Cell-Free DNA as a Biomarker in a Rodent Model of Chlorpyrifos Poisoning Causing Mitochondrial Dysfunction.

INTRODUCTION: Organophosphates (OPs) are a major public health problem worldwide due to ease of access and high toxicity lacking effective biomarkers and treatment. Cholinergic agents such as OPs and carbamates are responsible for many pesticide-related deaths. While the inhibition of AChE is thought to be the main mechanism of injury, there are other important pathways that contribute to the overall toxicity of OPs such as mitochondrial dysfunction. An existing gap in OP poisoning are biomarkers to gauge severity and prognosis. Cell-free DNA (cfDNA) are novel biomarkers that have gained increased attention as a sensitive biomarker of disease with novel use in acute poisoning. This study investigates alterations in cerebral mitochondrial function in a rodent model of chlorpyrifos poisoning with the use of cfDNA as a potential biomarker. METHODS: Twenty rodents were divided into two groups: Control (n = 10) and Chlorpyrifos (n = 10). Chlorpyrifos was administered through the venous femoral line with a Harvard Apparatus 11 Elite Syringe pump (Holliston, MA, USA) at 2 mg/kg. Animals were randomized to receive chlorpyrifos versus the vehicle (10% DMSO) for 60 min which would realistically present an acute exposure with continued absorption. At the end of the exposure (60 min), isolated mitochondria were measured for mitochondrial respiration along with measures of acetylcholinesterase activity, cfDNA, cytokines and western blot. RESULTS: The Chlorpyrifos group showed a significant decrease in heart rate but no change in the blood pressure. There was a significant increase in bulk cfDNA concentrations and overall decrease in mitochondrial respiration from brain tissue obtained from animals in the Chlorpyrifos group when compared to the Control group with no difference in acetylcholinesterase activity. In addition, there was a significant increase in both IL-2 and IL-12 in the Chlorpyrifos group. CONCLUSIONS: In our study, we found that the total cfDNA concentration may serve as a more accurate biomarker of OP exposure compared to acetylcholinesterase activity. In addition, there was an overall decrease in cerebral mitochondrial function in the Chlorpyrifos group when compared to the Control group.

A randomized and blinded trial of inhaled nitric oxide in a piglet model of pediatric cardiopulmonary resuscitation.

AIM: Inhaled nitric oxide (iNO) during cardiopulmonary resuscitation (CPR) improved systemic hemodynamics and outcomes in a preclinical model of adult in-hospital cardiac arrest (IHCA) and may also have a neuroprotective role following cardiac arrest. The primary objectives of this study were to determine if iNO during CPR would improve cerebral hemodynamics and mitochondrial function in a pediatric model of lipopolysaccharide-induced shock-associated IHCA. METHODS: After lipopolysaccharide infusion and ventricular fibrillation induction, 20 1-month-old piglets received hemodynamic-directed CPR and were randomized to blinded treatment with or without iNO (80 ppm) during and after CPR. Defibrillation attempts began at 10 min with a 20-min maximum CPR duration. Cerebral tissue from animals surviving 1-h post-arrest underwent high-resolution respirometry to evaluate the mitochondrial electron transport system and immunohistochemical analyses to assess neuropathology. RESULTS: During CPR, the iNO group had higher mean aortic pressure (41.6 ±â€¯2.0 vs. 36.0 ±â€¯1.4 mmHg; p = 0.005); diastolic BP (32.4 ±â€¯2.4 vs. 27.1 ±â€¯1.7 mmHg; p = 0.03); cerebral perfusion pressure (25.0 ±â€¯2.6 vs. 19.1 ±â€¯1.8 mmHg; p = 0.02); and cerebral blood flow relative to baseline (rCBF: 243.2 ±â€¯54.1 vs. 115.5 ±â€¯37.2%; p = 0.02). Among the 8/10 survivors in each group, the iNO group had higher mitochondrial Complex I oxidative phosphorylation in the cerebral cortex (3.60 [3.56, 3.99] vs. 3.23 [2.44, 3.46] pmol O2/s mg; p = 0.01) and hippocampus (4.79 [4.35, 5.18] vs. 3.17 [2.75, 4.58] pmol O2/s mg; p = 0.02). There were no other differences in mitochondrial respiration or brain injury between groups. CONCLUSIONS: Treatment with iNO during CPR resulted in superior systemic hemodynamics, rCBF, and cerebral mitochondrial Complex I respiration in this pediatric cardiac arrest model.

Alterations in cerebral and cardiac mitochondrial function in a porcine model of acute carbon monoxide poisoning.

OBJECTIVES: The purpose of this study is the development of a porcine model of carbon monoxide (CO) poisoning to investigate alterations in brain and heart mitochondrial function. DESIGN: Two group large animal model of CO poisoning. SETTING: Laboratory. SUBJECTS: Ten swine were divided into two groups: Control (n = 4) and CO (n = 6). INTERVENTIONS: Administration of a low dose of CO at 200 ppm to the CO group over 90 min followed by 30 min of re-oxygenation at room air. The Control group received room air for 120 min. MEASUREMENTS: Non-invasive optical monitoring was used to measure cerebral blood flow and oxygenation. Cerebral microdialysis was performed to obtain semi real time measurements of cerebral metabolic status. At the end of the exposure, both fresh brain (cortical and hippocampal tissue) and heart (apical tissue) were immediately harvested to measure mitochondrial respiration and reactive oxygen species (ROS) generation and blood was collected to assess plasma cytokine concentrations. MAIN RESULTS: Animals in the CO group showed significantly decreased Complex IV-linked mitochondrial respiration in hippocampal and apical heart tissue but not cortical tissue. There also was a significant increase in mitochondrial ROS generation across all measured tissue types. The CO group showed a significantly higher cerebral lactate-to-pyruvate ratio. Both IL-8 and TNFα were significantly increased in the CO group compared with the Control group obtained from plasma. While not significant there was a trend to an increase in optically measured cerebral blood flow and hemoglobin concentration in the CO group. CONCLUSIONS: Low-dose CO poisoning is associated with early mitochondrial disruption prior to an observable phenotype highlighting the important role of mitochondrial function in the pathology of CO poisoning. This may represent an important intervenable pathway for therapy and intervention.

In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning.

The objective of this study was to compare the use of hydroxocobalamin (B12a) and a succinate prodrug to evaluate for improvement in mitochondrial function in an in vitro model of cyanide poisoning. Peripheral blood mononuclear cells (PBMC) and human aortic smooth muscle cells (HASMC) incubated with 50 mM of sodium cyanide (CN) for five minutes serving as the CN group compared to controls. We investigated the following: (1) Mitochondrial respiration; (2) Superoxide and mitochondrial membrane potential with microscopy; (3) Citrate synthase protein expression. All experiments were performed with a cell concentration of 2-3 × 106 cells/ml for both PBMC and HASMC. There were four conditions: (1) Control (no exposure); (2) Cyanide (exposure only); (3) B12a (cyanide exposure followed by B12a treatment); (4) NV118 (cyanide followed by NV118 treatment). In this study the key findings include: (1) Improvement in key mitochondrial respiratory states with the succinate prodrug (NV118) but not B12a; (2) Attenuation of superoxide production with treatment of NV118 but not with B12a treatment; (3) The changes in respiration were not secondary to increased mitochondrial content as measured by citrate synthase; (4) The use of easily accessible human blood cells showed similar mitochondrial response to both cyanide and treatment to HASMC. The use of a succinate prodrug to circumvent partial CIV inhibition by cyanide with clear reversal of cellular respiration and superoxide production that was not attributed to changes in mitochondrial content not seen by the use of B12a.

Ex vivo use of cell-permeable succinate prodrug attenuates mitochondrial dysfunction in blood cells obtained from carbon monoxide-poisoned individuals.

The purpose of this study was to evaluate a new pharmacological strategy using a first-generation succinate prodrug, NV118, in peripheral blood mononuclear cells (PBMCs) obtained from subjects with carbon monoxide (CO) poisoning and healthy controls. We obtained human blood cells from subjects with CO poisoning and healthy control subjects. Intact PBMCs from subjects in the CO and Control group were analyzed with high-resolution respirometry measured in pmol O2 per second per 10-6 PBMCs. In addition to obtaining baseline respiration, NV118 (100 µM) was injected, and the same parameters of respiration were obtained for comparison in PBMCs. We measured mitochondrial dynamics with microscopy with the same conditions. We enrolled 37 patients (17 in the CO group and 20 in the Control group for comparison) in the study. PMBCs obtained from subjects in the CO group had overall significantly lower respiration compared with the Control group (P < 0.0001). There was a significant increase in respiration with NV118, specifically with an increase in maximum respiration and respiration from complex II and complex IV (P < 0.0001). The mitochondria in PBMCs demonstrated an overall increase in net movement compared with the Control group. Our results of this study suggest that the therapeutic compound, NV118, increases respiration at complex II and IV as well as restoration of mitochondrial movement in PBMCs obtained from subjects with CO poisoning. Mitochondrial-directed therapy offers a potential future strategy with further exploration in vivo.

Prophylaxis of mitochondrial dysfunction caused by cellular decompression from hyperbaric exposure.

Mitochondrial dysfunction occurring in response to cellular perturbations can include altered mitochondrial motility and bioenergetic function having intracellular heterogeneity. Exogenous mitochondrial directed therapy may correct these dysfunctions. Using in vitro approaches, we find that cell perturbations induced by rapid decompression from hyperbaric conditions with specific gas exposures has differential effects on mitochondrial motility, inner membrane potential, cellular respiration, reactive oxygen species production, impaired maintenance of cell shape and altered intracellular distribution of bioenergetic capacity in perinuclear and cell peripheral domains. Addition of a first-generation cell-permeable succinate prodrug to support mitochondrial function has positive overall effects in blunting the resultant bioenergy responses. Our results with this model of perturbed cell function induced by rapid decompression indicate that alterations in bioenergetic state are partitioned within the cell, as directly assessed by a combination of mitochondrial respiration and dynamics measurements. Reductions in the observed level of dysfunction produced can be achieved with application of the cell-permeable succinate prodrug.

Stiffness can mediate balance between hydrodynamic forces and avidity to impact the targeting of flexible polymeric nanoparticles in flow.

We report computational investigations of deformable polymeric nanoparticles (NPs) under colloidal suspension flow and adhesive environment. We employ a coarse-grained model for the polymeric NP and perform Brownian dynamics (BD) simulations with hydrodynamic interactions and in the presence of wall-confinement, particulate margination, and wall-adhesion for obtaining NP microstructure, shape, and anisotropic and inhomogeneous transport properties for different NP stiffness. These microscopic properties are utilized in solving the Fokker-Planck equation to obtain the spatial distribution of NP subject to shear, margination due to colloidal microparticles, and confinement due to a vessel wall. Comparing our computational results for the amount of NP margination to the near-wall adhesion regime with those of our binding experiments in cell culture under shear, we found quantitative agreement on shear-enhanced binding, the effect of particulate volume fraction, and the effect of NP stiffness. For the experimentally realized polymeric NP, our model predicts that the shear and volume fraction mediated enhancement in targeting has a hydrodynamic transport origin and is not due to a multivalent binding effect. However, for ultrasoft polymeric NPs, our model predicts a substantial increase in targeting due to multivalent binding. Our results are also in general agreement with experiments of tissue targeting measurements in vivo in mice, however, one needs to exercise caution in extending the modeling treatment to in vivo conditions owing to model approximations. The reported combined computational approach and results are expected to enable fine-tuning of design and optimization of flexible NP in targeted drug delivery applications.

Mitochondrial networking in human blood cells with application in acute care illnesses.

Mitochondria are dynamic organelles that adapt in response to environmental stresses or mutations. Dynamic processes involving mitochondria include their locomotion within cells and fusion and fission events in which mitochondrial join together or split apart. Various imaging strategies have been utilized to track mitochondrial dynamics. One common limitation of most of the methods available is that the time required to perform the technique and analyze the results prohibits application to clinical diagnosis and therapy. We recently demonstrated "whole-cell" mitochondrial analysis in a two-dimensional fashion with fluorescence microscopy. Our developed technique allows evaluation of whole-cell mitochondrial networking, including assessment of mitochondrial motility and rates of fission and fusion events using human blood cells (peripheral blood mononuclear cells (PBMCs)) on a clinically relevant timescale. We demonstrate this methodology in a cohort of healthy subjects as well as a cohort of hospitalized subjects having sepsis, an acute care illness. As there is increasing use of human blood cells as a proxy of organ mitochondrial function with respiration in various disease states, the addition of mitochondrial dynamics will now allow for more thorough clinical evaluation of mitochondrial networking in human disease with potential exploration of therapeutics.

Acute decompression following simulated dive conditions alters mitochondrial respiration and motility.

While barotrauma, decompression sickness, and drowning-related injuries are common morbidities associated with diving and decompression from depth, it remains unclear what impact rapid decompression has on mitochondrial function. In vitro diving simulation was performed with human dermal fibroblast cells subjected to control, air, nitrogen, and oxygen dive conditions. With the exception of the gas mixture, all other related variables, including absolute pressure exposure, dive and decompression rates, and temperature, were held constant. High-resolution respirometry was used to examine key respiratory states. Mitochondrial dynamic function, including net movement, number, and rates of fusion/fission events, was obtained from fluorescence microscopy imaging. Effects of the dive conditions on cell cytoskeleton were assessed by imaging both actin and microtubules. Maximum respiration was lower in fibroblasts in the air group than in the control and nitrogen groups. The oxygen group had overall lower respiration when compared with all other groups. All groups demonstrated lower mitochondrial motility when compared with the control group. Rates of fusion and fission events were the same between all groups. There were visible differences in cell morphology consistent with the actin staining; however, there were no appreciable changes to the microtubules. This is the first study to directly assess mitochondrial respiration and dynamics in a cell model of decompression. Both hyperbaric oxygen and air dive conditions produce deleterious effects on overall mitochondrial health in fibroblasts.

Fluorescence Microscopy Imaging Calibration for Quantifying Nanocarrier Binding to Cells During Shear Flow Exposure.

Targeted drug delivery is a fast growing industry in healthcare and technologies are being developed for applications utilizing nanocarriers as vehicles for drug transport. As the size scale of these particles becomes further reduced, advanced fluorescence microscopy and image analysis techniques become increasingly important for facilitating our understanding of nanocarrier binding and avidity, thereby establishing the basis for nanocarrier design optimization. While there is a significant body of published work using nanocarriers in vitro and in vivo, the advent of smaller particles that have typically been studied (~500 nm) limits the ability to attain quantitative measurements of nanocarrier binding dynamics since image acquisition and analysis methods are restricted by microscopy pixel size. This work demonstrates the use of a novel calibration technique based on radioisotope counting and fluorescence imaging for enabling quantitative determination of nanocarrier binding dynamics. The technique is then applied to assess the temporal profile of endothelial cell binding of two antibody targeted nanocarrier types in the presence of fluid shear stress. Results are provided for binding of nanoparticles smaller than a microscopy image pixel.