bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythromycin and Rho‐Kinase Activity: Another Pathway Responsible for its Gastrointestinal Motility‐Enhancing Effect
Authors: Belik Jaques , Welsh Chris , Shifrin Yulia , Pan Jingyi
Published: June 2021

Erythromycin enhances gastrointestinal motility and is used to treat feeding intolerance in neonates. Erythromycin is thought to enhance gastric emptying through activation of motilin receptors, but the mechanism is not completely understood and motilin does not induce gastric contraction in newborn rabbits. Hypothesizing that erythromycin promotes gastric emptying via motilin receptor independent pathways, we evaluated its effect on gastric and pyloric tissue. Adult rabbit and newborn rat tissue were studied, since motilin receptors are present in the former, but not in rodents. The in vivo acute erythromycin effect on newborn rodent gastric emptying was measured via ultrasound. Gastrointestinal muscle tone is directly dependent on rho‐kinase II (ROCK) activity, thus the phosphorylated MYPT/total MYPT expression ratio was utilized as a surrogate marker of its activity and measured in fundic and pyloric tissue following carbachol exposure in the absence or presence of erythromycin.

Results

Erythromycin promoted a similar pattern of ROCK activity changes in both species. Erythromycin exposure significantly increased fundic, but reduced pyloric tissue ROCK activity, thus favoring enhanced gastric emptying. Erythromycin ip injection in newborn rodents increased the 3 hour post‐feed stomach milk clearance from 60±2 % to 76±3 % (P<0.01).

Conclusion

Erythromycin enhances gastrointestinal motility via rho‐kinase pathway and this explains its effects in newborn rodents where the gastrointestinal tract motilin receptors are absent.

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Hypoxia, but not Hypercapnia Evokes the Arousal Response During Sleep in Mice
Authors: Ramirez Sanja , Rubens Daniel , Pillai Aiswarya Pillai , Allen Travis , Ramirez Jan
Published: June 2021

One of the most established risk factor for SIDS is an infant sleeping in prone position. This position exposes the child to hypoxia and hypercapnia, which should normally lead to an arousal response that wakes up the child. It is thought that the failure to arouse is an important first step in the events leading to the death of the child. Here we explored the differential roles of hypoxia and hypercapnia in the generation of the arousal response during natural sleep using CD‐1 mice as an animal model. Mice were observed in a full‐body plethysmograph during natural sleep while exposing the animals to hypoxic (100% N2) and hypercapnic stimuli (gas composition of 21% O2 5% CO2 balanced N2). We demonstrate that the hypoxic stimuli evoked a whole body arousal response in all mice within less than 15 seconds. The hypercapnic stimulus reliably evoked a respiratory response consisting of an increase in breathing frequency and tidal volume. However, mice lacked a whole body arousal response and did not wake up even if the hypercapnic stimulus was continued for 5 minutes. We conclude that in these mice the arousal response is triggered by hypoxia and not hypercapnia. Moreover, our study suggests that the presence of a hypercapnic ventilatory response is not automatically indicative for arousal. Ongoing studies explore the interaction between the hypercapnic and hypoxic response and the failed arousal seen in mice with inner ear defect. Our study has important implications in understanding the mechanisms that lead to failed arousal in SIDS.
Differential Effects of Systemic versus Intratracheal Lipopolysaccharide (LPS) Administration on Ventilatory Behavior in Spontaneously Breathing Adult C57BL/6 Male Mice
Authors: Ahmad Mohammed , Hyder Muzammil , Rice Keeley , Conyers Jonathan , Solomon Irene
Published: June 2021

Both intraperitoneal (ip; systemic) and intratracheal (IT) administration of the bacterial endotoxin LPS have been used to elicit neuroinflammation. While both methods are reported to induce time‐dependent changes in expression levels of pro‐inflammatory cytokines in the CNS, their effects on basal ventilatory activity remain to be characterized. Here, we report the ventilatory behavior (recorded using whole body plethysmography) before and after administration of LPS delivered either ip (5 mg/kg) or IT (0.5 mg/kg) in unanesthetized spontaneously breathing adult C57BL/6 male mice. Before LPS, ventilatory activity consisted of a fairly regular pattern of breathing, which included periodic short duration apneic pauses (as previously described in this strain) and occasional sighs. Both ip and IT LPS altered ventilatory activity; however, the effects were not identical. IP LPS (1) robustly increased breathing frequency and progressively decreased tidal volume and (2) reduced or eliminated the incidence of both apneic pauses and sighs. IT LPS (1) increased breathing frequency and progressively decreased tidal volume, but to a lesser extent than ip, (2) produced periods of dysrhythmic breathing, (3) prolonged the durations of the apneic pauses; and (4) increased the incidence of sighs. These findings indicate that while both methods of administration alter ventilatory activity, the resulting ventilatory patterns are markedly different. We suggest that differences in the extent of ip versus IT LPS‐induced neuroinflammatory changes in respiratory‐related brainstem and spinal cord regions mediate, at least in part, these different ventilatory behaviors.
Time Course of Altered Ventilatory Activity Evoked by Systemic Lipopolysaccharide (LPS) Administration in Spontaneously Breathing Adult C57BL/6 Male Mice
Authors: Hyder Muzammil , Conyers Jonathan , Ahmad Mohammed , Rice Keeley , Solomon Irene
Published: June 2021

Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) is commonly used to study inflammation‐associated behavioral and biochemical changes in rodents that are attributed to neuroinflammation. Little is known, however, about the time course of alterations in ventilatory activity following systemic LPS administration. Therefore, the present study was designed to evaluate the effects of a single bolus of high dose LPS (5 mg/kg, ip) or vehicle (saline) on basal ventilatory behavior (recorded using whole body plethysmography) in unanesthetized spontaneously breathing adult C57BL/6 male mice at 2‐, 6‐, and 24‐hr post administration. Before LPS, ventilatory activity was characterized by a regular pattern of breathing that included spontaneous apneic pauses (which have been previously described in this strain) and periodic sighs. A similar ventilatory pattern was seen following saline administration. After LPS, changes in ventilatory activity were noted as early as 2‐hr, with the primary changes consisting of: (1) an increase in breathing frequency of up to 50% above pre‐LPS levels at 2‐hr post‐LPS, after which a progressive attenuation of the frequency increase to 蠄25% was noted; (2) a decrease in the incidence of apneic pauses and sighs by 2‐hr post‐LPS, after which both ventilatory behaviors were further reduced or eliminated; and (3) a progressive decrease in tidal volume. These findings indicate that systemic LPS administration produces time‐dependent modulation of ventilatory activity, which we propose results from the time‐dependent LPS‐induced changes in cytokine expression in respiratory‐related brainstem and spinal cord regions.
Intratracheal Lipopolysaccharide (LPS) Administration Attenuates Acute Hypoxic Ventilatory Response (HVR) in Spontaneously Breathing Adult C57BL/6 Male Mice
Authors: Conyers Jonathan , Rice Keeley , Ahmad Mohammed , Hyder Muzammil , Solomon Irene
Published: June 2021

Intratracheal (IT) administration of lipopolysaccharide (LPS) not only produces acute lung injury but also induces time‐dependent changes in expression of pro‐inflammatory cytokines in respiratory‐related brainstem regions. The influence of IT LPS on control of breathing, including the acute hypoxic ventilatory response (HVR), has received little attention. Therefore, we examined the effects of IT LPS administration (0.5 mg/kg) at 2‐, 6‐, and 24‐hr post‐LPS on the HVR (10% O2 for 60‐90 s; recorded using whole body plethysmography) and recovery from acute hypoxia in unanesthetized spontaneously breathing adult C57BL/6 male mice. Before LPS, the HVR was characterized by a robust increase in breathing frequency (fb) and tidal volume (VT). IT LPS not only altered the basal breathing pattern but also markedly blunted the hypoxia‐induced increase in fb and produced a slight attenuation of the increase in VT. Specifically, the hypoxia‐induced increase in fb was reduced from a range of 100‐178% increase pre‐LPS to 21‐36% increase post‐LPS; the hypoxia‐induced increase in VT was reduced from a range of 135‐177% increase pre‐LPS to 100‐140% increase post‐LPS. The effect on fb was greatest at 6‐hr post‐LPS while that on VT was greatest at 24‐hr post‐LPS. During recovery from hypoxia, before LPS, there was an increase in the incidence of sighs and spontaneous apneic pauses; however, after LPS, these behaviors were also attenuated. These findings indicate that IT LPS alters the acute HVR and its recovery in a time‐dependent manner, with a dominant effect being related to frequency modulation.
Confronting the Effects of 0.9% Saline Administration on Breathing
Authors: Glausen Taylor , Cunningham Caitlin , DeRuisseau Lara
Published: June 2021

Saline (0.9%) is regularly used in hospitals and research settings as a resuscitation solution or as a conduit to administer compounds to patients or subjects. Preliminary data in our laboratory suggested dissimilar ventilation following short‐term i.p. saline administration (n=4) compared to non‐injected (n=6) mice (frequency: 111±15 vs. 126±4 breaths/min; tidal volume (TV): 0.31±0.02 vs. 0.35±0.01 mL/breath; minute ventilation (MV): 35.1±5.9 vs 43.9±1.4 mL/min). In addition, previous reports suggest that the addition of 0.9% saline could induce changes in pH, possibly altering breathing. We tested the hypothesis that ventilation would be higher in mice prior to saline administration compared to after saline administration. Utilizing unrestrained barometric plethysmography, we tested 18 week old CD‐1 male mice prior to intervention and following 7 days of 0.9% saline (i.p., 10mL/kg body mass) during exposure to room air. Values are reported as MEAN ± SEM; p<0.05; n=4. There were no differences when comparing PRE vs. POST saline in frequency (137±9 vs. 142±10 breaths/min, TV (0.46±.01 vs. 0.52±.02 mL/breath), or MV (62.5±3.8 vs. 72.5±7.7 mL/min). Conscious blood gas sampling showed no differences between 7 days of saline administration (pH: 7.46±0.00; PaCO2: 29.3±2.3) and 1 day of washout (pH: 7.39±0.03; PaCO2: 39.1±4.6). Our preliminary findings indicate no impact of saline administration on the pattern of breathing during exposure to room air. Funding: Le Moyne College Student Research Committee and McDevitt Center Research Fellowship in Natural Science (TG).
Unilateral Denervation of the Diaphragm Muscle Increases Central Drive Only During Ventilatory Behaviors
Authors: Gill Luther , Mantilla Carlos , Sieck Gary
Published: June 2021

Compensatory respiratory plasticity is critical to preserve breathing capacity after phrenic denervation (DNV), when the contribution of the ipsilateral phrenic motor neuron (PhrMn) pool is abolished and the force generating capacity of the diaphragm muscle (DIAm) is compromised. In rats, ventilation is maintained after DNV, and this is most likely mediated by an increase in central drive to the contralateral PhrMn pool. The extent of central drive to PhrMns was estimated by measuring the root mean square (RMS) of DIAm EMG at 75ms (RMS75) after the onset of activity. Transdiaphragmatic pressure (Pdi) was measured to estimate DIAm force. We hypothesized that after unilateral DNV central drive to the contralateral PhrMn pool increases across motor behaviors. Immediately following DNV, there was a significant increase in RMS75 during ventilatory behaviors (eupnea, and hypoxia‐hypercapnia). RMS75 did not change during non‐ventilatory behaviors (airway occlusion). These results indicate that after DNV there is: 1) an increase in central drive to the contralateral PhrMn pool during ventilatory behaviors such that Pdi was maintained; 2) central drive to the contralateral PhrMn pool was unchanged during more forceful (greater than ~50% Pdimax) non‐ventilatory behaviors, suggesting that central drive was near maximum. These results highlight the compensatory plasticity in the neural control of the DIAm and the importance of measuring multiple motor behaviors.
Coordination of the Respiratory Muscles in Hypercapnic and Hypoxic Environments
Authors: Jenkin Sarah , Milsom William
Published: June 2021

Breathing is dependent on the coordination of respiratory muscles. The timing and contribution of the respiratory muscles determine the balance between breathing frequency and tidal volume. At rest, the mammalian breathing cycle has three phases: active inspiration (I), expiratory braking (E1) and an expiratory pause (E2). With elevated respiratory drive, expiratory muscles may contract to produce active expiration. This study investigated how respiratory muscles are recruited under hypercapnic and hypoxic conditions. These stimuli modify the breathing response by primarily increasing tidal volume or breathing frequency, respectively. EMG electrodes were placed in the diaphragm, intercostal and abdominal muscles. Anesthetized and unanesthetized rats were then exposed to progressive hypercapnia and hypoxia. Progressive hypercapnia (0% to 10% CO2) induced active expiration at the end of the breathing cycle (E3 phase) at 8% and 10% inspired CO2 in unanesthetized rats. The placement in E3 correlated with an increase in tidal volume. Active expiration was not observed in anesthetized rats, even at the highest level of CO2. Progressive hypoxia (21% to 9% O2) never induced active expiration, in either unanesthetized or anesthetized rats indicating that passive expiration was sufficient even at the highest breathing frequencies. This further suggests that the control mechanisms that coordinate the respiratory muscles during hypercapnia and hypoxia are different and dependent on higher brain centers. Supported by the NSERC of Canada.
Na+ transport by small airway surface epithelia
Authors: Grishchenko Nikolay , Luan Xiaojie , Ianovski Juan
Published: June 2021

Background

Maintaining an adequate level of Airway Surface Liquid (ASL) is crucial for the normal functioning of the immune system of the lungs. A balance between the secretion and the absorption of ions and fluid by the airways surface epithelia (ASE) ensures proper amount of ASL for mucociliar clearance. ASE expresses Epithelial Na+ Channel (ENaC) and the Cl‐ channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Currently, there are two opposing hypotheses to explain how the interplay between CFTR and ENaC activity regulate ASL height. One theory proposes that each surface epithelial cell expresses both CFTR and ENaC and is able to secrete or reabsorb fluid and ions. A recent hypothesis proposes that CFTR and ENaC are expressed in different groups of cells. Cells within the pleats of ASE express CFTR and are mainly secretory while the cells of the folds express ENaC and are principally absorptive.

Question

Are the cells of ASE able to transport ions in both directions?

Methods

To detect transepithelial Na+ flux we used Scanning Ion Selective Techniquethe, which measures ion concentration gradient generated by the epithelia using ion selective microelectrodes.

We investigated the ion transport in the folds of the swine distal airways under normal Kreb's solution and in the presence of CFTR and ENaC modulators.

Results

ASE showed spontaneous basal Na+ transport from the lumen into the tissue. Adding the sodium azide blocked the basal secretion. Stimulation with forskolin or thapsigargin triggered rapid and reversal Na+ transport and was blocked by treatment with the CFTR blocker.

Conclusions

The epithelial cells of the fold regions of ASE are able to secret and absorb Na+. Our results allow us to reject the hypothesis of the monodirectional Na+ transportation in the surface epithelia.
Effects of Endoplasmic Reticulum (ER) Stress on Epithelial Injury and Fibrosis in Alveolar Epithelial Type II Cell (AT2)‐Specific Grp78 Knockout Mice
Authors: Zhou Beiyun , Flodby Per , Wang Hongjun , Liu Yixin , Minoo Parviz , Crandall Edward , Lee Amy , Borok Zea
Published: June 2021

To study the causal relationship between unfolded protein response (UPR) activation/ER stress and pulmonary fibrosis, we investigated epithelial injury and fibrosis in Grp78‐CKO1 mice (generated by crossing SFTPC‐rtTA, tetO‐cre and Grp78flox/flox mice) with AT2‐specific knockout (KO) of Grp78. Doxycycline (Dox)‐induced KO of the Grp78 allele resulted in decreased Grp78 mRNA by ~50% in AT2 cells and increased mRNA expression of ER stress markers (i.e., Grp94 (~16‐fold), Atf‐6 (~40‐fold), calreticulin (Crt, ~18‐fold) and CHOP (~2.7‐fold) in whole lung. Despite increased vimentin and collagen expression, Grp78‐CKO1 lungs did not display overt fibrosis, although Sircol assay demonstrated augmentation (~30%) of bleomycin‐induced collagen deposition. Since Grp78 was reduced only 50% in Grp78‐CKO1 mice, Grp78‐CKO2 mice were generated by crossing SftpccreERT2/creERT2 and Grp78flox/flox mice. Grp78 expression in AT2 cells was reduced by >80% in tamoxifen treated mice, with increased mRNA expression of Grp94 (~8‐fold), Atf‐6 (~4‐fold), Crt (~5‐fold) and especially high induction of CHOP (~16‐fold). Hematoxylin/eosin and trichrome staining demonstrated overt fibrosis in lungs of Grp78‐CKO2 mice. These results indicate that 1) ER stress‐mediated epithelial cell injury plays an important role in fibrogenesis and 2) development of fibrosis may be dependent on the degree of ER stress and activation of pro‐apoptotic UPR signaling.
Maternal Tobacco Smoke Exposure Alters Elastin Splicing at Exon 7 and 34 in the Lung of Rat Pups
Authors: Sainz Anthony , Zou Changjiang , Zhao James , Wang Yan , Albertine Kurt , Joss‐Moore Lisa
Published: June 2021

Maternal tobacco smoke (MTS) exposure reduces offspring lung function in humans and animal models. We showed that MTS increases static lung compliance in female, but not male, rat pups. Lung function depends on expression of alternatively spliced elastin transcripts. Using RNA‐seq, we identified alternative splicing at exons 7 and 34 of elastin transcripts in the immature rat lung. We hypothesized that levels of exon 7‐ and 34‐containing elastin transcripts would vary with lung development in male and female rat lung, and with MTS exposure, in female rat lung.

Pregnant rats were exposed to tobacco smoke (MTS) or room air (Control) and pups killed at term birth (d0‐immature lung), or cross‐fostered to a control dam and killed at day of life 21 (d21‐mature lung). We measured elastin mRNA transcripts that contained exon 7 (E+7) and exon 34 (E+34). (n=5/group).

Results: Between d0 and d21, lung E+7 decreased in control female (46±15%*) and male (61±14%*) rats, while E+34 increased in female (140±28%*) and was unchanged in male rats. Compared to age‐and sex‐matched controls, in females, MTS decreased E+7 at d0 (16±2%*), and increased E+7 at d21 (589±176%*). Similarly, in female pups, MTS decreased E+34 at d0 (25±16%*), and increased E+34 at d21 (200±51%*). In male lung, MTS did not affect levels of E+7 at d0, but increased E+7 at d21 (462±85%*). MTS did not affect E+34 in males. *p<0.05

Conclusions

Exon 7‐ and 34‐containing elastin transcripts in rat lung are developmentally regulated and sex‐specifically altered by MTS. We speculate that MTS‐induced differences in elastin alternative splicing during development may alter lung function and structure through disruption of the timing and integrity of elastic fiber assembly in the rat.

*Funding: APS and NHLBI; 1 R25 HL115473‐01
Characterization of Noribogaine at nAChRs and Effect on Nicotine Self‐Administration in Rats
Authors: Maillet Emeline , Qing Chang , Milon Nicolas , Hanania Taleen , Heghinian Mari , Garamszegi Nandor , Mash Deborah
Published: June 2021

Noribogaine, a polypharmacological drug with activities at opioid receptors, ionotropic nicotinic receptors, and serotonin reuptake transporters, is being investigated for treatment of substance abuse‐related disorders. In this study, noribogaine molecular activity at neuronal nicotinic acetylcholine receptors (nAChRs) was characterized. Noribogaine inhibited α3β4, α7 nAChRs, and endogenously expressed habenula‐type α3‐(α5, β2, β4) nAChRs populations. Anti‐addictive compounds ibogaine, 18MC‐ a synthetic derivative of coronaridine, and mecamylamine were tested in parallel. Then, noribogaine was tested for use as a treatment for nicotine dependence in a nicotine self‐administration study in rats in comparison to varenicline, an approved medication for smoking cessation. After initial food pellet training, followed by 26 sessions of nicotine self‐administration training, the rats were administered noribogaine (12.5, 25 or 50 mg/kg p.o.), noribogaine vehicle, varenicline or saline using a within‐subject design with a latin square test schedule. Noribogaine dose‐dependently decreased nicotine self‐administration by up to 64% of saline treated rats levels and was equi‐effective to varenicline. Noribogaine pharmacological profiling at the nAChRs receptor population and its ability to dose‐dependently attenuate drug‐taking behavior for nicotine supports future studies to assess its potential usefulness in multiple therapeutic arenas, including smoking cessation, substance abuse related disorders, and anxiety related disorders.
The Discriminative Stimulus Effects of Nicotine, Epibatidine, and Varenicline in Mice: Involvement of β2 Containing Nicotinic Acetylcholine Receptor Subtypes
Authors: Moura Fernando , McMahon Lance
Published: June 2021

Nicotine is a high efficacy β2 containing nicotinic acetylcholine receptor (β2 nAChR) agonist, whereas varenicline has low efficacy at β2 nAChRs. To better understand the receptor pharmacology of varenicline, male C57BL/6J mice (n=8) were trained to discriminate varenicline (3.2 mg/kg s.c.) under an FR10 schedule of food presentation. Separate groups of mice (n=8 per group) were trained to discriminate nicotine (1 mg/kg base weight s.c.) or the β2 nAChR agonist epibatidine (0.0032 mg/kg s.c.). Mice trained with nicotine and epibatidine satisfied the test criteria after a mean of 87 and 89 sessions, respectively, whereas varenicline‐trained mice required significantly more (i.e., 113) sessions. Varenicline was less effective than nicotine and epibatidine in maintaining stimulus control as evidenced by the number of training sessions required between tests. The nicotine and epibatidine dose‐effect functions did not vary as a function of training drug; the functions were steep and characterized by low inter‐subject variability. In contrast, the varenicline dose‐effect functions were shallow and highly variable among mice. Varenicline produced a maximum 63% and 61% drug‐appropriate responding in the nicotine and epibatidine discriminations, respectively. The nAChR antagonist mecamylamine (3.2 mg/kg) antagonized the effects of nicotine, epibatidine, and varenicline. In contrast, the β2 antagonist DHβE (3.2 mg/kg) antagonized the effects of nicotine and epibatidine, but not varenicline. These results suggest that the discriminative stimulus effects of varenicline are not as robust as those of nicotine and epibatidine and are not mediated by β2 nAChRs.

*Supported by USPHS grant DA25267
Characterization of a Nicotine Discriminative Stimulus in Rhesus Monkeys
Authors: Moerke Megan , McMahon Lance
Published: June 2021

In general, the discriminative stimulus effects of drugs are examined following s.c. or i.m. administration; this may not be predictive of the effects of drugs that are abused by other means. Nicotine is primarily inhaled in the form of cigarette smoke. Here, two drug discrimination assays were used to compare the effects of nicotine administered i.v. or s.c. in rhesus monkeys responding under an FR5 schedule of stimulus shock termination. In monkeys discriminating 1.78 mg/kg nicotine s.c. (n=5), the ED50 of nicotine was 0.41 mg/kg. In monkeys discriminating 0.032 mg/kg nicotine i.v. (n=4), the ED50 of nicotine was 0.0067 mg/kg. In 3 of these monkeys, a dose of 3.2 mg/kg nicotine s.c. was required to substitute for the i.v. discriminative stimulus; up to 5.6 mg/kg nicotine did not substitute in the fourth monkey. The nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine antagonized the discriminative stimulus effects of nicotine in both discriminations; 1 mg/kg mecamylamine produced rightward shifts of the nicotine dose‐effect function of 3.2‐fold in the s.c. discrimination and a minimum 15‐fold in the i.v. discrimination. A dose of 3.2 mg/kg of the β2 subtype‐selective nAChR antagonist dihydro‐β‐erythroidine (DHβE) did not significantly antagonize the discriminative stimulus effects of nicotine in the s.c. discrimination. However, when combined with the training dose (0.032 mg/kg) of nicotine in the i.v. discrimination, DHβE dose‐dependently decreased discriminative stimulus effects (ED50=0.17 mg/kg). These results indicate there is a critical difference in the involvement of the β2 subtype of nAChR between the two discriminations that cannot be explained by the difference in training dose. Supported by USPHS Grant DA25267.
An Altered Pattern of Neurotransmitter Receptor Expression Emerges during Adolescent Alcohol Consumption
Authors: Rhoads Dennis , Bolewska Patrycja , Hodges Megan , Martin Bryan , Mauterer Madelyn , Orlando Krystal
Published: June 2021

Adolescent rats develop a severe alcohol withdrawal syndrome when fed alcohol as part of a liquid diet. The present study sought to correlate quantitative changes in neurotransmitter receptors with the severity of alcohol withdrawal symptoms. Membranes were isolated from brain fractions from rats fed an alcohol‐containing liquid diet beginning at P28 and continuing on into the adolescent period. Membranes were probed for specific receptors by Western blotting. During the period of up to 10 days, excitatory glutamate receptors (both AMPA and NMDA subtypes) increase significantly. This up regulation of glutamate receptors coincides with a time when the primary withdrawal behavior is anxiety‐like. By 14 days, adenosine A1 receptors decline significantly reaching a level 50% of that of pair‐fed controls. There are no corresponding changes in adenosine A2a receptors. Down regulation of A1 coincides with a time when 90% of the rats experience convulsions upon alcohol withdrawal. A detergent resistant (raft) fraction was isolated from brain membranes to determine which of these receptors may be located in lipid rafts. Initial analyses indicate all 3 receptors of interest are at least partially localized to lipid rafts. We conclude that the most severe alcohol withdrawal symptoms may occur in adolescents when up regulation of excitatory glutamate receptors coincides with down regulation of A1 receptors and thus with loss of the ‘brakes’ adenosine might normally apply to the glutamate system. Further studies will explore the potential role of lipid raft dynamics in these changes.
Sex‐ and Drinking History Dysregulate ω‐3 and ω‐6 Pathways during the Onset of Liver injury in Very Heavy Drinking Alcohol Dependents
Authors: Song Ming , Vatsalya Vatsalya , Schwandt Melanie , Barve Shirish , Cave Matthew , George David , McClain Craig
Published: June 2021

Background/Aims

Heavy alcohol drinking causes liver injury. In this study we aim to study the relation between gender, drinking history and fatty acids and the severity of liver injury.

Methods

114 heavy drinking alcohol dependent (AD) patients in the 21–65 years old were recruited, including 74 males and 40 females. Patients were divided into two groups: non‐elevated liver‐injury (NELI) group with ALT蠄 40IU/L, and elevated liver‐injury (ELI) group with ALT >40IU/L. Chem20, ω‐3 and ω‐6 fatty acids were evaluated.

Results

In ELI group, 40 are males, 16 are females. Females showed more severe liver injury than males, as demonstrated by markedly higher serum AST and LDH. Drinking history measurement showed significant predictability for the fatty acids involved in the pro‐inflammatory and protective pathways. Most of the fatty acids in ω‐3 pathway were increased in males, but not in females in ELI group compared to those in NELI group. ω‐3 and ω‐6 pathway regulation showed differences in response with drinking history that was closely associated with its unique markers.

Conclusions

Variability in response to pro‐inflammatory and protective mechanism of fatty acid pathways represent susceptibility in the immune response and liver injury in patients with heavy alcohol drinking that may require specific medical management.
The Role of Voltage‐Operated Calcium Channels in Astrocyte Reactivity
Authors: Smith Jessica , Paez Pablo
Published: June 2021

Reactive astrogliosis is a universal response of astrocytes to brain injuries and diseases as diverse as trauma, infection, neurodegeneration, and ischemia. Reactive astrocytes undergo dramatic morphological changes (Wilhelmsson et al., 2006), and various alterations in gene expression have been observed (Sofroniew, 2009). It has long been debated whether reactive astrocytes are harmful or beneficial. In the past few years, both types of properties have been observed. Previous studies have shown that the plasma membranes of astrocytes depolarize when treated with high levels of potassium and this in turn activates voltage‐operated calcium channels. What we are looking to find out more about is the role that these channels play in astrocyte reactivity. We hypothesize that incubating primary astrocyte cultures with high levels of potassium will cause astrocytes to become reactive and that blocking the voltage‐operated calcium channels of these cells will inhibit their ability to become reactive. Our experiments involved incubating cell cultures for three days with different levels of potassium and performing immunocytochemistry with GFAP and S100 antibodies to determine the amount of reactivity. We found that high levels of potassium caused the astrocytes to become reactive and through the use of L‐type voltage‐operated calcium channel blockers Verapamil and Nifedipine, we were able to conclude that these channels are necessary for reactivity under these conditions. In summary, our results suggest that voltage‐operated calcium channels may play a fundamental role in the induction of reactive astrocytes, and indicate that the inhibition of these calcium channels may be an effective way to prevent astrocyte activation.
GPSM3 as a Therapeutic Target for Rheumatoid Arthritis
Authors: Gall Bryan , Setola Vincent , Watkins Colleen , Siderovski David
Published: June 2021

ABSTRACT: Rheumatoid arthritis (RA) is mediated by dysregulation of immune‐system cells. G Protein Signaling Modulator‐3 (GPSM3) is a GoLoco motif protein with an expression profile restricted to leukocytes and the greatest expression seen in monocytes and neutrophils ‐‐ two immune‐system cell lineages paramount to RA pathogenesis. In a collagen autoantibody‐induced model of arthritis, we showed that GPSM3 deficiency protects mice against inflammatory sequelae. GPSM3‐deficient monocytes exhibit disrupted chemotaxis ex vivo, suggesting a role for GPSM3 in cell migration during acute inflammatory arthritis. Notably, human polymorphisms in GPSM3 are associated with a decreased likelihood of developing RA. Given these data, we speculate that the GPSM3 SNPs result in decreased GPSM3 expression/function. To test this hypothesis, we genotyped 50 healthy volunteers, from whom we obtained buccal swabs and whole blood specimens. The SNP minor allele frequency in these volunteers was 0.3 (n = 50). qRT‐PCR analyses of whole blood‐isolated RNA from volunteers heterozygous for the GPSM3 SNPs showed a 32% decrease (p < 0.05) in GPSM3 transcript abundance compared to volunteers without the minor SNP allele (n = 5), suggesting that the minor allele associates with a decrease in GPSM3 expression. From these data, we conclude that GPSM3 SNPs could result in decreased transcript abundance in vivo, which may mimic the ‘protective’ phenotype observed in GPSM3‐deficient mice. Similar studies are underway with RA patients and demographically matched volunteers to examine whether GPSM3 SNP genotype affects (1) transcript levels, (2) protein immunoreactivity, and (3) disease severity in RA patients.
Effects Of Aqueous Extracts Of The Leaves Of Millettia Aboensis On The Liver Of Wistar Albino Rats
Authors: Onyegeme‐Okerenta Blessing
Published: June 2021

The effect of aqueous extracts of the leaves of Millitia aboensis on the liver of Wistar albino rats was investigated. The experimental animals was divided into six groups (groups 1‐6) based on their body weight. Groups 1 ‐ 5 were administered with 1000, 2000, 3000, 4000 and 5000 mg/kg body weight of the extract respectively while group 6 (positive control) was given normal feed and water. The administration of the extract was for 7days and thereafter, the rats were sacrificed and the effects of the plant extract on the liver investigated. Liver function tests and histological examinations of the liver were carried out. Results for liver enzyme assay showed that aspartate aminotransferase (AST) activity significantly increased (p<0.05) in the different groups as compared to the control group (200±4.06 IU/L). It was observed that enzyme activity increased with increase in the of the extract concentration. Activities of alanine aminotransferase (ALT) for group 1 to group 5 ranged from 49.9±0.64 IU/L to 80.2±0.71 IU/L. For alkaline phosphatase (ALP), activities of the enzyme for group 1 to group 5 ranged from 150.5±9.92 to 483±10.82. The results of this study suggest that aqueous leaf extract of M. aboensis was hepatotoxic to Wistar albino rats, and toxicity was dose dependant
Atropine‐mydriasis NO‐system Dependent, L‐NAME‐miosis, L‐arginine‐miosis, and Counteraction by Stable Gastric Pentadecapeptide BPC 157, in Living Rats
Authors: Kokot Antonio , Zlatar Mirna , Stupnisek Mirjana , Drmic Domagoj , Radic Radivoje , Seiwerth Sven , Sikiric Predrag
Published: June 2021

In living rats, atropine‐mydriasis would depend on NO‐related mechanisms in a particular way: both L‐NAME, a NOS‐blocker and L‐arginine, a NOS‐substrate wouldexhibit a miotic effect, not yet described; the
atropine‐mydriasis and L‐NAME‐ and L‐arginine‐miosis, could consequently be counteracted by BPC 157 due to its interactions with the NO‐system and sphincter function. We administraded BPC 157 (10µg, 10ng, 10pg/kg), L‐NAME (5mg/kg), and/or L‐arginine (100mg/kg) alone/together intraperitoneally in healthy rats (miosis‐studies) or at 30 min after 1% atropine/2drops/eye (mydriasis‐studies). BPC 157 does not alter normal pupil diameter while miosis occured promptly after both L‐NAME and L‐arginine, lasting for more than 3h, when they were given alone. When L‐NAME+L‐arginine were given together, this miosis was shorter, suggesting that their separate miotic effects are competitive and may antagonize each other. These L‐NAME‐/L‐arginine‐effects were both shortened in rats that received BPC 157. In case of maximal atropine‐mydriasis all of the agents, BPC 157, L‐NAME and L‐arginine, induce a counteraction, commencing sooner in BPC 157‐rats, but all of the agents lead to completely blunted atropine‐mydriasis returning to regular pupil size hours sooner than in atropine‐rats which received saline. Concluding, BPC 157 may counteracted those effects due to its interactions with the NO‐system and sphincter function. Supported by Grant 108‐1083570‐3635, Croatia.
Effects of Palmitoylethanolamide and Silymarin Combination Therapy in an Animal Model of Kidney Ischemia and Reperfusion
Authors: Bruschetta Giuseppe , Impellizzeri Daniela , Siracusa Rosalba , Di Paola Rosanna , Paterniti Irene , Prosdocimi Marco , Esposito Emanuela , Cuzzocrea Salvatore
Published: June 2021

Silymarin, a poliphenolic compound, has multiple pharmacological activities including antioxidant and anti‐inflammatory effects. Palmitoylethanolamide (PEA) has been extensively studied and its capability to reduce inflammatory processes and ischemia/reperfusion (I/R) damage in various organs is well established. The aim of this study was to investigate the efficacy and safety of silymarin combination with PEA in a mouse model of renal I/R. Mice were subjected to bilateral renal artery occlusion (30 min) and reperfusion (6 h) and received intraperitoneally silymarin (100, 30 and 10 mg/kg); PEA (1 mg/kg); PEA (1mg/kg)+ silymarin (10mg/kg) as a combination therapy 15 min before release of clamps. The treatment with silymarin reduced kidney dysfunction, histological damage, neutrophil infiltration and oxidative stress in a dose dependent manner. Furthermore, PEA +silymarin combination therapy showed a significant synergistic effect. Therefore, NF‐κB and apoptosis pathways were also significantly inhibited. Our results clearly demonstrate that PEA+silymarin combination therapy significantly attenuated the degree of renal inflammation.
The Role of Cilia in the Regulation of Olfactory Horizontal Basal Cells
Authors: Joiner Ariell , Green Warren , McIntyre Jeremy , Martens Jeffrey
Published: June 2021

The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the lifespan of an organism. The OE is composed of differentiated olfactory sensory neurons (OSNs), supporting cells, and two populations of basal stem cells, globose basal cells (GBCs) and horizontal basal cells (HBCs). While GBCs frequently divide and differentiate into OSNs and supporting cells to restore the OE, HBCs are more quiescent yet able to restore the OE following severe insult. In the mouse OE, immunohistochemical (IHC) analysis of Arl13b (a cilia‐localized protein) revealed that in addition to being expressed in olfactory cilia of OSNs, Arl13b‐labeled primary cilia are on HBCs. Due to the growing evidence that cilia play a role in stem cell formation and function, we hypothesized that primary cilia on HBCs regulate their stem cell properties. To test this hypothesis, we generated a triple transgenic mouse (K5rtTA;tetOCre;Ift88fl/fl) that incorporates an inducible tetracycline system, the K5 promoter that in the OE is specifically expressed in HBCs, and the Ift88 conditional allele whose deletion results in a loss of cilia. Upon four weeks of doxycycline treatment in these mice, there was a significant loss of HBC cilia with no change in the cell composition of the OE. However, IHC analysis of various OE cell‐specific markers revealed that three weeks following a methimazole‐induced injury to the OE of mice with depleted HBC cilia resulted in an altered recovery of the OE. These data suggest that cilia are critical for HBC differentiation during the restoration of the OE, making HBC cilia important for olfactory tissue maintenance.

Supported by R01DC009606 (to J.R.M.) and F31DC013496 (to A.M.J.)
Spine Fusion Using a Soft Elastomeric Nanofibrous Composite of Collagen, Poly (1,8‐octanediol‐co‐citrate) and Chondroitin 6‐sulfate
Authors: Wagner William , Morykwas Michael , Argenta Louis , Campbell Douglas , Wang Rui
Published: June 2021

Degradable biomaterials with tunable mechanical, compositional and structural properties have been developed for fusion of bone; however few have demonstrated enhanced repair rates or formation of quality bone compared to autologous bone grafts. The purpose of this study was to fabricate and test a new nanocomposite to mimic extracellular matrix and accelerate spine fusion. An electrospun composite of Type I collagen, chondroitin 6‐sulfate and Poly (1,8‐octanediol‐co‐citrate), termed BRM, was fabricated and tested in a sheep model of lumbar vertebrae fusion. By SEM, BRM had mean fiber diameters of 528±46nm (mean±SEM). Osteoblasts were cultured on BRM and had significantly greater (P<0.05) proliferation compared to electrospun collagen. BRM or autologous bone was placed into PEEK cages and surgically implanted to evaluate posterior lumbar interbody fusion. Fusion sites were monitored by CT and evaluated over a 6 weeks period followed by histologic evaluation. At 6 weeks, mean CT units were 259±45 for BRM and 231±45 (mean±SEM) for autologous bone (noninjured site was 614). Histologic evaluation demonstrated integration and replacement of BRM and significant osteoblastic activity at fusion sites. BRM promoted osteoblastic activity and was bioresorbed without extensive inflammation. BRM resulted in accelerated spine fusion and demonstrated comparable effectiveness as autologous bone graft. The viscoelasticity of BRM and the presence of growth factor binding sites on collagen and chondroitin sulfate may act in concert to promote bone formation.
Loss of PPARγ Promotes Mitochondrial Dysfunction through Downregulation of PGC1α
Authors: Kang Bum‐Yong , Yeligar Samantha , Bijli Kaiser , Adesina Sherry , Kleinhenz Jennifer , Murphy Tamara , Sutliff Roy , Hart C
Published: June 2021

Rationale

Pulmonary hypertension (PH) is an important clinical problem that causes significant morbidity and mortality despite existing therapies. Recent evidence indicates that metabolic derangements in pulmonary vascular wall cells stimulate mitochondrial (MT) dysfunction that promotes proliferative vascular remodeling. We previously showed that expression of peroxisome proliferator‐activated receptor gamma (PPARγ) was reduced in murine hypoxia‐induced PH and that reductions in PPARγ were sufficient to stimulate Nox4 expression, H2O2 production, and human pulmonary artery smooth muscle cell (HPASMC) proliferation.

Objective

We hypothesize that PPARγ coactivator 1 alpha (PGC1α, which stimulates MT biogenesis), is a critical PPARγ‐regulated target that modulates MT function, reactive oxygen species (ROS) production, and the proliferative PASMC phenotype in PH.

Methods and Results

To examine the molecular pathways stimulated by reductions in PPARγ, siPPARγ or GW9662 (PPARγ antagonist) were used to inhibit PPARγ in HPASMCs. Treatment with GW9662 for 72 hrs reduced the oxygen consumption rate (OCR) and generated a bioenergetic profile comparable to those observed in hypoxia. siPPARγ reduced PGC1α and stimulated MT H2O2 production. Similarly, depletion of PGC1α with siRNA stimulated Nox4 expression and HPASMC proliferation.

Conclusion

These findings suggest that PPARγ regulates PGC1α, MT function, ROS production, Nox4 expression, and HPASMC proliferation.

Supported by Atlanta VA Merit Review 1I01BX001910, NIH HL 102167 (RLS and CMH), AHA‐SDG 13SDG14150004 (BYK), 11SDG5440016 (KB), 13SDG13930003, and NIAAA K99 AA021803 (SMY).
Serotoninergic Antagonism in the Retrotrapezoid Nucleus Prevents the Expiratory Long‐Term Facilitation Induced by Acute Intermittent Hypoxia
Authors: Lemes Eduardo , Colombari Eduardo , Zoccal Daniel
Published: June 2021

Serotonin (5‐HT) plays an important role in the development of sympathetic and phrenic long‐term facilitation (LTF) elicited by acute intermittent hypoxia (AIH). Herein, we evaluated the role of 5‐HT in the retrotrapezoid nucleus (RTN) on the emergence of active expiratory pattern in response to AIH. Using anesthetized (urethane; 1.2 g/Kg, ip) male Holztmann rats (280‐290 g), we analyzed the changes in the arterial pressure, heart rate and in the diaphragmatic and abdominal (ABD) activities elicited by single and repeated (3 times, 5 min apart) bilateral microinjections of 5‐HT (1mM, 50nl) in the RTN. We also analyzed the effects of bilateral microinjections of ketanserin (5‐HT2A antagonist; 10 µM, 50 nl) in the RTN on the cardiorespiratory changes induced by AIH (10 times of 6‐7% O2, for 45 s, each 5 min). Repeated, but not single, microinjections of 5‐HT in the RTN (n=7) elicited a sustained increase in the abdominal expiratory activity (ΔABD: 85±33%, 60 min after microinjections, P<0.05) that was prevented by previous microinjections of ketanserin. The 5HT2A receptor antagonism in the RTN during AIH exposure also prevented the increase in ABD activity evoked by AIH (ΔEMGABD: 40±9 vs ‐1±1% 60 min after AIH, respectively vehicle and ketanserin; P<0.05). The other cardiorespiratory parameters were not affected by the 5‐HT and the ketanserin microinjections in the RTN. Our results indicate that, in anesthetized conditions, the activation of serotoninergic receptors in the RTN is sufficient to promote a sustained increase in abdominal activity and required for the emergence of expiratory LTF induced by AIH. Supported by FAPESP, CNPq, CAPES and PROPe.
Developmental Nicotine Exposure Alters The Respiratory Frequency Response to Muscarine
Authors: Wollman Lila , Haggerty Jarl , Pilarski Jason , Levine Richard , Fregosi Ralph
Published: June 2021

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters development of the brainstem circuits that control breathing. Previous work has shown that DNE desensitizes nicotinic acetylcholine receptors (nAChRs) and alters the respiratory motor response to exogenous nicotine in neonatal rats. In the brainstem‐spinal cord (BSSC) split‐bath preparation, nicotine applied to the medullary compartment increases the frequency of respiratory related bursting recorded from the fourth cervical ventral root (C4VR), but this effect is markedly blunted by DNE. Because ACh is the endogenous ligand for both nAChRs and muscarinic acetylcholine receptors (mAChRs), and mAChRs are expressed on PreBotzinger neurons, we hypothesized that DNE also alters muscarinic modulation of C4VR frequency in this system. In neonatal rats, postnatal day 0‐6, 1 mM muscarine, a mAChR agonist, applied to the medullary compartment in the BSSC split‐bath preparation increased burst frequency recorded from C4VR in 4/4 control animals and, in some cases, the pattern of bursting became irregular. Muscarine applied to the medulla decreased burst frequency in 4/4 DNE animals. These preliminary data show that chronic stimulation of nAChRs also alters the development of muscarinic synaptic transmission by yet to be determined mechanisms.
The Pontine Respiratory Group (PRG) Partially Mediates Clinical Opioid‐Induced Respiratory Depression in Adult Rabbits
Authors: Miller Justin , Stuth E , Hopp F , Zuperku E , Stucke A
Published: June 2021

Recent studies in adult dogs suggest that the parabrachial nucleus, a part of the PRG, plays an important role in the respiratory depression by clinical concentrations of intravenous (IV) opioids (Prkic et al 2012). This study investigated whether a similar area could be identified in adult rabbits. Each rabbit (2.2 – 4.0 kg) was anesthetized with 1‐1.5 MAC sevoflurane, tracheotomized, ventilated and decerebrated. The PRG was functionally identified by microinjecting 70 nl of 50 µM AMPA in a grid‐wise fashion with a glass multi‐barrel electrode from 1‐3 mm caudal of the inferior colliculi and 1‐4 mm lateral of midline into the dorso‐ventral center of neuronal activity. The area of maximal tachypnea as determined from the phrenic neurogram was on average 0.6 mm caudal/2.7 mm lateral/7.2 mm from the dorsal surface (n=24). Bilateral microinjection of the mu‐opioid agonist [D‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO, 100 µM, 700nl) into this region led to a significant decrease in breathing frequency (‐45±9% ; P < 0.001; n=10) via increased expiratory duration (TE= 343±43%; P < 0.001; n=10). This was completely reversed (P > 0.05; n=8) by local microinjection of the opioid‐antagonist naloxone (1mM, 840 nl). In a separate set of animals, bilateral local injection of naloxone into the same area partially reversed the bradypnea caused by systemic opiate administration (0.5 mcg/kg/min IV remifentanil) by attenuating the opiate induced increase in expiration duration (ΔTE= ‐127%; P < 0.05; n=11). These studies suggest that in adult rabbits, PRG mu‐opioid receptors contribute to the bradypnea induced by systemically administered opioids at clinically relevant doses through an increase in expiratory duration.

Supported by VA grant I01BX000721.