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.

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.

Reactive oxygen species (ROS) are critical in a broad spectrum of cellular processes including signaling, tumor progression, and innate immunity. The essential nature of ROS signaling in the immune systems of Drosophila and zebrafish has been demonstrated; however, the role of ROS, if any, in mammalian adaptive immune system development and function remains unknown. The current work provides the first clear demonstration that thymus specific elevation of mitochondrial superoxide (O2·−) disrupts normal T-cell development to impair function of the mammalian adaptive immune system. To assess the effect of elevated mitochondrial superoxide in the developing thymus, we used a T-cell specific knockout of manganese superoxide dismutase (i.e. SOD2) and have thus established a murine model to examine the role of mitochondrial superoxide in T-cell development. Conditional loss of SOD2 led to increased superoxide, apoptosis, and developmental defects in the T-cell population resulting in immunodeficiency and susceptibility to influenza A virus (IAV), H1N1. This phenotype was rescued with mitochondrially targeted superoxide scavenging drugs. These new findings demonstrate that loss of regulated levels of mitochondrial superoxide lead to aberrant T-cell development and function, and further suggest that manipulations of mitochondrial superoxide levels may significantly alter clinical outcomes resulting from viral infection.

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O₂∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O₂∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O₂∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O₂∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

Patients diagnosed with post-traumatic stress disorder (PTSD) are at a significantly elevated risk of developing comorbid inflammatory conditions, but the mechanisms underlying this predilection remain unclear. Our previous work has shown that T-lymphocytes exposed to elevated levels of norepinephrine (NE) displayed a pro-inflammatory signature reminiscent of an autoreactive phenotype. With this, we hypothesized that the increased sympathetic tone observed during psychological trauma may be promoting pro-inflammatory T-lymphocytes, which causes a predisposition to comorbid inflammatory conditions. Here, we examined the consequences of psychological trauma on splenic T-lymphocytes using a mouse model of repeated social defeat stress. Social defeat led to anxiety-like and depression-like behavior as has been previously described. The spleens of socially-defeated mice showed significant elevations of NE, tyrosine hydroxylase (TH), and acetylcholinesterase (ACHE) levels, which appeared to be due in part to increased expression within T-lymphocytes. Additionally, T-lymphocytes from stressed animals showed higher levels of pro-inflammatory cytokines and mitochondrial superoxide. Interestingly, in this model system, close associations exist within splenic T-lymphocytes amid the autonomic, inflammatory, and redox environments, but these only weakly correlate with individual behavioral differences among animals suggesting the psychological and physiological manifestations of trauma may not be tightly coupled. Last, we describe, for the first time, elevations in calprotectin levels within T-lymphocytes and in circulation of psychologically stressed animals. Calprotectin correlated with both behavioral and physiological changes after social defeat, suggesting the potential for a new biological marker and/or therapeutic target for psychological trauma and its inflammatory comorbidities.