Chronic Stimuli Research Articles

LYMPHOCYTE TRAFFICKING FOLLOWING ACUTE STRESS AND ALTITUDE HYPOXIA IN LOW AND HIGH HEMATOCRIT SHEEP

The object of the present study was to investigate small and large lymphocytestrafficking in sheep with low and high hematocrit values following shearing,exposure to moderate altitude and transport to low altitude. Twenty out of 101 Ilede France ewes (1-7 years old) were used in the present experiment. All ewes ofthe flock were artificially inseminated in May 2015 following estrussynchronization. The animals were allocated into two groups following threefoldmeasurements of hematocrit in all ewes as follows: I- low hematocrit group (n=10)and II - high hematocrit group (n=10). The ewes were transported to the PetrohanPass (1440 m above sea level) in June 2015 immediately after shearing, conductedat the experimental farm of the Institute of Animal Science, Kostinbrod (500 mabove sea level). Blood samples were collected before shearing, immediately aftershearing, 3 h after shearing, at 14 d following exposure to moderate altitude,immediately after transport to low altitude and following 7d of stay at low altitude.All leukocyte subpopulations were counted microscopically. In the current studywe presented the percentage of lymphocytes only, including small and large(reactive) lymphocytes. High and low hematocrit ewes had different percentage ofsmall lymphocytes when exposed to various acute and chronic stress stimuli. Therewere significant differences in the percentage of large (reactive) lymphocytesbetween low and high hematocrit ewes following blood collection and immediatelyafter shearing. The observed difference in small lymphocyte dynamics among thegroups in response to different stress stimuli was attributed to hematocrit relateddifferences in the time course and magnitude of lymphocyte distribution at earlyand late phases of stress. The results were interpreted to mean that the differencesin lymphocyte trafficking between the two groups of sheep in response to stresswere related to possible difference in the share of aerobic and glycolytic pathwaysfor energy supply.

Selection of endurance capabilities and the trade-off between pressure and volume in the evolution of the human heart

Chimpanzees and gorillas, when not inactive, engage primarily in short bursts of resistance physical activity (RPA), such as climbing and fighting, that creates pressure stress on the cardiovascular system. In contrast, to initially hunt and gather and later to farm, it is thought that preindustrial human survival was dependent on lifelong moderate-intensity endurance physical activity (EPA), which creates a cardiovascular volume stress. Although derived musculoskeletal and thermoregulatory adaptations for EPA in humans have been documented, it is unknown if selection acted similarly on the heart. To test this hypothesis, we compared left ventricular (LV) structure and function across semiwild sanctuary chimpanzees, gorillas, and a sample of humans exposed to markedly different physical activity patterns. We show the human LV possesses derived features that help augment cardiac output (CO) thereby enabling EPA. However, the human LV also demonstrates phenotypic plasticity and, hence, variability, across a wide range of habitual physical activity. We show that the human LV's propensity to remodel differentially in response to chronic pressure or volume stimuli associated with intense RPA and EPA as well as physical inactivity represents an evolutionary trade-off with potential implications for contemporary cardiovascular health. Specifically, the human LV trades off pressure adaptations for volume capabilities and converges on a chimpanzee-like phenotype in response to physical inactivity or sustained pressure loading. Consequently, the derived LV and lifelong low blood pressure (BP) appear to be partly sustained by regular moderate-intensity EPA whose decline in postindustrial societies likely contributes to the modern epidemic of hypertensive heart disease.

Abstract 902: Leveraging Natural Cardiomyocyte Variability to Investigate Downregulation of β-1 Adrenoceptors Following Dobutamine Treatment

In heart failure, the most consistent effect on the myocardial beta-adrenergic pathway is a downregulation of β-1 adrenoceptors (β1AR). This loss of receptors impairs the ability of the heart to respond to increases in cardiac demand, and can lead to decreased myocyte function even at rest. As a model of the process that leads to downregulation of β1AR, we measured intracellular Ca 2+ transients and unloaded sarcomere shortening in electrically stimulated isolated adult rat cardiomyocytes undergoing 30 minutes of exposure to 40 nM dobutamine. These experiments were facilitated by a custom device containing micro-patterned wells that orient and immobilize the cardiomyocytes, allowing for rapid and consistent measurements over an extended time period. After functional measurements, cells are picked up individually with a computerized micro-aspirator and subjected to single-cell RT-qPCR to quantify abundance of transcripts of interest. In this way, the physiological responses of individual cells to β1AR stimulus can be correlated to gene expression levels. We found a surprising diversity in the single-cell responses to dobutamine exposure. Although average cardiomyocyte contractility increased significantly, several apparently healthy cells registered no change or even a decrease in sarcomere shortening in the presence of dobutamine. Gene expression was quantified in cardiomyocytes representing a spectrum of sensitivities to dobutamine (n = 7). The transcript for β1AR was significantly downregulated in all cells after 30 minutes, indicating consistent adaptation to chronic stimulus. However, abundance of the gene encoding PKA was negatively correlated with dobutamine-triggered augmentation of Ca 2+ release (p-value: 0.0201). In other words, PKA expression was reduced in cells that experienced potent increases in Ca 2+ release after dobutamine treatment. These data suggest the presence of gene regulatory circuits that modulate individual components of the β1AR pathway independent of overall β1AR abundance. These circuits may be critical to restoring β1AR sensitivity in the failing heart.

Protein arginine methyltransferase biology in humans during acute and chronic skeletal muscle plasticity.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins. While dysregulation of PRMTs has been documented in a number of the most prevalent diseases, our understanding of PRMT biology in human skeletal muscle is limited. This study served to address this knowledge gap by exploring PRMT expression and function in human skeletal muscle in vivo and characterizing PRMT biology in response to acute and chronic stimuli for muscle plasticity. Fourteen untrained, healthy men performed one session of sprint interval exercise (SIE) before completing four bouts of SIE per week for 6 wk as part of a sprint interval training (SIT) program. Throughout this time course, multiple muscle biopsies were collected. We found that at basal, resting conditions PRMT1, PRMT4, PRMT5, and PRMT7 were the most abundantly expressed PRMT mRNAs in human quadriceps muscle. Additionally, the broad subcellular distribution pattern of PRMTs suggests methyltransferase activity throughout human myofibers. A spectrum of PRMT-specific inductions, and decrements, in expression and activity were observed in response to acute and chronic cues for muscle plasticity. In conclusion, our findings demonstrate that PRMTs are present and active in human skeletal muscle in vivo and that there are distinct, enzyme-specific responses and adaptations in PRMT biology to acute and chronic stimuli for muscle plasticity. This work advances our understanding of this critical family of enzymes in humans.NEW & NOTEWORTHY This is the first report of protein arginine methyltransferase (PRMT) biology in human skeletal muscle in vivo. We observed that PRMT1, -4, -5, and -7 were the most abundant PRMT mRNAs in human muscle and that PRMT proteins exhibited a broad subcellular localization that included myonuclear, cytosolic, and sarcolemmal compartments. Acute exercise and chronic training evoked PRMT-specific alterations in expression and activity. This study reveals a hitherto unknown complexity to PRMT biology in human muscle.

Increased pain and inflammatory sensitivity in growth hormone-releasing hormone (GHRH) knockout mice

Growth hormone (GH) and GH-releasing hormone (GHRH), in addition to metabolic and endocrine effects, play a role in the modulation of pain and inflammation. We aimed to elucidate the consequences of GHRH deficiency on acute nociceptive stimulation and on both acute and chronic inflammatory stimuli in a mouse model of GH deficiency. Mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO, -/-) were compared to wild type (GHRH +/+) mice.Responsiveness to acute nociceptive stimulation and to acute inflammatory stimulation was evaluated by conventional hot plate apparatus and formalin test, respectively. We also evaluated responsiveness to colonic inflammation induced both in vivo, after dextran sodium sulfate (DSS) treatment, or ex vivo, by incubating colon segments with bacterial lipopolysaccaride (LPS). Macroscopical and histological examinations were performed, prostaglandin (PG) E2 and 8-iso-PGF2α levels and cyclooxigenase (COX)-2 and tumor necrosis factor (TNF)-α gene expression were measured.Compared to controls, -/- mice showed decreased response latency during the hot plate test, and increased licking/biting time in formalin test, particularly in the second phase of inflammation. DSS treated -/- mice showed a significant increase of colonic inflammation compared to controls. Moreover DSS treatment increased PGE2 and 8-iso-PGF2α levels, along with COX-2 and TNF-α gene expression more markedly in colon specimens of -/- mice compared to controls. LPS-induced PGE2 and 8-iso-PGF2α production from colonic segments incubated ex vivo was also increased in -/- mice.Generalized GHRH gene ablation increases sensitivity to thermal pain and both acute and persistent inflammatory stimuli in male mice.

An overview of a biopsychosocial model of epigenetics and pain catastrophizing

AbstractIn an earlier Special Issue in the Journal of Applied Biobehavioral Research (JABR), Gatchel (Journal of Applied Biobehavioral Research, 2017, 22[1], e12088) addressed the construct of pain catastrophizing (PC). The present article is meant to extend those reviews within a biopsychosocial context, as well as update recent research on PC with a specific concentration on genetic factors. An overview of biological factors as they relate to PC and epigenetics are reviewed first (brain areas associated with pain and how they adapt neurochemically to chronic noxious stimuli, polymorphism of various genes, etc.), proceeded by the discussion of psychological (depression, anxiety, and the genomic link to neuroticism) and social influences (reason people engage in PC, age's impact on neuronal restructuring) as they corroborate the argument of PC's link to genetic factors. Finally, this article concludes by providing future directions for research concerning PC such as examining the efficacy of Pain Neurobiology Education, as well as gene therapy.

Exercise exerts an anxiolytic effect against repeated restraint stress through 5-HT2A-mediated suppression of the adenosine A2A receptor in the basolateral amygdala

Repeated or chronic stressful stimuli induce emotion- and mood-related abnormalities, such as anxiety and depression. Conversely, regular exercise exerts protective effects. Here, we found that exercise recovered anxiety-like behaviors, as measured using the open field and elevated plus maze tests in an anxiety mouse model. In addition to behavioral improvement, exercise enhanced the synaptic density of the 5-hydroxytryptamine 2A receptor (5-HT2AR), but not the 5-HT1AR in the basolateral amygdala (BLA) region in this mouse model. Furthermore, global treatment with a selective 5-HT2AR antagonist (MDL11930) generated an anxiety phenotype. Thus, synaptic recruitment of 5-HT2AR in BLA neurons may mediate the anxiolytic effects of exercise. The exercise regimen also reduced adenosine A2A receptor (A2AR)-mediated protein kinase A (PKA) activation, and the anxiolytic effect of the exercise was blunted by local activation of A2AR within the BLA using CGS21680, a selective A2AR agonist. Particularly, A2AR-mediated PKA activity was shown to be dependent on 5-HT2AR signaling in the BLA. These results imply that repeated stress upregulates A2AR-mediated adenosine signaling to facilitate PKA activation, whereas regular exercise inhibits A2AR function by increasing 5-HT2AR in the BLA. Accordingly, this integrated modulation of 5-HT and adenosine signaling, via 5-HT2AR and A2AR respectively, may be a mechanism underlying the anxiolytic effect of regular exercise.

2034-P: The Role of 14-3-3zeta in the “Beiging” of White Adipose Tissue

Adipocyte hypertrophy and hyperplasia is a hallmark of obesity, and treatments that induce the oxidation of lipid stores may represent a potential therapy. When stimulated by cold or beta-adrenergic agonists, adipocytes in inguinal white adipose tissue (iWAT) can convert to beige cells, which resemble brown adipocytes. This process can reduce body weight in rodents and improve glucose homeostasis through increased Ucp1-dependent lipid oxidation. 14-3-3?, a molecular scaffold we found to be essential for adipogenesis, regulates the enzymatic activities of tryptophan and tyrosine hydroxylases, both of which influence beiging. Thus, the aim of the current study is to investigate whether 14-3-3? influences the beiging process. Compared to control mice, acute (3 hr) and chronic (72 hr) cold (4 C) exposure in male transgenic mice over-expressing 14-3-3? led to improved cold tolerance due to significantly increased Ucp1 mRNA and protein in iWAT. Following chronic exposure, they were also able to maintain their body weight by increasing their food intake. Consistent with these data, analysis of adipocyte area revealed a decrease in the size of inguinal adipocytes in transgenic mice, suggesting increased lipid oxidation. In contrast, gonadal adipocytes were larger in transgenic mice, which may explain their ability to maintain their body weight. Systemic 14-3-3? knockout mice had significantly lower levels of Ucp1 mRNA in iWAT and BAT but did not display differences in tolerance to acute cold. Depletion of 14-3-3? by siRNA in brown adipocytes did not impair isoproterenol-mediated induction of Ucp1 mRNA, suggesting that 14-3-3? is not required for Ucp1 expression in this cell type. In future studies, we will examine if 14-3-3? influences the responsiveness of iWAT to chronic β-adrenergic stimuli and assess the impact of 14-3-3? overexpression in brown adipocyte function. Collectively, our results point to a novel role of 14-3-3? in the beiging of inguinal adipocytes and increase our understanding of how beiging is regulated. Disclosure K. Diallo: None. G.E. Lim: None. Funding Canadian Institutes of Health Research; Fonds de la recherche en santé du Québec; Centre de recherche du CHUM; Montreal Diabetes Research Center

Pain without inflammation in rheumatic diseases.

Chronic pain is a common symptom in rheumatic diseases, and the patient with pain and no signs of inflammation poses a challenge to the physician. Notably, all rheumatic diseases have components of non-inflammatory pain and a higher prevalence of fibromyalgia compared to the overall population. Hypothetically, a chronic pain stimulus may have stronger impact in a chronic inflammatory state, and the process towards a pain condition may be influenced by individual predisposition for development of chronic pain. In addition, the features of peripheral and central pain processing may be exacerbated by inflammation, and disturbed pain processing may be a feature contributing to widespread pain. We herein review and describe the prevalence of chronic pain and different pain modalities in the most common rheumatic diseases. In addition, the background mechanisms of non-inflammatory pain in rheumatic diseases are discussed. Finally, we here review the current strategies for pain management, with a special focus on non-inflammatory pain. The key message is that pain management should be individualized and based on a thorough pain analysis with investigation of the pain modality, localization and pain intensity. Other factors to consider are the underlying rheumatic disease and treatment, the patient's mental and physical health status and psychological factors.

Protective effect of Shaoyao Decoction against colorectal cancer via the Keap1–Nrf2–ARE signaling pathway

Ethnopharmacological relevanceUlcerative colitis is one of the three high risk factors for colorectal cancer. Studies have found that about 20% of cancers are caused by repeated chronic inflammatory stimuli over a long period of time. Ulcer-related colorectal cancer is one of the main causes of death in patients with ulcerative colitis. At present, surgery is the first choice for the treatment of colorectal cancer, combined with radiotherapy and chemotherapy, which have serious side effects. However, reportedly, a compound prescription of Chinese traditional medicine Shaoyao Decoction (SYD) commonly used to treat damp-heat dysentery has anti-colorectal cancer effect. Thus this study described the effect of SYD to AOM/DSS-induced colon cancer model. Aim of the studyIn this study, modern biomedical approaches were employed for investigating the protective/preventive effects of SYD in mice with azoxymethane (AOM)/DSS-induced CRC. Materials and methodsThe mice pretreated with AOM/DSS were randomly allocated to SYDL, SYDM, SYDH group and SASP (sulfasalazine) group. Mice without AOM/DSS treatment were randomly divided into PBS control group and SYD control group. ResultsIt was found that SYD inhibited the production of inflammatory cytokines, TNF-α, IL-1β, superoxide dismutase (SOD), and malonaldehyde (MDA), and increased the antioxidant indices, as measured by the mRNA expression of GR, TR, HO-1, γ-GCSc, γ-GCSm, NQO-1, UGT1A1, and UGT1A10 in AOM-treated mice. Particularly, the expressions rates of NF-κB and Ki-67 in the SYD-treated experimental groups were significantly lower than those in the model group, indicating that the proliferative ability of the CRC tissues was weaker in the SYD-treated experimental groups. Moreover, the positive levels of Nrf2 in the SYD-treated experimental groups were slightly higher than those in the model group, suggesting that SYD exhibited antioxidant activity. ConclusionsTo sum up, our results suggest that SYD inhibits the development of acute/chronic colitis and prevents colitis-associated CRC by suppressing inflammation and preventing oxidative stress-induced cellular damage.

Flavonoids: Broad Spectrum Agents on Chronic Inflammation.

Flavonoids are major plant constituents with numerous biological/pharmacological actions both in vitro and in vivo. Of these actions, their anti-inflammatory action is prominent. They can regulate transcription of many proinflammatory genes such as cyclooxygenase-2/inducible nitric oxide synthase and many cytokines/chemokines. Recent studies have demonstrated that certain flavonoid derivatives can affect pathways of inflammasome activation and autophagy. Certain flavonoids can also accelerate the resolution phase of inflammation, leading to avoiding chronic inflammatory stimuli. All these pharmacological actions with newly emerging activities render flavonoids to be potential therapeutics for chronic inflammatory disorders including arthritic inflammation, meta-inflammation, and inflammaging. Recent findings of flavonoids are summarized and future perspectives are presented in this review.

MON-327 A Case of Parathyroid Carcinoma in a Patient with Late Stage Chronic Kidney Disease

Introduction: Parathyroid carcinoma (PC) is rare, representing 0.3-5% of primary hyperparathyroidism cases. Its pathophysiology is incompletely understood. Established predisposing factors are lacking, with the exception of known genetic mutation and possibly radiotherapy. The rarity of PC and the fact that its clinical and biochemical features overlap benign parathyroid disease make pre-operative diagnosis challenging. Often the diagnosis is made post-operatively through histopathology. It has been hypothesized that chronic kidney disease may predispose to PC, given chronic parathyroid stimulus related to hypocalcemia and hyperphosphatemia. Here we describe an unusual case of parathyroid cancer in a man with long-standing secondary hyperparathyroidism related to stage IV chronic kidney disease. Case report: A 70-year-old man with hypertension and stage IV chronic kidney disease (CKD) presented for treatment ofsuspected tertiary hyperparathyroidism. His intact parathyroid hormone (iPTH) was 384 (8-54 pg/ml) and serum calcium 11.3 (8.6-10.2 mg/dl). He initially opted not to pursue surgery. Five months later, he presented to theemergency department with shortness of breath and new onset of hoarseness. He was diagnosedwith left vocal cord paralysis. Neck ultrasound showed a 1.1 X 1.4 x 1.4 cm hypoechoic nodule posterior and inferior to the lower left thyroid gland with internal color flow, and he was referred to surgery. Pre-operative serumcalcium was 10.1 with iPTH 774. Intra operatively, the mass was adherent to the surrounding musculature, thyroid and esophagus. Biopsy suggested parathyroid cancer. He underwent an en bloc resection of the left lower parathyroid mass, left superior parathyroidectomy, and ipsilateral hemithyroidectomy. Intra-operative iPTH dropped from 747 to 245. Pathology was consistent witha 3 cm parathyroid carcinoma. The posterior margin was focally positive for tumor. PTH remained elevated post-operativelyat 249 with a corrected serum calcium of 8.6. PET CT was without evidence of metabolically active disease,and neck ultrasound was unremarkable.The patientdeclined HRPT2 mutation testing. Conclusion: The vast majority of PC cases present with primary hyperparathyroidism. The first case of PC diagnosed in secondary hyperparathyroidism was published in 1982. Most patients with long-standing CKD develop polyclonal hyperplasia. In some cases, a monoclonal form of nodular growth has been reported. This monoclonal transformation may involve somatic mutations, such as in the tumor suppressor gene WT1. Whether such cases could progress to malignancy is conceivable, although as yet unproven. Biochemical monitoring for PC recurrence in patients with underlying secondary hyperparathyroidism is challenging. PTH levels do not normalize after surgery, and PTH resistance may delay the development of recurrence-associated hypercalcemia.

Inhibition of miRNA-27b enhances neurogenesis via AMPK activation in a mouse ischemic stroke model.

Stroke is a leading cause of death and disability, but treatment options remain limited. Recent studies have suggested that cerebral ischemia‐induced neurogenesis plays a vital role in post‐stroke repair. Overactivation of AMP‐activated protein kinase (AMPK), a master sensor of energy balance, has been reported to exacerbate neuron apoptosis, but the role of chronic AMPK stimulus in post‐stroke recovery remains unclear. MicroRNAs have emerged as regulators of neurogenesis and have been reported to be involved in neurological function. In this study, we verified that miR‐27b directly targets AMPK and inhibits AMPK expression. In cultured neural stem cells, miR‐27b inhibitor improved proliferation and differentiation via the AMPK signaling pathway, but did not have an obvious effect on cell viability under oxygen and glucose deprivation conditions. In a mouse middle cerebral artery occlusion model, administration of miR‐27b inhibitor significantly enhanced behavioral function recovery and spatial memory. Up‐regulation of neurogenesis was observed both in the subventricular zone and in the hippocampal dentate gyrus. Collectively, our data suggest that miR‐27b inhibition promotes recovery after ischemic stroke by regulating AMPK activity. These findings may facilitate the development of novel therapeutic strategies for stroke.

The immunopathology of lung fibrosis: amphiregulin-producing pathogenic memory T helper-2 cells control the airway fibrotic responses by inducing eosinophils to secrete osteopontin.

Fibrosis is defined as excessive deposition of the extracellular matrix (ECM) in the parenchyma of various organs, and sometimes leads to irreversible organ malfunction such as idiopathic pulmonary fibrosis (IPF), a fatal disorder of the lung. Chronic inflammatory stimuli induce fibrotic responses in various organs. Various immune cells, including T helper (Th) cells in the lung, protect the host from different harmful particles, including pathogenic microorganisms. However, the dysregulation of the function of these immune cells in the lung sometimes causes inflammatory diseases, such as lung fibrosis. In this review, we will introduce an outline of the cellular and molecular mechanisms underlying the pathogenic fibrotic responses in the lung. We will also introduce the concept of the "Pathogenic Th population disease induction model," in which unique subpopulations of certain Th cell subsets control the pathology of immune-mediated inflammatory diseases. Finally, we introduce our recent findings, which demonstrate that amphiregulin-producing pathogenic memory Th2 cells control airway fibrosis through the osteopontin produced by inflammatory eosinophils. The identification of this new pathogenic Th cell population supports the concept of "Pathogenic Th population disease induction model", and will provide novel strategies for treating intractable diseases, including lung fibrosis.

Chronic systemic inflammatory challenge induced by LPS promotes cognitive impairment

Neuroinflammation is a major component in the progression of neurodegenerative diseases such as dementia, although is not its main initiator. Its been demonstrated that inflammatory activation of microglia and astrocytes promotes protein aggregation of amyloid plaques and tau tangles, leading to neuronal death through other mechanisms, such as excitotoxicity. Although many studies investigated the effect of systemic inflammation on the progression of dementia and amyloid plaques in genetic mouse models of Alzheimer's disease, is not clear if systemic inflammation can induce dementia per se.Methods90‐day Wistar rats were exposed to chronic inflammatory challenges with intraperitoneal injections of LPS in two doses (500 μg/kg and 250 μg/kg) or saline once a week, for a 16‐week period. Rats were euthanasied 24 h after last LPS injection. Behavioral evaluation was conducted through Open Field (OF) and Object Location Memory‐Test (OLM) for spatial memory assessment. Hippocampal S100B content was measured by ELISA, nuclear Nf‐κB was measured by western blot, and L‐[3H] glutamate uptake was analyzed.ResultsBoth groups receiving LPS presented impaired spatial memory in the 24 h OLM, which is characteristic of hippocampal degeneration in animal models of dementia, while OF shown no locomotor impairment. S100B protein remained unchanged between groups, and glutamate uptake was reduced only in LPS 500 μ/kg group. Nuclear fraction of Nf‐κB was increased in both LPS groups.DiscussionHere, we demonstrate that chronic peripheral inflammatory stimuli promote cognitive impairment in the absence of other underlying neurodegenerative causes, such as genetic background. Increased Nf‐κB in nucleus indicates hippocampal neuroinflammation, despite LPS challenge being peripheral. Besides, glutamate uptake was reduced in the higher dose of LPS, indicating a possible excitotoxic damage. Conversely, S100B content, which is an important biomolecule in inflammatory signaling, was not affected by LPS.Support or Funding InformationThis study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Apoio a Pesquisa do Estado do Rio Grande do Sul (FAPERGS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A high‐throughput microfluidic microphysiological system (PREDICT‐96) to recapitulate hepatocyte function in dynamic flow conditions

Microphysiological systems (MPSs) are dynamic cell culture platforms that incorporate mechanical and chemical cues to recapitulate organ‐specific functions and architecture. Recent advances in microfabrication technologies and processes have enabled the development of enhanced in vitro models that capture human physiology more accurately while increasing throughput and reducing material‐based technical artifacts. A common candidate for MPS development is liver due to its crucial role in drug metabolism, inflammation and human disease. While several liver‐on‐chip models exist, few studies have combined long term culture with dynamic inputs, such as media flow, in a high‐throughput format. Herein, we have developed an oxygen permeable, high‐throughput (96‐well form factor), thermoplastic MPS (PREDICT‐96 array) integrated with an on‐board, ultra‐low volume, recirculating pumping system (PREDICT‐96 pump) to facilitate media flow in the system; collectively, these technologies comprise the PREDICT‐96 platform. To demonstrate usability of the PREDICT‐96 array, we introduce PHHs using standard handling procedures for multi‐well plates and maintain viable culture for up to 14 days. By incorporating media flow to mimic in vivo mass transport, we observe a robust increase in metabolic and secretory functions of PHHs when compared to static culture. The PHHs demonstrate reproducible baseline metabolic activity and secretion markers which underscores the utility of the system for drug screening applications. Furthermore, long‐term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and resolve cellular responses to chronic stimuli over time. The low‐volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi‐cellular co‐cultures and integration with high‐throughput analytical tools.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(235) Homeostatic Regulation of Intrinsic Plasticity in Peripheral Nociceptors

Stabilization of neuronal activity is crucial for normal nervous system regulation. When changes in the environment chronically alter neuronal activity, homeostatic plasticity creates compensatory shifts (i.e., regulation of intrinsic excitability) to return the system to a physiological level. This plasticity helps prevent circuits from becoming either hyper- or hypoexcitable. Despite this protective mechanism, evidence suggests that the development and maintenance of many chronic pain conditions depend on chronically altered sensory afferent activity. This led us to consider the possibility that malfunction of homeostatic mechanisms may contribute to chronic pain. Since homeostatic plasticity has been primarily studied in the central nervous system, we first tested whether this plasticity also occurs in peripheral nociceptors [i.e., small diameter dorsal root ganglia (DRG) sensory afferent neurons]. We hypothesized that mouse and human primary sensory afferents undergo homeostatic regulation of intrinsic plasticity, evidenced by compensatory changes in neuronal excitability in response to chronic stimuli. Using a combination of pharmacologic and optogenetic approaches with whole-cell electrophysiology, we previously presented that sustained, 24-hour KCl depolarization of dissociated mouse and human DRG neurons led to a compensatory decrease in excitability of small diameter cells. This suggested that putative nociceptors undergo homeostatic regulation of intrinsic excitability. In an effort to demonstrate bidirectionality of this homeostatic plasticity, in this study we used a 24-hour incubation with lidocaine and TTX. We found no change in excitability, likely due to the already quiescent nature of DRG neurons in vitro. Ongoing experiments are being performed to determine whether mechanisms of this plasticity are altered in vivo and during chronic pain. This work was supported by grants R01 NS042595 (RG) and T32DA007261 (LM).

Lumps and Bumps of the Gingiva: A Pathological Miscellany

Lesions of the gingivae are amongst the commonest lesions seen in patients and the vast majority are reactive hyperplasias, related to a number of chronic irritant stimuli. However, there are a number of entities that have a predilection for the gingivae, which are much less common in other parts of the oral cavity. The purpose of this paper is to discuss the clinical and histological differential diagnoses when presented with a lump on the gingivae, including the approach to diagnosis and diagnostic pitfalls.

Heat attenuates sensitivity of mammalian cells to capsaicin.

The transient receptor potential (TRP) channels are thermo-sensors, and transient receptor potential vanilloid (TRPV)1 and V4 are widely expressed in primary afferent neurons and nonneuronal cells. Although heat acclimation is considered as changes of thermoregulatory responses by thermo-effectors to heat, functional changes of TRP channels in heat acclimation has not been fully elucidated. Here, we investigated whether heat acclimation induces capsaicin tolerance. NIH3T3 cells were incubated at 39.5°C. We determined the expression level of TRPV1 and TRPV4 messenger RNA (mRNA), performed cellular staining of TRPV1 and TRPV4, and investigated actin assembly and activation of the extracellular signal-regulated kinase (ERK). Exposure to moderate heat decreased the levels of TRPV1 but not TRPV4 mRNA. It also induced stress fiber formation and the intensity of TRPV1 seemed to be decreased by chronic heat stimuli. In addition, heat acclimation attenuated the capsaicin-induced activation of ERK. Heat acclimation may induce capsaicin tolerance via the downregulation of TRPV1.

The Physiopathological Crossroads of Aging

Stress, inflammation and Plasminogen activator inhibitor 1 (PAI-1) are key mechanisms throughout the development of aging, constituting a crossroad in the most frequent pathologies that accompany it. Among metabolic processes, obesity, metabolic syndrome and type 2 diabetes mellitus are included and Alzheimer’s disease among the neurodegenerative processes. Stress is a mechanism of defense of the organism against exogenous and endogenous actions called stressors. In the case of low intensity stimuli, the organism responds with actions aimed at a physiological adaptation (Homeostasis). On the other hand, when a high intensity (experimental level) or chronic stimulus (oxidative stress) is repeated, structural and functional changes are observed in different organs with activation of the hypothalamus-pituitary-adrenal axis, the renin angiotensin system and the sympathetic nervous system, stimulating the production of hormones that release cytokines with proin-flammatory/antiinflammatory properties that play an important role in the previously mentioned pathologies, as well as a marked increase in PAI-1, a gene regulated by stress and by cytokines, with manifest action at the origin of thromboembolic disease, so frequent in aging. The objective of this review is to highlight the importance of the binomial stress and PAI-1 in aging and in the pathologies that accompany it. Because PAI-1 is part of the pathology and complications in aging, some authors suggest the study of PAI-1 inhibitors to achieve its physiological levels, as part of the treatment of these diseases.