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Kaplan-Meier curves for 60-day survival following exposure to 7.4 Gy ⁶⁰Co gamma TBI. There was no significant difference in the survival time between the groups by log-rank test (P > 0.05). The MNDD for groups 1 to 4 were 15.0, 15.0, 15.0, and 15.5, respectively.
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More evidence is needed to support recommendations for medical management of acute radiation syndrome (ARS) and associated infections resulting from a radiological/nuclear event. While current guidelines recommend the administration of antibiotics to chemotherapy patients with febrile neutropenia, the clinical benefit is unclear for acute radiation...
Citations
... If needed, NHPs were administered a booster (0.1-0.3 ml im) of Ketamine hydrochloride prior to irradiation to reduce potential movement. Positioned in opposite directions on the irradiation platform, two NHPs were exposed to cobalt-60 total-body gamma radiation simultaneously at a dose of 7.2 Gy (dose rate of 0.6 Gy/min) 43 . When any two animals' abdominal lateral separation measurements were not within + /− 1 cm, the two animals were irradiated individually. ...
The identification and validation of radiation biomarkers is critical for assessing the radiation dose received in exposed individuals and for developing radiation medical countermeasures that can be used to treat acute radiation syndrome (ARS). Additionally, a fundamental understanding of the effects of radiation injury could further aid in the identification and development of therapeutic targets for mitigating radiation damage. In this study, blood samples were collected from fourteen male nonhuman primates (NHPs) that were exposed to 7.2 Gy ionizing radiation at various time points (seven days prior to irradiation; 1, 13, and 25 days post-irradiation; and immediately prior to the euthanasia of moribund (preterminal) animals). Plasma was isolated from these samples and was analyzed using a liquid chromatography tandem mass spectrometry approach in an effort to determine the effects of radiation on plasma proteomic profiles. The primary objective was to determine if the radiation-induced expression of specific proteins could serve as an early predictor for health decline leading to a preterminal phenotype. Our results suggest that radiation induced a complex temporal response in which some features exhibit upregulation while others trend downward. These statistically significantly altered features varied from pre-irradiation levels by as much as tenfold. Specifically, we found the expression of integrin alpha and thrombospondin correlated in peripheral blood with the preterminal stage. The differential expression of these proteins implicates dysregulation of biological processes such as hemostasis, inflammation, and immune response that could be leveraged for mitigating radiation-induced adverse effects.
... All animals were kept in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-International. Housing requirements, sensory and dietary enrichment details have been previously described in elsewhere (25,26). Single housing was utilized for the animals for this study, and the justification for single housing was described earlier (27). ...
Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8–7.2 Gy (LD29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. In collaboration with the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at −80°C, and shipped to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges.
... All animals were kept in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Housing requirements, sensory and dietary enrichment details have been described in detail previously (24,25). Single housing was utilized for the animals for this study and justification for single housing is described earlier (26). ...
Radiosensitivity differs in humans and possibly in closely related nonhuman primates. The reasons for variation in radiosensitivity are not well known. In an earlier study, we examined gene expression (GE) pre-radiation in peripheral blood among male (n = 62) and female (n = 60) rhesus macaques (n = 122), which did or did not survive (up to 60 days) after whole-body exposure of 7.0 Gy (LD66/60). Eight genes (CHD5, CHI3L1, DYSF, EPX, IGF2BP1, LCN2, MBOAT4, SLC22A4) revealed significant associations with survival. Access to a second rhesus macaque cohort (males = 40, females = 23, total n = 63) irradiated with 5.8–7.2 Gy (LD29-50/60) and some treated with gamma-tocotrienol (GT3, a radiation countermeasure) allowed us to validate these gene expression changes independently. Total RNA was isolated from whole blood samples and examined by quantitative RT-PCR on a 96-well format. cycle threshold (Ct)-values normalized to 18S rRNA were analyzed for their association with survival. Regardless of the species-specific TaqMan assay, similar results were obtained. Two genes (CHD5 and CHI3L1) out of eight revealed a significant association with survival in the second cohort, while only CHD5 (involved in DNA damage response and proliferation control) showed mean gene expression changes in the same direction for both cohorts. No expected association of CHD5 GE with dose, treatment, or sex could be established. Instead, we observed significant associations for those comparisons comprising pre-exposure samples with CHD5 Ct values ≤ 11 (total n = 17). CHD5 Ct values ≤ 11 in these comparisons were mainly associated with increased frequencies (61-100%) of non-survivors, a trend which depending on the sample numbers, reached significance (P = 0.03) in males and, accordingly, in females. This was also reflected by a logistic regression model including all available samples from both cohorts comprising CHD5 measurements (n = 104, odds ratio 1.38, 95% CI 1.07–1.79, P = 0.01). However, this association was driven by males (odds ratio 1.62, 95% CI 1.10–2.38, P = 0.01) and CHD5 Ct values ≤ 11 since removing low CHD5 Ct values from this model, converted to insignificance (P = 0.19). A second male subcohort comprising high CHD5 Ct values ≥ 14.4 in both cohorts (n = 5) appeared associated with survival. Removing these high CHD5 Ct values converted the model borderline significant (P = 0.051). Based on the probability function of the receiver operating characteristics (ROC) curves, 8 (12.3%) and 5 (7.7%) from 65 pre-exposure RNA measurements in males, death and survival could be predicted with a negative and positive predictive value ranging between 85–100%. An associated odds ratio reflected a 62% elevated risk for dying or surviving per unit change (Ct-value) in gene expression, considering the before-mentioned CHD5 thresholds in RNA copy numbers. In conclusion, we identified two subsets of male animals characterized by increased (Ct values ≤ 11) and decreased (Ct values ≥ 14.4) CHD5 GE copy numbers before radiation exposure, which independently of the cohort, radiation exposure or treatment appeared to predict the death or survival in males.
... Several parameters were used as guidelines for moribundity including inappetence, minimal, or no response to stimuli, severe anemia, weakness, etc. [25]. An on-call veterinary technician/veterinarian was available 24 h a day in the event of an emergency situation [28]. ...
Ionizing radiation exposure is known to induce molecular and cellular injury, inflicting a cascade of potentially catastrophic events leading to tissue and organ damage. Metabolomic analysis allows for the identification and quantification of small molecules downstream of genomic changes induced by radiation exposure. We aimed to characterize metabolomic changes that underscore the prefinal stage of lethally irradiated rhesus nonhuman primates (NHPs). Peripheral blood was drawn at baseline, post-exposure, as well as at the preterminal stage in NHPs (immediately prior to death in moribund NHPs) that did not survive exposure with 7.2 Gy total-body radiation (LD70/60). Herein, we analyzed global metabolomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in plasma samples of NHPs collected at various timepoints in relation to irradiation. The overall goal was to identify metabolic shifts present immediately prior to death. Our findings showed radiation induced significant time-dependent metabolic perturbations when compared to pre-irradiation profiles, particularly in glycerophospholipid metabolism and steroid hormone biosynthesis and metabolism pathways. These findings provide valuable insights for identifying biomarkers for lethality, which may be helpful for triage during a mass casualty scenario.
... Fifteen naïve rhesus NHPs (7 males and 8 females) were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-International accredited facility. Prior to conducting the study, animals were quarantined for six weeks (46). Details regarding animal care, housing, health monitoring, and enrichment have been described in detail earlier (47,48). ...
Currently, no radioprotectors have been approved to mitigate hematopoietic injury after exposure to ionizing radiation. Acute ionizing radiation results in damage to both hematopoietic and immune system cells. Pre-exposure prophylactic agents are needed for first responders and military personnel. In this study, the ability of gamma-tocotrienol (GT3), a promising radioprotector and antioxidant, to ameliorate partial-body radiation-induced damage to the hematopoietic compartment was evaluated in a nonhuman primate (NHP) model. A total of 15 rhesus NHPs were divided into two groups, and were administered either GT3 or vehicle 24 h prior to 4 or 5.8 Gy partial-body irradiation (PBI), with 5% bone marrow (BM) sparing. Each group consisted of four NHPs, apart from the vehicle-treated group exposed to 5.8 Gy, which had only three NHPs. BM samples were collected 8 days prior to irradiation in addition to 2, 7, 14, and 30 days postirradiation. To assess the clonogenic ability of hematopoietic stem and progenitor cells (HSPCs), colony forming unit (CFU) assays were performed, and lymphoid cells were immunophenotyped using flow cytometry. As a result of GT3 treatment, an increase in HSPC function was evident by an increased recovery of CFU-granulocyte macrophages (CFU-GM). Additionally, GT3 treatment was shown to increase the percentage of CD34+ cells, including T and NK-cell subsets. Our data further affirm GT3’s role in hematopoietic recovery and suggest the need for its further development as a prophylactic radiation medical countermeasure.
... Animals that were deemed moribund using criteria listed on the approved protocol were scheduled for euthanasia based on the veterinarian's discretion. An on-call veterinary technician/veterinarian was available 24 h a day in the event of an emergency situation [26]. ...
Ionizing radiation exposure is known to induce molecular and cellular injury, inflicting a cascade of potentially catastrophic events leading to tissue and organ damage. Metabolomic analysis allows for the identification and quantification of small molecules downstream of genomic changes induced by radiation exposure. We aimed to characterize metabolomic changes that underscore the prefinal stage of lethally irradiated rhesus nonhuman primates (NHPs). Peripheral blood was drawn at baseline, post-exposure as well as at the preterminal stage of NHPs (immediately prior to death in moribund NHPs), which did not survive exposure with 7.2 Gy total-body radiation (LD70/60). Herein, we analyzed global metabolomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in plasma samples of NHPs collected at various timepoints in relation to irradiation. The overall goal was to identify metabolic shifts present immediately prior to death. Our findings, for the first time, show that radiation induced significant time dependent metabolic perturbations when compared to pre-irradiation samples, particularly in the glycerophospholipid metabolism and steroid hormone biosynthesis and metabolism pathways. These findings provide valuable insights for identifying biomarkers for lethality, which may be helpful for triage during a mass casualty scenario.
... When an animal reached a state of moribundity, the animal was euthanized. Moribundity was used as a surrogate for mortality, and animals were euthanized in order to minimize pain and distress [32]. The following parameters were used as guidelines for moribundity: significant weight loss (10%) from baseline; inappetence (complete anorexia for 2 days and deteriorating conditions); minimal or absence of response to stimuli, severe anemia (<13% hematocrit due to acute blood loss or <40 g/dL hemoglobin) and core body temperature below 96.6°F following a period of febrile neutropenia (such as >103°F and <500 neutrophils/µl); weakened/inability to obtain feed or water; severe thrombocytopenia (<10,000 platelets/µl) or other signs of severe organ dysfunction with poor prognosis as determined by the veterinarian such as dyspnea or severe cyanosis; sustained vomiting or diarrhea, obstruction, intussusception, and peritonitis; renal failure as determined by clinical chemistry and urinalysis; sustained CNS depression, seizures, or paralysis of one or more extremities; non-healing wounds, repeated self-trauma, and severe skin infections; and severe organ system dysfunction with poor prognosis. ...
... When an animal reached a state of moribundity, the animal was euthanized. Moribundity was used as a surrogate for mortality, and animals were euthanized in order to minimize pain and distress [32]. The following parameters were used as guidelines for moribundity: significant weight loss (10%) from baseline; inappetence (complete anorexia for 2 days and deteriorating conditions); minimal or absence of response to stimuli, severe anemia (<13% hematocrit due to acute blood loss or <40 g/dL hemoglobin) and core body temperature below 96.6°F following a period of febrile neutropenia (such as >103°F and <500 neutrophils/µl); weakened/inability to obtain feed or water; severe thrombocytopenia (<10,000 platelets/µl) or other signs of severe organ dysfunction with poor prognosis as determined by the veterinarian such as dyspnea or severe cyanosis; sustained vomiting or diarrhea, obstruction, intussusception, and peritonitis; renal failure as determined by clinical chemistry and urinalysis; sustained CNS depression, seizures, or paralysis of one or more extremities; non-healing wounds, repeated self-trauma, and severe skin infections; and severe organ system dysfunction with poor prognosis. ...
Background:
Animal models are vital for the development of radiation medical countermeasures for the prophylaxis or treatment of acute radiation syndrome and for the delayed effects of acute radiation exposure. Nonhuman primates (NHPs) play an important role in the regulatory approval of such agents by the United States Food and Drug Administration following the Animal Rule. Reliance on such animal models requires that such models are well characterized.
Methods:
Data gathered from both male and female animals under the same conditions and gathered concurrently are limited; therefore, the authors compared and contrasted here the radiosensitivity of both male and female NHPs provided different levels of clinical support over a range of acute, total-body gamma irradiation, as well as the influence of age and body weight.
Results:
Under matched experimental conditions, the authors observed only marginal, but clearly evident differences between acutely irradiated male and female NHPs relative to the measured response endpoints (rates of survival, blood cell changes and cytokine fluctuations). These differences appeared to be accentuated by level of exposure as well as by nature of clinical support.
Conclusion:
Additional studies with both sexes under various experimental conditions and different radiation qualities run concurrently are needed.
The identification and validation of radiation biomarkers is critical for assessing the radiation dose received in exposed individuals and for developing radiation medical countermeasures that can be used to treat acute radiation syndrome (ARS). Additionally, a fundamental understanding of the effects of radiation injury could further aid in the identification and development of therapeutic targets for mitigating radiation damage. In this study, blood samples were collected from fourteen male nonhuman primates (NHPs) that were exposed to 7.2 Gy ionizing radiation at various time points (seven days prior to irradiation; 1, 13, and 25 days post-irradiation; as well as immediately prior to the euthanasia of moribund animals (preterminal)). Plasma was isolated from these samples and was analyzed using a liquid chromatography tandem mass spectrometry approach in an effort to determine the effects of radiation on plasma proteomic profiles. Of particular interest was to determine if the expression of certain proteins reacted to radiation in a way that would act as a predictor for health decline leading to a preterminal phenotype. Our results suggest that radiation induced a diverse temporal pattern among protein expression that displayed prominent changes within NHP proteomic plasma profiles. Of these significantly altered proteins, several play important roles in certain biological processes such as hemostasis, inflammation, and immune response.
There are currently four radiation medical countermeasures that have been approved by the United States Food and Drug Administration to mitigate hematopoietic acute radiation syndrome, all of which are repurposed radiomitigators. The evaluation of additional candidate drugs that may also be helpful for use during a radiological/nuclear emergency is ongoing. A chlorobenzyl sulfone derivative (organosulfur compound) known as Ex-Rad, or ON01210, is one such candidate medical countermeasure, being a novel, small-molecule kinase inhibitor that has demonstrated efficacy in the murine model. In this study, nonhuman primates exposed to ionizing radiation were subsequently administered Ex-Rad as two treatment schedules (Ex-Rad I administered 24 and 36 h post-irradiation, and Ex-Rad II administered 48 and 60 h post-irradiation) and the proteomic profiles of serum using a global molecular profiling approach were assessed. We observed that administration of Ex-Rad post-irradiation is capable of mitigating radiation-induced perturbations in protein abundance, particularly in restoring protein homeostasis, immune response, and mitigating hematopoietic damage, at least in part after acute exposure. Taken together, restoration of functionally significant pathway perturbations may serve to protect damage to vital organs and provide long-term survival benefits to the afflicted population.
