Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical ...production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological “normoxic” limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard “normoxic” oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen's sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed “consumptive oxygen depletion” (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.
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•Oxygen (O2) is an often overlooked variable in cell and tissue culture systems.•We discuss differences between in vivo and in vitro O2 diffusion.•Alternative culturing technology to enhance O2 delivery are highlighted.•Guidelines to improve cell culture quality and minimize O2 fluctuations are proposed.
The field of free radical biology originated with the discovery of superoxide dismutase (SOD) in 1969. Over the last 5 decades, a plethora of research has been performed in species ranging from ...bacteria to mammals that has elucidated the molecular reaction, subcellular location, and specific isoforms of SOD. However, while humans have only begun to study this class of enzymes over the past 50 years, it has been estimated that these enzymes have existed for billions of years, and may be some of the original enzymes found in primitive life. As life evolved over this expanse of time, these enzymes have taken on new and different functional roles potentially in contrast to how they were originally derived. Herein, examination of the evolutionary history of these enzymes provides both an explanation and further inquiries into the modern-day role of SOD in physiology and disease.
Post-traumatic stress disorder (PTSD) is a mental health disorder that arises after experiencing or witnessing a traumatic event. Despite affecting around 7% of the population, there are currently no ...definitive biological signatures or biomarkers used in the diagnosis of PTSD. Thus, the search for clinically relevant and reproducible biomarkers has been a major focus of the field. With significant advances of large-scale multi-omic studies that include genomic, proteomic, and metabolomic data, promising findings have been made, but the field still has fallen short. Amongst the possible biomarkers examined, one area is often overlooked, understudied, or inappropriately investigated: the field of redox biology. Redox molecules are free radical and/or reactive species that are generated as a consequence of the necessity of electron movement for life. These reactive molecules, too, are essential for life, but in excess are denoted as "oxidative stress" and often associated with many diseases. The few studies that have examined redox biology parameters have often utilized outdated and nonspecific methods, as well as have reported confounding results, which has made it difficult to conclude the role for redox in PTSD. Herein, we provide a foundation of how redox biology may underlie diseases like PTSD, critically examine redox studies of PTSD, and provide future directions the field can implement to enhance standardization, reproducibility, and accuracy of redox assessments for the use of diagnosis, prognosis, and therapy of this debilitating mental health disorder.
Neutrophils are key effector cells of the innate immune system, serving as a first line of defense in the response to injury and playing essential roles in the wound healing process. Following ...myocardial infarction (MI), neutrophils infiltrate into the infarct region to propagate inflammation and begin the initial phase of cardiac wound repair. Pro-inflammatory neutrophils release proteases to degrade extracellular matrix (ECM), a necessary step for the removal of necrotic myocytes as a prelude for scar formation. Neutrophils transition their phenotype over time to regulate MI inflammation resolution and stabilize scar formation. Neutrophils contribute to the evolution from inflammation to resolution and scar formation by serving anti-inflammatory and repair functions. As anti-inflammatory cells, neutrophils contribute ECM proteins during scar formation, in particular fibronectin, galectin-3, and vimentin. The diverse and polarizing functions that contribute to MI wound repair make this innate immune cell a viable target to improve MI outcomes. Thus, understanding the signaling involved in neutrophil physiology in the context of MI may help to identify novel therapeutic targets.
•Neutrophils play key roles during repair after myocardial infarction (MI).•Neutrophils transition from pro-inflammatory to anti-inflammatory to reparative over the MI time continuum.•Targeting neutrophil signaling is an unexplored avenue for therapeutic intervention.
In the field of psychiatry, biological markers are rarely, if ever, used in the diagnosis of mental health disorders. Clinicians rely primarily on patient histories and behavioral symptoms to ...identify specific psychopathologies, which makes diagnosis highly subjective. Moreover, therapies for mental health disorders are aimed specifically at attenuating behavioral manifestations, which overlooks the pathophysiological indices of the disease. This is highly evident in posttraumatic stress disorder (PTSD) where inflammation and immune system perturbations are becoming increasingly described. Further, patients with PTSD possess significantly elevated risks of developing comorbid inflammatory diseases such as autoimmune and cardiovascular diseases, which are likely linked (though not fully proven) to the apparent dysregulation of the immune system after psychological trauma. To date, there is little to no evidence that demonstrates current PTSD therapies are able to reverse the increased risk for psychological trauma-induced inflammatory diseases, which suggests the behavioral and somatic consequences of PTSD may not be tightly coupled. This observation provides an opportunity to explore unique mechanisms outside of the brain that contribute to the long-term pathology of PTSD. Herein, we provide an overview of neuroimmune mechanisms, describe what is known regarding innate and adaptive immunity in PTSD, and suggest new directions that are needed to advance the understanding, diagnosis, and treatment of PTSD moving forward.
•Posttraumatic stress disorder (PTSD) is associated with systemic inflammation.•PTSD significantly increases the risk of developing comorbid inflammatory diseases.•Current PTSD therapies may not attenuate the apparent inflammation.•Underlying mechanisms of immune dysfunction in PTSD are unknown.•We propose areas of research needed to comprehensively understand PTSD pathology.
T lymphocytes, as part of the adaptive immune system, possess the ability to activate and function in extreme cellular microenvironments, which requires these cells to remain highly malleable. One ...mechanism in which T lymphocytes achieve this adaptability is by responding to cues from both reactive oxygen and nitrogen species, as well as metabolic flux, which together fine-tune the functional fate of these adaptive immune cells.
To date, examinations of the redox and metabolic effects on T lymphocytes have primarily investigated these biological processes as separate entities. Given that the redox and metabolic environments possess significant overlaps of pathways and molecular species, it is inevitable that perturbations in one environment affect the other. Recent consideration of this redox-metabolic couple has demonstrated the strong link and regulatory consequences of these two systems in T lymphocytes.
The redox and metabolic control of T lymphocytes is essential to prevent dysregulated inflammation, which has been observed in cardiovascular diseases such as hypertension. The role of the adaptive immune system in hypertension has been extensively investigated, but the understanding of how the redox and metabolic environments control T lymphocytes in this disease remains unclear.
Herein, we provide a discussion of the redox and metabolic control of T lymphocytes as separate entities, as well as coupled to one another, to regulate adaptive immunity. While investigations examining this pair together in T lymphocytes are sparse, we speculate that T lymphocyte destiny is shaped by the redox-metabolic couple. In contrast, disrupting this duo may have inflammatory consequences such as hypertension.
•Repeated social defeat stress (RSDS) reproducibly produces peripheral inflammation.•Peripheral inflammation is not coupled to social interaction (SI) testing parameters.•SI-defined susceptible and ...resilient categorization does not reflect peripheral inflammation.•Anxiety-like behavioral parameters are linked to peripheral inflammation in RSDS.•Peripheral inflammation is more predictive of anxiety-like than social behavior in RSDS.
Post-traumatic stress disorder (PTSD) is a psychiatric illness that results in an increased risk for a variety of inflammatory diseases. The exact etiology of this increased risk is unknown, and thus several animal models have been developed to investigate the neuroimmune interactions of PTSD. Repeated social defeat stress (RSDS) is an established preclinical model of psychological trauma that recapitulates certain behavioral and inflammatory aspects of human PTSD. Furthermore, RSDS has been utilized to subgroup animals into susceptible and resilient populations based on one specific behavioral phenotype (i.e., social interaction). Herein, we conducted an extensive investigation of circulating inflammatory proteins after RSDS and found significant elevations in various cytokines and chemokines after exposure to RSDS. When categorizing animals into either susceptible or resilient populations based on social interaction, we found no inflammatory or other behavioral differences between these subgroups. Furthermore, correlative analyses found no significant correlation between social interaction parameters and inflammation. In contrast, parameters from the elevated zero maze (EZM) demonstrated significant associations and clustering to five circulating cytokines. When animals were subdivided into susceptible and resilient populations solely based upon combined EZM performance, significant inflammatory differences were evident between these groups. Strikingly, these circulating inflammatory proteins displayed a stronger predictive ability of EZM performance compared to social interaction test performance. These findings provide new insights into inflammatory markers associated with RSDS, and the utility of EZM to effectively group RSDS-exposed mice into populations with differential levels of peripheral inflammation.
Norepinephrine (NE) produces multifaceted regulatory patterns in T-lymphocytes. Recently, we have shown that NE utilizes redox signaling as evidenced by increased superoxide (O2●-) causally linked to ...the observed changes in these cells; however, the source of this reactive oxygen species (ROS) remains elusive. Herein, we hypothesized that the source of increased O2●- in NE-stimulated T-lymphocytes is due to disruption of mitochondrial bioenergetics. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T-lymphocytes stimulated with NE and assessed O2●- levels, mitochondrial metabolism, cellular proliferation, and cytokine profiles. We demonstrate that the increase in O2●- levels in response to NE is time-dependent and occurs at later points of T-lymphocyte activation. Moreover, the source of O2●- was indeed the mitochondria as evidenced by enhanced MitoSOX Red oxidation as well as abrogation of this signal by the addition of the mitochondrial-targeted O2●--scavenging antioxidant MitoTempol. NE-stimulated T-lymphocytes also demonstrated decreased mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2●-. The effects of NE in regards to redox signaling appear to be adrenergic receptor-dependent as specific receptor antagonists could reverse the increase in O2●-; however, differential receptors regulating these processes were observed in CD4+ versus CD8+ T-lymphocytes. Finally, mitochondrial O2●- was shown to be mechanistic to the NE-mediated T-lymphocyte phenotype as supplementation of MitoTempol could reverse specific changes in cytokine expression observed with NE treatment. Overall, these studies indicate that mitochondrial metabolism and O2●--mediated redox signaling play a regulatory role in the T-lymphocyte response to NE.
Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have ...demonstrated that the CSAR to capsaicin transient receptor potential (TRP) vanilloid 1 agonist is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10-12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10-12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.
Cytokine levels throughout the perinatal period Moore, Tiffany A.; Case, Adam J.
Journal of maternal-fetal and neonatal medicine/Journal of maternal-fetal & neonatal medicine
35, Issue:
25
Journal Article
Peer reviewed
Open access
Dysregulation of inflammatory processes is linked to perinatal complications yet a comprehensive description of cytokine levels throughout the perinatal period is lacking. We report prospective, ...serial levels of 29 unique cytokines measured in maternal blood during pregnancy, in the cord blood at birth, and in the neonatal blood.
Pregnant women (n = 140) for recruited from a Midwest tertiary medical center. Blood was obtained at five timepoints: 12-20 weeks, 24-28 weeks, and at labor in the women, umbilical cord at birth, 24-72 h in the newborn. Cytokine levels were analyzed using an electrochemiluminescence-based immunoassay.
Levels for 29 cytokines were measured. The data were separated into two groups: pregnancies with (n = 82) and without major complications (n = 53) (preterm birth, preeclampsia, diabetes mellitus). Eighteen cytokines showed significant changes over time (p < .002). The majority of the cytokines were highest in the newborn. No differences in cytokine levels between complication groups were noted at any timepoint.
This is the first known study to report prospective, serial cytokine levels throughout the perinatal period for pregnancies with/without complications. No differences in maternal cytokine levels between those with/without complications were detected; studies with a larger sample size would be needed to validate our current findings. Results also suggest cytokine dysregulation may be more localized to the placenta making it difficult to measure and predict during pregnancy using maternal systemic blood specimens.
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