bluuebunny/crossref_metadata_2025 huggingface dataset:
An Acute Sustained Hypoxic Stress is Sufficient to Cause Respiratory Muscle Weakness in the Mouse
Authors: O'Leary Andrew J. , O'Halloran Ken D.
Published: November 2023

Respiratory muscle weakness is a strong predictor of poor outcome in patients. Acute hypoxia (AH) is a feature of respiratory conditions such as acute respiratory distress syndrome (ARDS) and ventilator‐associated lung injury (VALI). However, the effects of AH on the respiratory muscles are largely unknown despite the clinical relevance. We assessed the effects of AH exposure on ventilation, respiratory muscle contractile function and gene expression.

Adult male C57BL6/J mice (n=8 per group) were exposed to 1, 4 or 8 hours of hypoxia (FiO2 = 0.10) or normoxia (FiO2 = 0.21). Ventilation was assessed using whole‐body plethysmography during gas exposure. Sternohyoid, diaphragm and EDL muscles were excised post‐mortem. Respiratory muscle contractile performance was analysed ex‐vivo. Muscle gene expression was examined using qRT‐PCR.

Minute ventilation was increased (p<0.01, two‐way ANOVA & Bonferroni post hoc test) after 10mins of AH compared with normoxia, returning to levels equivalent to normoxia by 20mins and remaining similar to normoxia thereafter. AH decreased tetanic force production in the sternohyoid (12.80 ± 1.15 vs. 9.72 ± 1.06 N/cm2, p=0.0683, unpaired t‐test) and in the diaphragm (29.53 ± 3.15 vs. 20.88 ± 1.98 N/cm2, p=0.0334). AH differentially altered PGC1α, NFκB1 and selenoprotein N1 mRNA levels in the diaphragm while decreased NRF1, NFκB1, junctophilin 1 & 2, ryanodine receptor 1, calsequestrin 1, dihydropyridine receptor, and selenoprotein N1 mRNA levels were observed in the sternohyoid (p<0.05, one‐way ANOVA & Tukey's post hoc test). No significant changes were observed in the mRNA expression of PPARα, PGC1β, or NRF2 in either muscle in response to AH exposure. UCP‐3 gene expression was increased (p<0.05, one‐way ANOVA & Tukey's post hoc test) in AH diaphragm, sternohyoid and EDL (limb) muscle.

Diaphragm weakness is reported in mechanically ventilated patients, which is primarily attributed to inactivity of the muscle per se, although this is controversial. A potential role of hypoxia in the development and/or exacerbation of this weakness is unclear. Here we show that AH is sufficient to cause respiratory muscle weakness with resultant decreased mechanical power outputs. Functional changes may relate to altered mitochondrial function, atrophy, and/or SR Ca2+ release. Following the acute hypoxic ventilatory response at 10mins, ventilation in AH remains at normoxic levels thereafter (most likely due to a decreased metabolic O2 demand during AH exposure), suggesting that the altered gene expression and muscle weakness observed are not due to enhanced respiratory muscle activity but relate to hypoxic stress per se. These findings highlight a potentially critical role for hypoxia in the respiratory muscle dysfunction observed in acute respiratory‐related disorders and mechanically ventilated patients.

Support or Funding Information

Supported by the Department of Physiology, UCC and the UCC Strategic Research Fund.
Developmental Window Exists in the Hypoxic Ventilatory Response of Juvenile and Adult Male Rats Following Carotid Sinus Nerve Transection
Authors: Coffee Gregory A. , Getsy Paulina M. , Lewis Stephen J.
Published: November 2023

The carotid bodies (CB) are peripheral chemoreceptors that respond to hypoxia by stimulating breathing and increasing blood pressure in order to restore oxygen (O2) levels. The carotid body activates chemosensory afferent terminals of the carotid sinus nerve (CSN), which stimulate the brainstem to increase ventilation. Data from our lab suggests a critical developmental window exists in which the hypoxic ventilatory response (HVR) switches from neonatal to adult. This hypoxic ventilatory response (HVR) seen in male adult rats is blunted after bilateral carotid sinus nerve transection (CSNX). Our objective is to determine the HVR of juvenile male Sprague Dawley rats before and after bilateral CSNX. To test this we performed CSNX at postnatal (P) age P21, and exposed the rats to hypoxia (10% O2) using the BUXCO unstrained, awake, whole‐body plethysmography system four days post surgery. Our results suggest the HVR seen in control juvenile rats is blunted compared to adult rats (n= 12 juvenile and n = 9 adult). Also, after CSNX the juvenile rats exhibit a more diminished HVR compared to adult rats (n = 3 juvenile and n = 9 adult). This data suggests that in control male rats, a critical developmental window may exist in which the carotid body becomes fully sensitized to low blood O2, thus allowing the greater HVR seen in the adult. In addition, our data shows that a secondary means of responding to hypoxia after CSNX may not have functionally developed in the juvenile rats.

Support or Funding Information

NIH PO1‐HL1018710
Chronic Intermittent Hypoxia Altered Stability of Rhythm Generation but Increased Robustness of Respiratory Network
Authors: Dashevskiy Tatiana , Garcia Alfredo J. , Ramirez Jan‐Marino
Published: November 2023

Stable respiratory rhythmic activity generated within the pre‐Bötzinger complex (preBötC), the presumed site for the generation of inspiration, depends on a sparsely connected excitatory network that is composed of silent, tonic firing and intrinsically bursting neurons. The stability of respiratory rhythmic activity is decreased following 10 days of exposure of neonatal mice to chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea. Combining computational modeling and electrophysiological recordings from the preBötC which was isolated in transverse slices, we explored the mechanisms underlying the CIH‐induced stability decrease. Our experiments revealed that signal transmission from individual respiratory neurons reduces network synchronization, thus causing irregularities in the respiratory rhythm. We explored three possible scenarios that can affect the network synchronization: 1) changes of intrinsic membrane properties, 2) increases in synaptic inhibition and 3) downregulation of synaptic excitation. Using a variety of blockers (DNQX, picrotoxin and riluzole) we tested experimentally the effect of persistent sodium current blocker, down regulation of excitation and inhibition in CIH treated animals and control. Our experimental findings suggested that CIH‐mediated irregularities in rhythm generation can be explained by the up‐regulation of persistent sodium current, suggesting that the contribution of persistent sodium dependent bursting neurons may have been altered. We thus examined the network dynamics in a theoretical model of the preBötC in which synaptic inhibition and excitation, as well as heterogeneous intrinsic membrane properties were modified according to our experimental data. We found that manipulating the persistent sodium current, synaptic inhibition and excitation changed the ratio of intrinsically bursting, tonic spiking and silent neurons within the network. Theoretical analysis provided an estimation of the equilibrium state of the network, which allowed us to calculate the optimal ratio between silent, intrinsically tonic spiking, and bursting neurons. Our experimental and computational studies suggest that the CIH‐mediated irregularities in rhythm generation is due to an up‐regulation of persistent sodium current which results in the recruitment of additional active neurons that disrupt rhythm generation. As a result of CIH the composition of the network is reconfigured which causes a loss of rhythmic stability but increases robustness to environmental challenge such as hypoxia.
To Sigh Or Not To Sigh: Role Of Glia In Sigh Generation
Authors: Dashevskiy Tatiana , Bloom Jacob , Ramirez Jan‐Marino
Published: November 2023

The respiratory network generates two distinct rhythmic activities: normal respiration (eupnea), which is relatively fast and sighs that occur at a very slow, but regular frequency. Understanding how the slow periodicity of sighs is generated has been challenging. Here, we propose that glia cells play a critical role in setting up the periodicity of sighs. In mouse transverse brain slice preparations containing the respiratory network, specifically the pre‐Bötzinger complex (PreBötC) we recorded Ca2+ signals over a time interval of 2 to 5 min to capture sighs, and subsequently applied aCSF solution containing 0KCl to differentiate glia from neurons. Neurons drastically decrease their Ca2+ signal, while glia increase intracellular Ca2+ in response to 0KCl. Among the glia cells we observed Ca2+ signals that were synchronized with the periodic sighs. In fact, the phase shift analysis between sighs and Ca2+ signal from non‐neuronal cells showed that on average Ca2+ spikes preceded sigh activity. Furthermore, glia toxins inhibit sighs while eupneic activity was preserved. Based on our pharmacological manipulations in vitro and in vivo we propose the hypothesis that release of glial ATP through activation of P2Y receptors activates glial and neuronal Gq/11 protein. Gq/11 in turn increases glial and neuronal intracellular Ca2+, which in turn leads to the release of glial ATP, thus locking the cycle. Our experimental data are consistent with our computational approach that models the neuronal‐glial interaction. The network model predicts that the presence of IP3‐like channels is necessary to produce “sigh” behavior and to mimic transitions between activity states caused by neuromodulators. We analyze the model dynamics and describe dose‐response curve to neuromodulator such as norepinephrine. Our theoretical data are not only similar to our in vivo and in vitro experimental data, but the model also predicts that depending on the modulatory state of the respiratory center, the network can exhibit bi‐stable behavior, meaning that sighs can be triggered between “on” and “off” states by short perturbation. We detected two different mechanisms that support sigh bi‐stability. The first mechanism is based on the presence of a saddle equilibrium and the range of Ca2+ parameters where the observed bistability is limited by sub‐critical Andronov‐Hopf bifurcation and homoclinic bifurcation for periodic cycle. The second mechanism is based on the presence of a saddle periodic orbit and the parameter range is defined by sub‐critical Andronov‐Hopf bifurcation and saddle‐node for periodic orbits bifurcation. This theoretical prediction is consistent with our experiments in in vivo anesthetized mice: Freely breathing anesthetized mice that did not spontaneously produce sighs were stimulated by repeated, brief pulses of hypoxia, which elicited sustained periodic sighs.

Support or Funding Information

NIH: F32 HL121939‐01
Noradrenergic signaling in the retrotrapezoid nucleus differentially modulates breathing in anesthetized rats by alpha‐1 and alpha‐2 receptor dependent mechanisms
Authors: Oliveira Luiz M , Mulkey Daniel K , Moreira Thiago , Takakura Ana C
Published: November 2023

Noradrenaline is a potent modulator of breathing that can increase or decrease respiratory activity by alpha‐1 or alpha‐2 receptors activation, respectively. The retrotrapezoid nucleus (RTN) is a region located in the rostral aspect of ventrolateral medulla and is known to contribute to central chemoreception, inspiration and active expiration. The aims of this study are to further investigate sources of catecholaminergic inputs to the RTN, as well as identify cardiorespiratory effects produced by activation of adrenergic receptors in this region. We identified a considerable number of the retrograde tracer FluorGold labeled cells (41 ± 8%) in the A7 region, suggesting the A7 is a major source of noradrenergic drive to the RTN. In urethane‐anesthetized, vagotomized and artificial ventilated male Wistar rats (260–290 g, n= 8–10/group), mean arterial pressure (MAP), diaphragm (DiaEMG) and abdominal (AbdEMG) muscle activities were recorded. Unilateral injection of noradrenaline produced a transient dose‐dependent (from 0.25 – 1 mM) inhibition of DiaEMG frequency (Δ = −10 ± 4; −14 ± 7 and −27 ± 11% of control) and amplitude (Δ = −41 ± 4.1; −48 ± 3% and −65 ± 2% of control), without changing MAP and AbdEMG. Unilateral injection of the alpha‐2 adrenergic and imidazoline receptors agonist moxonidine (20 μM) also produced a reduction in DiaEMG amplitude and frequency. The inhibition in the inspiratory activity produced by noradrenaline or moxonidine was completely blocked by the pretreatment with yohimbine (alpha‐2 receptors antagonist, 100 μM) in the RTN. We also found that unilateral RTN injection of phenylephrine (Phe, alpha‐1 adrenergic receptor agonist, 10 μM) increased DiaEMG amplitude by 62 ± 8% and frequency by 75 ± 18% and was able to elicit active expiration. The stimulatory effect on breathing produced by Phe was completely blocked by prior injection of prazosin (alpha‐1 receptor antagonist, 200 μM) into the RTN. We conclude that i) the pontine A7 region is a major source of cathecolamines to the RTN; ii) activation of alpha‐2 adrenergic receptors in the RTN inhibits respiratory activity; and iii) activation of alpha‐1 adrenergic receptors in the RTN increases inspiratory and expiratory activity. In related work, we have characterized the cellular and molecular mechanisms underlying noradrenaline modulation of RTN chemoreceptors (see abstract by Kuo et al.).

Support or Funding Information

FAPESP, CNPq, CAPES/PROEX
Airway Deflation Activated Receptors Are Stimulated by Hypertonic Saline
Authors: Guardiola Juan , Proctor Mary , Yu Jerry
Published: November 2023

Recently, deflation activated receptors (DARs) in the lung have been identified as a group of airway sensory receptors. DARs often share their afferents with rapidly adapting receptors (RARs) as well as slowly adapting pulmonary stretch receptors (SARs) to form a sensory unit. However, the afferent properties of DARs have not been well characterized. It has been reported that airway RAR units are stimulated, whereas typical SAR units (units do not respond to lung deflation) are not stimulated by hypertonic saline. Since RAR units are often share afferents with DARs, possibly, it is the DARs that are stimulated by hypertonic saline. The present study determines whether DARs can be stimulated by increased osmolarity. In anesthetized, open‐chest and artificially ventilated rabbits, we recorded single unit activities from typical SAR units and SAR units that respond to lung deflation (units contain DARs). We examined responses of these two groups to injection of hypertonic saline (8.1% sodium chloride, 0.1 ml) into receptive fields in the lung. Hypertonic saline decreased the activity of typical SARs, although not substantially. At baseline, activity of typical SAR units was 40.3 ± 5.4 imp/s (n=12). It decreased to 34.8 ± 4.7 imp/s (p<0.05) at 17.5 seconds after injection of hypertonic saline. On the other hand, hypertonic saline stimulated deflation activated SAR units significantly. Baseline activity increased from 15.9 ± 2.2 imp/s to its speak of 43.4 ± 10.0 imp/s (n=10, p<0.01) at 10.5 seconds after injection. The activity returned to the control level within a minute. Our results show that behavior of deflation activated and typical SAR units was different. The former, but not the latter, was stimulated by hypertonic saline. We conclude that DARs are stimulated by hypertonic saline.

Support or Funding Information

(Supported by a VA Merit Review Award PULM‐029‐10S)
Neurovascular Coupling is Coordinated by The Activation of Nitric Oxide Production in Astrocytes
Authors: Puebla Mariela D , Muñoz Manuel F , Figueroa Xavier
Published: November 2023

Changes in neuronal activity must be coupled to proportional variations in the local cerebral blood flow to keep brain function. Then, an increase in synaptic activity leads to vasodilation of local parenchymal arterioles, which is achieved by a mechanism known as neurovascular coupling. Astrocytes mediate this communication mechanism through the propagation to the astrocytic end‐feet of an intracellular Ca2+ signaling initiated by neurotransmitters such as glutamate. ATP release through hemichannels formed by connexin 43 (Cx43) is thought to coordinate this signaling process. As astrocytes express the Ca2+‐dependent nitric oxide (NO)‐synthetizing enzymes eNOS and nNOS, and NO activates Cx43 hemichannels, we evaluated the participation of NO in the control of neurovascular coupling. Hemichannel activity was measured by recording ethidium uptake. In addition of hemichannels activity, changes in intracellular Ca2+ concentration and ATP release were also evaluated in primary cultures of astrocytes and vasomotor activity of cortex arterioles was recorded in rat brain slices. Stimulation with glutamate or the glutamate metabotropic receptor agonist, 1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (t‐ACPD), induced hemichannel activation, an increase in astrocytic Ca2+ concentration, ATP release and vasodilation of parenchymal arterioles; responses that were blocked by the inhibition of NO production with NG‐nitro‐L‐arginine (L‐NA). Blockade of hemichannels with the connexin blocking peptide 37, 43Gap27 inhibited the increase in Ca2+, but not ATP release. Interestingly, the inhibition of purinergic receptor with pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) also blocked the activation of connexin hemichannel and the Ca2+ signaling observed in response to glutamate. In addition, the increase in ethidium uptake, intracellular Ca2+ concentration and ATP release induced by glutamate were abolished by the calcium homeostasis modulator 1 (CALHM1) channel blocker, ruthenium red. Consistent with the participation of NO in this response, glutamate and SNAP, a NO donor, evoked the S‐nitrosylation of CALHM1 channels. These results suggest that NO production plays a central role in the control of astrocyte‐mediated neurovascular coupling through activation of CALHM1 channels by S‐nitrosylation and the further opening of Cx43 hemichannel via ATP release and purinergic receptor activation

Support or Funding Information

FONDECYT 1150530
GABAB receptor plasticity in corticotrophin releasing hormone neurons in the paraventricular nucleus during chronic unpredictable stress
Authors: Gao Yonggang , Zhou Jing‐Jing , Li De‐Pei
Published: November 2023

GABAB (γ‐aminobutytic acid type B) receptor is important in the regulation of neuronal excitability. Chronic stress leads to hyperactivity of corticotrophin releasing hormone (CRH) neurons. However, the role of GABAB receptors in the regulation of CRH neurons in paraventricular nucleus (PVN) of the hypothalamus in chronic stress is not known. Here, we determined pre‐ and postsynaptic GABAB receptor function in identified PVN‐CRH neurons expressing eGFP driven by crh premotor‐driven eGFP. Whole cell patch‐clamp recording were performed on PVN‐CRH neurons in brain slices from unstressed rats and rats with chronic unpredictable mild stress (CUMS). Bath application of the GABAB receptor agonist baclofen induced a significant lesser reduction of firing activity in PVN‐CRH neurons in CUMS than in unstressed rats. Furthermore, baclofen induced lesser outward currents in PVN‐CRH neurons in CUMS than in unstressed rats. Baclofen‐induced currents were eliminated by inclusion of GDP‐β‐s in internal recording solution or blocking G protein‐coupled inwardly‐rectifying K (GIRK) channels with tertiapin. GABAB1 receptor expression level in the PVN was significantly decreased in CUMS than in unstressed rats. Baclofen produced significant greater inhibition of the frequency of glutamatergic excitatory postsynaptic currents (EPSCs) and GABAergic inhibitor postsynaptic currents (IPSCs) without affecting the amplitude of EPSCs and IPSCs in PVN‐CRH neurons in CUMS and unstressed rats. These data suggest that down‐regulated postsynaptic GABAB receptor contributes to suppressing GABAB‐mediated inhibition of PVN‐CRH neurons in chronic stress.

Support or Funding Information

NIMH grants MH096086.
Androgenic Regulation of Sex Differences in Cortical Thickness of the Developing Mouse Brain
Authors: Miyasaki Dillon , Ramos Layla , Tsai Houng‐Wei
Published: November 2023

The cerebral cortex plays an important role in complex cognitive and social behaviors. Considerable evidence suggests that many of those are sexually dimorphic, but the neural mechanism underlying sex differences in cortical function still remains unclear. An earlier study showed greater cortical thickness in three areas of the parietal cortex of adult male mice compared with females, which seemed to be influenced by gonadal steroids, not sex chromosomal complement. Since activation of androgen receptor (AR) by perinatal testosterone secreted by the developing testes increases the in the number of calbindin‐positive neurons in the sexually dimorphic nucleus of the male preoptic area, a hypothalamic region that controls male copulatory behaviors, we thus hypothesized that AR might also be responsible for masculinization of the mouse cerebral cortex. To test our hypothesis, we collected the whole brains (n=4–6 per group) of wild‐type male and female mouse pups as well as their littermates with testicular feminization mutation (Tfm), lacking functional AR, on postnatal days 21–24. Their brains were post‐fixed in 4% paraformaldehyde, serially sectioned, and stained with crystal violet. After the slides were cover‐slipped, measurement of cortical thickness in the primary somatosensory cortex, secondary somatosensory cortex, secondary motor cortex, and entorhinal cortex was taken under a light microscope using ImageJ. Among these brain regions, our preliminary data showed a significant effect of experimental group on cortical thickness in the entorhinal cortex only (P = 0.01). While wild‐type male mice had a thicker entorhinal cortex than wild‐type females, the cortical thickness in Tfm mice was intermediate between the wild‐type males and females and did not differ significantly from either of those groups. Our preliminary data suggests an important role for the AR in sexual differentiation of brain structure in the mouse entorhinal cortex during development.

Support or Funding Information

This work was supported by National Institutes of Health Grant SC3‐GM102051.
Sex‐specific Effect of Androgen on SFSWAP Expression in the Developing Mouse Cortex and Hippocampus
Authors: Ridder Aaron , Lee YuanYu , Tsai Houng‐Wei
Published: November 2023

The cortex and hippocampus both are important for the control of complex cognitive and social behaviors, and considerable evidence suggests that these functions are sexually dimorphic. However, neural mechanisms underlying these differences are still not well understood. We have previously used immunoblotting to detect splicing factor suppressor of white apricot (SFSWAP) protein at a molecular weight of 105 kDa in the developing mouse cortex/hippocampus. We then measured cortical/hippocampal SFSWAP levels in male and female mice collected on the day of birth (PN0), and 7 (PN7), and 14 (PN14), and 21 (PN21) days after birth and found higher SFSWAP expression in females than males at all ages except PN0. Since androgens masculinize the brain nuclei of the hypothalamus and amygdala underlying male‐specific behaviors, we thus hypothesized that sexually dimorphic SFSWAP expression in the cerebral cortex/hippocampus might be similarly regulated by testosterone. To test our hypothesis, we treated male and female pups with either vehicle (0.05 ml sesame oil) or testosterone propionate (TP, 0.1 mg dissolved in 0.05 ml sesame oil) daily for three days starting on PN21. Their cortex/hippocampus (n= 6 per group) was collected 3–5 h after the last injection, followed by protein extraction and immunoblotting for SFSWAP. While a trend of female‐biased SFSWAP upregulation was found in vehicle‐treated groups, our results showed that TP decreased SFSWAP levels in female pups (0.67 ± 0.07 vs. 1.00 ± 0.08, p<0.05), not males. The observation of pharmacological effects of testosterone on SFSWAP downregulation in females suggests that SFSWAP expression might be mediated directly by androgen receptor or indirectly by estrogen receptors after it is converted to estradiol locally by aromatase. Further, the sex‐specific androgenic regulation of SFSWAP expression in the cortex/hippocampus could be due to the sex chromosome complement and/or the organizational effect of perinatal steroids occurring around birth.

Support or Funding Information

This work was supported by National Institutes of Health Grant SC3‐GM102051.
Saporin Inhibits Adonis‐biotin Stimulation of Proliferation of Cells of Caudate Nucleus of Newborn Lambs
Authors: McBride Shaemion , Ibe Basil Obijiaku , Rajaee Arezoo , Koos Brian
Published: November 2023

The subventricular zone of the caudate nucleus (CN) contains proliferating progenitor stem cells that may play a role in the greater plasticity of the developing brain. CN cells involved in this reorganizational capacity have not been defined. Rodents are used extensively to study developmental disorders, while larger mammals are seldom studied. We hypothesized that the reorganizational ability of the CN involves the proliferation of CN cells. Primary cells of CN of newborn lambs 3–8 days old were used to study the proliferative potential of CN in vitro. We used Adonis‐biotin, a cell permeable Adonis analog and the ribosome‐inactivating protein conjugate Streptavidin‐SAP (Saporin) as a control for cell growth stimulating properties of the Adonis‐biotin. Adonis‐biotin and Saporin modulate cell proliferation by interacting with cell DNA. Cells in culture were treated with different concentrations (nM); 0.1, 1.0, and 10.0 of Adonis‐biotin, Saporin and equimolar mixture of the Adonis‐biotin and Saporin and the culture medium as control for cell proliferation. Treated cells were incubated at 37°C for 4 days. Cell proliferation was determined each day by cell counting. Effect of these treatments on cell proliferation was also studied by DNA synthesis and immunocytochemistry with DAPI staining. Adonis‐biotin stimulation of cell proliferation was both concentration and time dependent. Treatment of cells with Saporin specifically inhibited cell proliferation such that the co‐treatment with Adonis‐biotin did not abrogate the inhibitory effect of Saporin on CN cell proliferation, suggesting that Saporin had profound toxicity to the cell. This observation was supported by DAPI staining where Adonis‐biotin, but not Saporin, treatment stimulated cell proliferation. Taken together this study shows the involvement of CN cells in the plasticity of basal ganglia of the developing brain.

Support or Funding Information

Grant #20842 from Department of Obstetrics and Gynecology, UCLA.
Pain Related Behaviors in Mice Expressing Sickle Hemoglobin: Modulation by Bivalent Ligands
Authors: Rodríguez‐Deliz Carla Leticia , Puig Guillermo , Cataldo Giuseppe , Simone Donald
Published: November 2023

Sickle cell disease (SCD) is a genetic disorder that produces abnormal hemoglobin, leading to vaso‐occlusion. The hallmark of this condition is pain. Little is known about the mechanisms of SCD, therefore, treatment remains inefficient. Several types of transgenic mice have been developed in order to study therapeutic options for SCD, including the Townes mice. In this study, we aimed to characterize pain‐related behaviors in the Townes sickle mouse, as well as identify the onset of SCD‐associated hyperalgesia. In addition, we aimed to study the effect of a new drug, MCC22, on such pain‐related behaviors. For this, we measured paw withdrawal frequency (PWF) and mechanical threshold, as well as grip force to compare between control (AA), sickle trait (AS) and sickle cell (SS) mice. MCC22 was administrated through intraperitoneal (IP) injection and the mechanical tests were conducted at 15, 30 and 60 minutes to evaluate its effects. We observed that sickle mice had significantly higher PWF and lower thresholds and grip force when compared to AA and AS mice. Administration of MCC22 reduced PWF in SS mice. Decreases in relevant pain behaviors suggest that MCC22 may prove to be a novel effective therapeutic in SCD.
Cytoprotective and Anti‐Glycation Defenses in Porcine Brain after Cardiac Arrest and Cardiocerebral Resuscitation
Authors: Nguyen Anh Quynh , Ryou Myoung‐Gwi , Hollrah Roger A , Williams Arthur G , Scott Gary F , Olivencia‐Yurvati Albert H , Mallet Robert T
Published: November 2023

Background

Cardiac arrest (CA) is often lethal, and survivors often face sequelae that greatly impact quality of life due to brain injury inflicted by ischemia‐reperfusion. Effective cardiocerebral resuscitation (CCR) is essential for survival and recovery from CA. The complexity of the injury cascades ignited by CA and the presence of the blood‐brain barrier challenge the development of pharmacological interventions to protect the brain. Our goal is to identify a blood‐brain barrier‐permeable intervention that mitigates CA‐induced brain damage and, thus, fosters neurological recovery. Pyruvate, a cellular metabolite, energy substrate and antioxidant, has been found to be neuroprotective in a rat stroke model via induction of the cytoprotective cytokine erythropoietin (EPO) [Stroke 2012; 43:1101–1107].

Hypothesis

Pyruvate treatment during CCR and post resuscitation decreases brain injury by upregulating cellular defense mechanisms including hypoxia inducible factor‐1α (HIF‐1α), EPO, the product of HIF‐1 α's gene program, and glyoxalase‐1, the principal component of the brain's defenses against the powerful glycating agent and glycolytic byproduct, methylglyoxal.

Methods

Yorkshire swine (25–35 kg, both genders) were subjected to sham protocol (n = 6) or pacing‐induced CA (n = 12). CCR was administered by precordial chest compressions (100/min) at 10–14 min CA, and transthoracic countershocks were applied to restore sinus rhythm. NaCl (n = 6) or Na‐pyruvate (n = 6) was infused iv (0.1 mmol/kg/min) during CCR and the first 60 min recovery. At 4 h recovery, brain was cross‐perfused with 0.9% NaCl, and then brain biopsies were freeze‐clamped for biochemical analysis or fixed in 10% formalin for immunohistochemistry.

Results

In hippocampus, activity of the oxyradical‐sensitive TCA cycle enzyme aconitase fell by 50% (P < 0.05) during CA + CPR with NaCl and Na‐pyruvate treatment, while no appreciable difference was detected in the cerebellum. There were no statistically significant differences in HIF‐1α or EPO contents in the hippocampus among the sham and CA groups. Similar trends were observed in the cerebellum except for EPO content, which fell by 40% (P<0.05) regardless of treatment. In contrast, pyruvate increased glyoxalase‐1 activity threefold in cerebral cortex (P<0.01).

Conclusion

Cardiac arrest inactivates aconitase and depletes cytoprotective EPO in ischemia‐sensitive brain regions. Although pyruvate intervention during CPR and the initial 60 min recovery did not produce sustained protection or upregulation of aconitase or EPO, it produced robust augmentation of glyoxalase‐1, thereby bolstering the brain's defenses against the glycating metabolite methylglyoxal.

Support or Funding Information

NINDS Support: NS076975
Chronic Mild Stress Increases Baseline Renal Sympathetic Nerve Activity and the Response to Acute Stressors in Conscious Rats
Authors: Das Shibandri , Li Cailian , Chen Haiping , Rossi Noreen F.
Published: November 2023

Depression is a non‐traditional risk factor for cardiovascular morbidity and mortality. Increased sympathetic tone is also recognized as deleterious to cardiovascular health. Chronic mild unpredictable stress paradigm (CMS) is a validated model of depression that has been associated with cardiovascular dysfunction in rats. We hypothesized that rats exposed to CMS will have higher arterial pressure and renal sympathetic nerve activity (RSNA) and enhanced responses to acute stressors. Male Sprague Dawley rats were equipped with telemetry transmitters for hemodynamic monitoring and subjected to 4 weeks of CMS or standard housing (control). During the last week, rats were equipped with telemetry transmitters for renal nerve recording. Baseline RSNA was obtained a minimum of 3 days after completion of CMS protocol and the rats were subjected to air jet stress and nasopharygeal reflex testing. Baseline arterial pressure was higher in CMS rats. Baseline RSNA was 0.80 ± 0.05 mV.s in control vs 1.20 ± 0.14 mV.sec in CMS rats (P < 0.05). Peak responses to either air jet stress or nasopharyngeal stimulation were significantly higher in the CMS rats. Although the latencies of the responses were shorter and the duration of the responses were longer in the CMS group, these did not achieve significance. The area under the curve (AUC = latency × duration) for air jet stress was 82% greater for air jet stress and 362% greater for nasopharygeal stimulation in the CMS vs control rats (P < 0.05 for both vs control). These results support the concept that rats subjected to a chronic unpredictable stress paradigm that models depression display higher basal sympathetic nerve activity and a more robust response to acute stress. This enhanced sympathetic tone and neuroexcitability may contribute to increased cardiovascular risk.

Support or Funding Information

VA Merit Grant to NFR
Altered Activity in Efferent Projection Sites of the Brain Orexin System is Associated with Poor Cardiovascular Adaptability in an Animal Model of Hypertension and Attention Deficit/Hyperactivity Disorder (ADHD)
Authors: Ahmari Niousha , Simons Chloe , Watkins Jaqueline , Long Maureen , Febo Marcelo , Zubcevic Jasenka , Hayward Linda
Published: November 2023

The neuropeptide orexin has recently been shown to be dysregulated in the spontaneously hypertensive rat (SHR), an animal model of essential hypertension. The SHR has also been proposed as a model of ADHD. The present study was undertaken to determine whether regional differences in brain activation could be linked to differences in behavior or cardiovascular reactivity in the SHR when compared to normotensive rats (WKY; n=3–10/group). Manganese‐enhanced MRI of naïve animals identified that multiple efferent projection sites of the orexin system, including the hippocampus (CA1 and CA3, P<0.01) and the midbrain (central gray, dorsal raphe, ventral tegmental nucleus) had significantly (P<0.05) lower activity in the SHR vs WKY, with the exception of locus coeruleus which had elevated activity (P<0.02). In object recognition and social interaction tests, the SHRs generally spent less time interacting than WKYs. On the elevated plus maze test, SHR spent twice the time in the open arm field. This was paralleled by a similar initial rise in blood pressure (22+/−1 mmHg) and heart rate between strains (165+/−10 bpm). By the end of the 5 minute test however, WKY values returned to baseline while the SHR blood pressure remained elevated (+3 vs +20 mmHg). These results support the link between heightened activity in locus coeruleus (a primary target of the orexin system), hypertension, and behavioral markers of ADHD, and raise a novel prospect that reduced activity in other midbrain regions may also be linked to altered system function/adaptation in the SHR.

Support or Funding Information

UF College of Veterinary Medicine Research Award (ML) & AHA 14SDG18300010 (JZ)
Predicting muscle sympathetic nerve activity and mean arterial blood pressure responses to mental stress
Authors: Wakeham Travis R , Fonkoue Ida A , Wang Min , Carter Jason R , Durocher John J
Published: November 2023

Mental stress consistently increases mean arterial pressure (MAP), but changes in muscle sympathetic nerve activity (MSNA) during mental stress can vary widely between subjects. The MAP responsiveness to mental stress has been reported to be augmented in both prehypertensive and obese adults when compared to normotensive and normal weight adults, respectively. Cardiovascular reactivity to mental stress has been suggested as a predictor of hypertensive risk (i.e., cardiovascular reactivity hypothesis), yet the major contributing factors to the MSNA and MAP responses to mental stress remain unclear. Predictive independent variables include factors such as resting blood pressure, sex, and body mass index (BMI). Therefore, we used a regression selection method with an algorithm that employed both forward selection and backward elimination to determine the best explanatory variable for the changes in MSNA (ΔMSNA) and MAP (ΔMAP) during mental stress. Predictive independent variables included resting systolic arterial blood pressure (SAP), diastolic arterial blood pressure (DAP), mean arterial blood pressure (MAP), heart rate, sex, age, and BMI in 97 adults (67 males and 30 females). Heart rate, MSNA, and MAP were simultaneously and continuously recorded during a 5–10 min supine baseline and 5 min of mental stress (via mental arithmetic). The strongest predicting variable for ΔMSNA responses to mental stress was BMI (p<0.02), which explained 3.5% of the variance; BMI and ΔMSNA were significantly correlated (p<0.04). In contrast, the strongest predicting variable for ΔMAP responses to mental stress was resting SAP (p<0.02), which explained 5.6% of the variance; resting SAP and ΔMAP responses to mental stress were significantly correlated (p<0.01). We conclude that BMI is the predominant factor that contributes to the ΔMSNA response to mental stress, while resting SAP is the most important contributing factor to ΔMAP responses. Our findings reinforce the importance of both BMI and resting blood pressure in neural cardiovascular reactivity studies.

Support or Funding Information

This project was supported by a Michigan Technological University Research Excellence Fund Seed Grant to J. Durocher.
Central projections to medulla for autonomic adjustments to exercise in rats
Authors: Koba Satoshi , Kato Momone , Hanai Eri , Watanabe Tatsuo
Published: November 2023

While research efforts have been made on determination of central regions responsible for autonomic adjustments to exercise, neurocircuitries between the regions are little known. Rostral ventrolateral medulla (RVLM), which contains sympathetic premotor neurons, is reportedly activated by exercise. In this study, we attempted to identify central efferent pathways which project to the RVLM and play a role in cardiovascular regulation during exercise. In the first set of experiments, immunofluorescence staining was performed on coronal brain sections of treadmill‐exercised rats which had preliminarily received microinjection of cholera toxin B subunit (CTb, a monosynaptic retrograde tracer) into the RVLM. A significant number of RVLM‐CTb‐labelled cells with immunoreactivity of Fos, a marker of activated neuronal cells, was found in paraventricular hypothalamic nucleus (PVN) and caudal periaqueductal gray (cPAG) of the exercised rats. These data suggest that PVN neurons sending efferent projections to the RVLM (PVN‐RVLM neurons) as well as cPAG neurons projecting to the RVLM are activated by voluntary exercise. In the second set of experiments, effects of selective stimulation of PVN‐RVLM neurons on blood pressure in anesthetized rats were investigated. Adeno‐associated virus (AAV) vector encoding channelrhodopsin2 had been ejected into the PVN of rats. Photostimulation of the RVLM of the AAV‐ejected rats caused a pressor response (N=4). These data suggest that activation of PVN‐RVLM neurons causes sympathoexcitation. Taken together, PVN‐RVLM neurons are considered a pathway which plays a role in evoking sympathoexcitatory response to exercise in rats.

Support or Funding Information

Supported by JSPS KAKENHI Grant number 15H05367 (SK) and 26670112 (SK).
Exercise Training Increases Baroreflex Control of Heart Rate and Decreases Muscle Sympathetic Nerve Activity in Chronic Chagasic Cardiomyopathy Patients
Authors: Sarmento Adriana Oliveira , Antunes‐Correa Ligia Moraes , Lobo Denise Moreira Lima , Carvalho Bruna Elisa Piovezani , Trombetta Ivani Credidio , Alves Maria Janieire Nazaré Nunes , Rondon Maria Urbana Pinto Brandão , Rondon Eduardo , Vieira Marcelo Luiz Campos , Mady Charles , Negrao Carlos Eduardo , Ianni Barbara Maria
Published: November 2023

Asymptomatic or mild symptomatic chronic chagasic cardiomyopathy (CCC) patients even with preserved ventricular function already have autonomic dysfunction. It is well known that decreased baroreflex control of heart rate (HR) and increased muscle sympathetic nerve activity (MSNA) increases mortality of patients with ventricular dysfunction to mortality, and that exercise training (ET) improves cardiovascular autonomic function of these patients. However, the effects of ET on baroreflex control of HR and MSNA in CCC patients with preserved ventricular function are unknown. We hypothesized that ET would increase baroreflex control of HR and decrease MSNA of these patients. Twenty CCC patients with electrocardiographic alterations and ejection fraction (EF)>55% were randomly divided into 2 groups: exercise‐trained (n=8; 47.8±2.5 yrs) and untrained (n=12, 51.7±2.4 yrs). Baroreflex control of HR (ECG) was assessed by stepwise intravenous infusions of phenylephrine and sodium nitroprusside and analyzed by linear regression. MSNA was measured using microneurography. BP was monitored on a beat‐to‐beat basis. Peak VO2 was determined by cardiopulmonary exercise test and cardiac function by echocardiography. ET consisted of three 60‐minute exercise sessions per week on a cycle ergometer for four months. There were no significant differences in gender, age and body mass index among groups. Under baseline conditions (before training), HR, BP, Peak VO2, MSNA and baroreflex control of HR were similar among groups. ET decreased resting HR (67.8±3.6 vs 59.7±3.7 bpm, P=0.009) and increased peak VO2 (25.5±2.6 vs 29.6±1.5 ml/kg/min, P=0.04) whereas there was no change in BP. ET increased baroreflex control of HR in response to phenylephrine infusion (0.24±0.1 vs 0.46±0.1, P=0.01). Furthermore, ET also reduced MSNA (31.8±3.3 vs 22.4±2.8 bursts/min, P=0.04). In conclusion, ET enhanced cardiovascular autonomic function in CCC patients with electrocardiographic alterations and preserved ventricular function. These findings highlight importance of including ET in the treatment of these patients which may lead to a better prognosis of them.

Support or Funding Information

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP ‐ #2011/17533‐6d) and, in part, by Fundação Zerbini. Adriana O Sarmento was supported by Conselho Nacional de Pesquisa (CNPq, #142179/2013‐2). Ligia M Antunes‐Correa was supported by FAPESP (#2013/15651‐7). Denise ML Lobo was supported by Conselho Nacional de Pesquisa (CNPq #140265/2013‐9). Carlos E Negrao and Maria UPB Rondon were supported by CNPq (#301867/2010‐0 and #308068/2011‐4, respectively).
Integration of Clinical Anatomy Labs in a Clinical Presentation‐based Curriculum: A Tool to Improve Long‐Term Retention of Anatomy
Authors: Nausheen Fauzia , Aleyd Ghaith , Suskind Robert , Tenore Alfred , Yakub Mohsin
Published: November 2023

In an active learning Clinical Presentation (CP) based curriculum, medical students spend most of their learning time in various activities like: discussions of CPs, clinical cases, flipped classrooms and self‐directed study. In light of a general inclination to lessen course hours, this has resulted in a significant decline of dissection time in human anatomy: a time‐honored basic science discipline. As a result there have been great concerns regarding traditional and modern approaches to teaching anatomy and its impact on student's knowledge of medical students which may affect their future competencies as physicians. In an attempt to improve long‐term retention of anatomy knowledge, California University of Science and Medicine, School of Medicine (CalMed‐SOM) has designed a blended curriculum that contains clinical anatomy, anatomical‐radiological correlations, anatomical characterizations of surgical procedures and ultra‐sonographic living anatomy labs. This approach in the teaching of anatomy teaching will allow anatomical principles to be appreciated and applied to the clinical concepts taught during the CPs and clinical cases of CalMed‐SOM's active learning curriculum. To achieve our goal, students will be divided into small groups, each of which will rotate through four different anatomy laboratory stations which will be integrated with the CP of that week and will address the gross anatomy, procedural anatomy, and ultra‐sonographic and radiologic components of the Clinical Anatomy Labs. Students will be given an online test before and after lab sessions of each course to assess the amount of knowledge gained. At the end of each semester, the student will again be assessed with the same pre and post exams administered during the previous courses in order to evaluate their retention of knowledge. In an attempt to explore Clinical Anatomy Lab experience of medical students toward the various methods of teaching anatomy we have constructed a questionnaire which will be administered at the end of each course. Data will be collected regarding student's experience, satisfaction and performance in each course. The results will be used as a guide to tune the curriculum and the teaching of Clinical Anatomy. We believe that this integrated, active approach will better prepare our students for their clinical encounters in the clerkship years.
Immune modulating activity of Maillard reaction products from milk proteins with lactose by nitric oxide reduction on macrophage
Authors: Kim Da Hyun , Chun Su‐Hyun , Lee Kyung Won , Lee Kwang‐Won
Published: November 2023

Caseins and whey proteins constitute most of the milk proteins. Also, lactose is the most abundant sugar in milk. The Maillard reaction makes the chemical compounds using reducing sugars and amino acids by non‐enzymatic reactions. The macrophages are participating in the initiation of inflammation, and they are activated by numerous inflammation factors, lipopolysaccharide (LPS), cytokines and other chemical factors. The activated macrophages synthesize nitric oxides for further anti‐inflammation processes. Using sodium caseinate and whey protein concentrate with lactose, we made Maillard reaction products (MRPs) as lactose‐sodium caseinate (LSC) and lactose‐whey protein concentrate (LWPC). The reaction was conducted with 120°C for 20 minutes in 0.1M sodium phosphate buffer solution, proteins (sodium caseinate or whey protein concentrate) and lactose ratio was 1:2 (w:w). As a result, the fluorescence of MRPs was increased and we confirmed by SDS‐PAGE that high molecular weight proteins were generated. Also, an intermediate in the Maillard reaction, hydroxymethylfurfural (HMF) was increased. Macrophage cells were produced more nitric oxide with sodium caseinate and whey protein concentrate, but lactose MRPs of each proteins are reduce nitric oxide level below the LPS‐induced nitric oxide level. Using RT‐PCR method, cytokine levels of IL‐1β and IL‐8 were also measured.