Detection of Acute Radiation Sickness: A Feasibility Study in Non-human Primates Circulating miRNAs for triage in Radiological Events

Development of biomarkers capable of estimating absorbed dose is critical for effective triage of affected individuals after radiological events. Levels of cell-free circulating miRNAs in plasma were compared for dose-response analysis in non-human primates (NHP) exposed to lethal (6.5 Gy) and sub-lethal (1 and 3 Gy) doses over a 7 day period. The doses and test time points were selected to mimic triage needs in the event of a mass casualty radiological event. Changes in miRNA abundance in irradiated animals were compared to a non-irradiated cohort and a cohort experiencing acute inflammation response from exposure to lipopolysaccharide (LPS). An amplification-free, hybridization-based direct digital counting method was used for evaluation of changes in microRNAs in plasma from all animals. Consistent with previous murine studies, circulating levels of miR-150-5p exhibited a dose- and time-dependent decrease in plasma. Furthermore, plasma miR-150-5p levels were found to correlate well with lymphocyte and neutrophil depletion kinetics. Additionally, plasma levels of several other evolutionarily and functionally conserved miRNAs were found altered as a function of dose and time. Interestingly, miR-574-5p exhibited a distinct, dose-dependent increase 24 h post irradiation in NHPs with lethal versus sub-lethal exposure before returning to the baseline level by day 3. This particular miRNA response was not detected in previous murine studies but was observed in animals exposed to LPS, indicating distinct molecular and inflammatory responses. Furthermore, an increase in low-abundant miR-126, miR-144, and miR-21 as well as high-abundant miR-1-3p and miR-206 was observed in irradiated animals on day 3 and/or day 7. The data from this study could be used to develop a multi-marker panel with known tissue-specific origin that could be used for developing rapid assays for dose assessment and evaluation of radiation injury on multiple organs. Furthermore this approach may be utilized to screen for tissue toxicity in patients who receive myeloablative and therapeutic radiation. Radiation Treatment: Male and female rhesus macaques (Macaca mulatta) NHPs were exposed to 1 (N = 6), 3 (N = 8), or 6.5 Gy (N = 5). The day of irradiation was defined as Day 0. At the time of irradiation, the age of the animals ranged from approximately 2 to 5 years. On the day prior to irradiation (Day −1), the body weights ranged from 3.4 to 5.9 kg and from 3.8 to 6.5 kg, for males and females, respectively. All animals were declared healthy before inclusion and were evaluated for mortality and clinical signs (twice daily). Detailed examinations, including body weight and temperature, were performed, prior to animal assignment, during the week prior to irradiation, and on Days 1, 3, and 7. On day 0, the animals were subjected to a single uniform total body dose of gamma radiation from a 60Co source (Theratron 1000), with a targeted dose rate of 50 cGy/min. Sequential anteroposterior and posteroanterior exposures with whole body irradiation were used. Animals were not sedated during the irradiation and were breathing ambient air. Dosimeters (nanoDot™; Landuer Glenwood, IL) were placed on the animals for confirmation of exposure, however there was no real-time dosimetry performed via Farmer's chamber. In this study, all animals reached scheduled termination on Day 7. LPS Treatment: In order to assess the specificity of miRNA biomarkers to ionizing radiation and to rule out or distinguish common systemic inflammation response indicators, 4 cohorts of 2 rhesus macaques (M. mulatta) NHPs each, with a body weight between 4.0–4.9 kg, were subjected to lipopolysaccharide (LPS) injections, known to induce a systemic cellular immune response. Intravenous injection of LPS mimics inflammation and is used to assess marker specificity to irradiation. Briefly, on Day 1 of the study, an intravenous injection of LPS (Sigma Aldrich, Catalog number: 020m4062, serotype 0127:B8) reconstituted in sterile saline (0.9% sodium chloride) was administered (0.0, (vehicle-only sham), 0.7, 2.1, 2.8 mg/kg), without sedation or anesthesia. Blood samples were collected by venipuncture, without sedation or anesthesia, into a Vacutainer® containing lithium heparin as an anti-coagulant for hematology once during the week prior to dosing and on Days 2 and 3. Blood samples for immunological assessment were processed to plasma. Plasma was prepared and frozen (−80°C) for miRNA analysis. Blood samples were collected, without sedation or anesthesia, by venipuncture into a Vacutainer® containing lithium heparin as an anti-coagulant for hematology three occasions prior to irradiation (within 2 weeks prior to irradiation) and daily for the first seven days after irradiation. Plasma was isolated and frozen (−80°C) prior to miRNA analysis. An amplification-free, hybridization based direct digital counting method developed by NanoString Technologies was used for evaluation of changes in circulating miRNAs. However, in this assay, available human probes were used as miRNAs are evolutionarily conserved between human and NHPs. RNA was extracted from 200 µL plasma using Qiagen miRNeasy kit. After lysis, three synthetic oligonucleotides (spike-in oligos) were added. Purified RNA was eluted in 100 µL water and concentrated to 20 µL. The digital multiplexed nanoString nCounter miRNA expression assay was performed with RNA present in 3 µL (10–20 ng cell free RNA).