Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology

Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: Rb (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and Cm (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl2 reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program’s modelling demonstrated that CoCl2 affected Rb as it begins to drastically decrease earlier than α or Cm, although ARPE-19 cells’ viability was not compromised. Using seahorse technology, all three concentrations of CoCl2 significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells’ viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters.

Unreliable Early Neuroprognostication After Severe Carbon Monoxide Poisoning Is Likely Due to Cytopathic Hypoxia: A Case Report and Discussion

A 17-year-old girl was found unconscious in a running vehicle. She developed very severe acute respiratory distress syndrome (which was treated with rescue high-frequency oscillation), hemodynamic instability, acute kidney injury, rhabdomyolysis, and remained comatose with a Glasgow Coma Scale score of 3 and gasping respirations for 67 hours (when the Glasgow Coma Scale score improved to 6, with tachypnea to Paco 2 28 and pH 7.5). By 92 hours, she was obeying commands, and she was extubated at 96 hours, shortly after which she was conversing with family and texting on her phone. A magnetic resonance imaging (MRI) scan 6 days after being found showed subacute infarctions affecting the medial aspect of the globus pallidus bilaterally as well as a small cortical/subcortical infarction in the right parietal lobe. At a 7-week follow-up, she had no delayed-onset signs of brain injury. This case demonstrated that neurologic prognostication after carbon monoxide poisoning may be unreliable for more than 72 hours after injury. We discuss that it is possible that the mitochondrial dysfunction induced by carbon monoxide was responsible for a functional coma without irreversible brain injury, similar to the mechanism of cytopathic hypoxia in multiple-organ dysfunction that allows some other organ recovery without necrosis in survivors.

Liposomal NAD+ prevents diminished O2consumption by immunostimulated Caco-2 cells

Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O2, a phenomenon that has been termed “cytopathic hypoxia.” We sought to use an in vitro “reductionist” model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD+/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O2 consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-α, interleukin-1β, and interferon-γ. Immunostimulated cells consumed O2 at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO−) decomposition catalyst; urate, an ONOO− scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)- N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O2 uptake induced by cytomix was associated with decreased cellular levels of NAD+/NADH. The decrease in cellular NAD+/NADH content and the decrease in O2 uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD+ was added to the cultures during immunostimulation. Empty liposomes also increased O2 uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD+. These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O2 due to NAD+/NADH depletion secondary to activation of PARP by ONOO− or other oxidants.