or Funding Information

N0014‐10‐0761; N00014‐13‐1‐0742
The Effect of Retrieval Practice After Sleep on Student Learning
Authors: Perry Carolyn , Easteal Ron
Published: November 2023

How educators can improve retention and learning of course material, especially in the anatomical sciences, is a topic of growing concern. It has been shown, both within controlled experimental scenarios and within classroom settings, that information retrieval through repeated practice testing, as opposed to repeated studying, is an efficient way of enhancing information retention and recall. In addition, it has been shown that sleep, a few hours after learning, enhances future retrieval of both declarative and procedural memory. Having previously implemented retrieval practice in third year human anatomy laboratories, we wondered if the inclusion of retrieval practice the morning after students learned new material, would result in an additional benefit to students' retention and recall. Students were to review the new material presented in that day's lecture a few hours before bed and complete a series of questions, on the material reviewed, that they could access and answer online. The next morning, the students were provided with an additional set of questions on the material learned the previous day. They were also provided with feedback that allowed them to compare their responses to the correct answers. With data collection ongoing, comparisons in midterm and final, written and laboratory examinations will be made between students who participated in laboratory retrieval practice only and students who, in addition, participated in retrieval practice after sleep. This will allow us to test whether retrieval practice after sleep enhances learning over and above that seen with regular retrieval practice. As previous studies have tested immediate retrieval practice (at the end of lecture), the inclusion of an initial retrieval test after sleep could have important implications for the timing of the commencement of retrieval practice. In addition, we will be able to test the effects of sleep consolidation alone by comparing students' performance on the quiz taken the same night as the material is reviewed and the quiz taken the next morning after sleep.
Previous Learning Experiences in the Anatomical Sciences
Authors: Taylor Melissa
Published: November 2023

The subject of anatomy is not a pre‐requisite for admission to Indiana University School of Medicine. Nevertheless, many students do take some form of anatomy while an undergraduate, and some also have taken a high school anatomy course. This study examines what kind of experience the first year medical students enrolled in an anatomy course at the Indiana University‐Bloomington School of Medicine have had with the anatomical sciences ‐ this can include high school, undergraduate, graduate, and also informal experience with anatomy. A survey was distributed to first year medical students, which consisted of two different cohorts: students who started their first year in 2015 who were enrolled in a non‐integrated curriculum, and first year students starting in 2016 who were enrolled in an integrated curriculum. Results were examined for trends and comparisons between the two cohorts. It was found that the 2016 cohort had more experience with anatomy than the 2015 group did. The majority of students from both cohorts who had no prior experience with anatomy said they believed they were at a disadvantage for not having taken anatomy before medical school. This data is useful to see what kind of instruction students have had before. It can also possibly be used as a persuasive argument for the inclusion of an anatomy pre‐requisite for acceptance into medical school.