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Recent advances in medical countermeasures (MCMs) has been dependent on the Food and Drug Administration (FDA) animal rule (AR) and the final guidance document provided for industry on product development. The criteria outlined therein establish the path for approval under the AR. The guidance document, along with the funding and requirements from the federal agencies provided the basic considerations for animal model development in assessing radiation effects and efficacy against the potential lethal effects of acute radiation injury and the delayed effects of acute exposure. Animal models, essential for determining MCM efficacy, were developed and validated to assess organ-specific, potentially lethal, radiation effects against the gastrointestinal (GI) and hematopoietic acute radiation syndrome (H-ARS), and radiation-induced delayed effects to lung and associated comorbidities of prolonged immune suppression, GI, kidney and heart injury. Partial-body irradiation models where marginal bone marrow was spared resulted in the ability to evaluate the concomitant evolution of multiple organ injury in the acute and delayed effects in survivors of acute radiation exposure. There are no MCMs for prophylaxis against the major sequelae of the ARS or the delayed effects of acute exposure. Also lacking are MCMs that will mitigate the GI ARS consequent to potentially lethal exposure from a terrorist event or major radiation accident. Additionally, the gap in countermeasures for prophylaxis may extend to mixed neutron/gamma radiation if current modelling predicts prompt exposure from an improvised nuclear device. However, progress in the field of MCM development has been made due to federal and corporate funding, clarification of the critical criteria for efficacy within the FDA AR and the concomitant development and validation of additional animal models. These models provided for a strategic and tactical approach to determine radiation effects and MCM efficacy.
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Síndrome Aguda da Radiação , Contramedidas Médicas , Liberação Nociva de Radioativos , Síndrome Aguda da Radiação/prevenção & controle , Animais , Modelos Animais de Doenças , Estados Unidos , United States Food and Drug AdministrationRESUMO
Bile acids (BAs) have been established as ubiquitous regulatory molecules implicated in a large variety of healthy and pathological processes. However, the scope of BA heterogeneity is often underrepresented in current literature. This is due in part to inadequate detection methods, which fail to distinguish the individual constituents of the BA pool. Thus, the primary aim of this study was to develop a method that would allow the simultaneous analysis of specific C24 BA species, and to apply that method to biological systems of interest. Herein, we describe the generation and validation of an LC-MS/MS assay for quantification of numerous BAs in a variety of cell systems and relevant biofluids and tissue. These studies included the first baseline level assessment for planar BAs, including allocholic acid, in cell lines, biofluids, and tissue in a nonhuman primate (NHP) laboratory animal, Macaca mulatta, in healthy conditions. These results indicate that immortalized cell lines make poor models for the study of BA synthesis and metabolism, whereas human primary hepatocytes represent a promising alternative model system. We also characterized the BA pool of M. mulatta in detail. Our results support the use of NHP models for the study of BA metabolism and pathology in lieu of murine models. Moreover, the method developed here can be applied to the study of common and planar C24 BA species in other systems.
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Ácidos e Sais Biliares/análise , Bile/química , Hepatócitos/química , Animais , Ácidos e Sais Biliares/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Macaca mulatta , Espectrometria de Massas em TandemRESUMO
Chronic respiratory diseases are among the leading causes of deaths worldwide and major contributors of morbidity and global disease burden. To appropriately investigate lung disease, the respiratory airways must be fixed in their physiological orientation and should be inflated prior to investigations. We present an inflation-fixation method that enables lipidomic investigations of whole lung samples and resected biopsy specimens by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Formalin-inflation enables sample preparation to parallel standard clinical and surgical procedures, in addition to greatly reducing the complexity of analysis, by decreasing the number of analytes in the MALDI plume and reducing adduct formation in the resulting mass spectra. The reduced complexity increased sensitivity and enabled high-resolution imaging acquisitions without any loss in analyte detection at 10 and 20 µm scans. We present a detailed study of over 100 lipid ions detected in positive and negative ion modes covering the conducting and respiratory airways and parts of the peripheral nervous tissue running through the lungs. By defining the resolution required for clear definition of the alveolar space and thus the respiratory airways we have provided a guideline for MSI investigations of respiratory diseases involving the airways, including the interstitium. This study has provided a detailed map of lipid species and their localization within larger mammalian lung samples, for the first time, thus categorizing the lipidome for future MALDI-MSI studies of pulmonary diseases.
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Técnicas Histológicas , Lipídeos/análise , Pulmão/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Biópsia , Macaca mulatta , MasculinoRESUMO
The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.
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Síndrome Aguda da Radiação/sangue , Cromatografia Líquida/métodos , Citrulina/sangue , Trato Gastrointestinal/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/sangue , Limite de Detecção , Modelos Lineares , Masculino , Camundongos , Modelos Animais , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Porco Miniatura , Estados UnidosRESUMO
Acute, high-dose radiation exposure results in life-threatening acute radiation syndrome (ARS) and debilitating delayed effects of acute radiation exposure (DEARE). The DEARE are a set of chronic multi-organ illnesses that can result in early death due to malignancy and other diseases. Animal models have proven essential in understanding the natural history of ARS and DEARE and licensure of medical countermeasures (MCM) according to the FDA Animal Rule. Our lab has developed models of hematopoietic (H)-ARS and DEARE in inbred C57BL/6J and Jackson Diversity Outbred (JDO) mice of both sexes and various ages and have used these models to identify mechanisms of radiation damage and effective MCMs. Herein, aggregate data from studies conducted over decades in our lab, consisting of 3,250 total-body lethally irradiated C57BL/6 young adult mice and 1,188 H-ARS survivors from these studies, along with smaller datasets in C57BL/6J pediatric and geriatric mice and JDO mice, were examined for lifespan and development of thymic lymphoma in survivors up to 3 years of age. Lifespan was found to be significantly shortened in H-ARS survivors compared to age-matched nonirradiated controls in all four models. Males and females exhibited similar lifespans except in the young adult C57BL/6J model where males survived longer than females after 16 months of age. The incidence of thymic lymphoma was increased in H-ARS survivors from the young adult and pediatric C57BL/6J models. Consistent with our findings in H-ARS, geriatric mice appeared more radioresistant than other models, with a lifespan and thymic lymphoma incidence more similar to nonirradiated controls than other models. Increased levels of multiple pro-inflammatory cytokines in DEARE bone marrow and serum correlated with shortened lifespan and malignancy, consistent with other animal models and human data. Of interest, G-CSF levels in bone marrow and serum 8-11 months after irradiation were significantly increased in females. Importantly, treatment with granulopoietic cytokine MCM for radiomitigation of H-ARS did not influence the long-term survival rate or incidence of thymic lymphoma in any model. Taken together, these findings indicate that the lifespan of H-ARS survivors was significantly decreased regardless of age at time of exposure or genetic diversity, and was unaffected by earlier treatment with granulopoietic cytokines for radiomitigation of H-ARS.
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PURPOSE: A question echoed by the National Biodefense Science Board (NBSB) in 2010, remains a reasonable question in 2023; 'Where are the Countermeasures?'. A critical path for development of medical countermeasures (MCM) against acute, radiation-induced organ-specific injury within the acute radiation syndrome (ARS) and the delayed effects of acute radiation exposure (DEARE) requires the recognition of problems and solutions inherent in the path to FDA approval under the Animal Rule. Keep Rule number one in mind, It's not easy. CONSIDERATIONS: The current topic herein is focused on defining the nonhuman primate model(s) for efficient MCM development relative to consideration of prompt and delayed exposure in the context of the nuclear scenario. The rhesus macaque is a predictive model for human exposure of partial-body irradiation with marginal bone marrow sparing that allows definition of the multiple organ injury in the acute radiation syndrome (ARS) and the delayed effects of acute radiation exposure (DEARE). The continued definition of natural history is required to delineate an associative or causal interaction within the concurrent multi-organ injury characteristic of the ARS and DEARE. A more efficient development of organ specific MCM for both pre-exposure and post-exposure prophylaxis to include acute radiation-induced combined injury requires closing critical gaps in knowledge and urgent support to rectify the national shortage of nonhuman primates. The rhesus macaque is a validated, predictive model of the human response to prompt and delayed radiation exposure, medical management and MCM treatment. A rational approach to further development of the cynomolgus macaque as a comparable model is urgently required for continued development of MCM for FDA approval. CONCLUSION: It is imperative to examine the key variables relative to animal model development and validation, The pharmacokinetics, pharmacodynamics and exposure profiles, of candidate MCM relative to route, administration schedule and optimal efficacy define the fully effective dose. The conduct of adequate and well-controlled pivotal efficacy studies as well as safety and toxicity studies support approval under the FDA Animal Rule and label definition for human use.
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Síndrome Aguda da Radiação , Contramedidas Médicas , Exposição à Radiação , Animais , Humanos , Síndrome Aguda da Radiação/etiologia , Modelos Animais de Doenças , Macaca mulatta , Exposição à Radiação/efeitos adversos , Exposição à Radiação/análiseRESUMO
Computed Tomography (CT) plays an important role in monitoring radiation-induced Pulmonary Fibrosis (PF), where accurate segmentation of the PF lesions is highly desired for diagnosis and treatment follow-up. However, the task is challenged by ambiguous boundary, irregular shape, various position and size of the lesions, as well as the difficulty in acquiring a large set of annotated volumetric images for training. To overcome these problems, we propose a novel convolutional neural network called PF-Net and incorporate it into a semi-supervised learning framework based on Iterative Confidence-based Refinement And Weighting of pseudo Labels (I-CRAWL). Our PF-Net combines 2D and 3D convolutions to deal with CT volumes with large inter-slice spacing, and uses multi-scale guided dense attention to segment complex PF lesions. For semi-supervised learning, our I-CRAWL employs pixel-level uncertainty-based confidence-aware refinement to improve the accuracy of pseudo labels of unannotated images, and uses image-level uncertainty for confidence-based image weighting to suppress low-quality pseudo labels in an iterative training process. Extensive experiments with CT scans of Rhesus Macaques with radiation-induced PF showed that: 1) PF-Net achieved higher segmentation accuracy than existing 2D, 3D and 2.5D neural networks, and 2) I-CRAWL outperformed state-of-the-art semi-supervised learning methods for the PF lesion segmentation task. Our method has a potential to improve the diagnosis of PF and clinical assessment of side effects of radiotherapy for lung cancers.
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Processamento de Imagem Assistida por Computador , Fibrose Pulmonar , Animais , Processamento de Imagem Assistida por Computador/métodos , Pulmão/diagnóstico por imagem , Macaca mulatta , Fibrose Pulmonar/diagnóstico por imagem , Fibrose Pulmonar/etiologia , Tomografia Computadorizada por Raios XRESUMO
The hematopoietic system is highly sensitive to stress from both aging and radiation exposure, and the hematopoietic acute radiation syndrome (H-ARS) should be modeled in the geriatric context separately from young for development of age-appropriate medical countermeasures (MCMs). Here we developed aging murine H-ARS models, defining radiation dose response relationships (DRRs) in 12-month-old middle-aged and 24-month-old geriatric male and female C57BL/6J mice, and characterized diverse factors affecting geriatric MCM testing. Groups of approximately 20 mice were exposed to â¼10 different doses of radiation to establish radiation DRRs for estimation of the LD50/30. Radioresistance increased with age and diverged dramatically between sexes. The LD50/30 in young adult mice averaged 853 cGy and was similar between sexes, but increased in middle age to 1,005 cGy in males and 920 cGy in females, with further sex divergence in geriatric mice to 1,008 cGy in males but 842 cGy in females. Correspondingly, neutrophils, platelets, and functional hematopoietic progenitor cells were all increased with age and rebounded faster after irradiation. These effects were higher in aged males, and neutrophil dysfunction was observed in aged females. Upstream of blood production, hematopoietic stem cell (HSC) markers associated with age and myeloid bias (CD61 and CD150) were higher in geriatric males vs. females, and sex-divergent gene signatures were found in HSCs relating to cholesterol metabolism, interferon signaling, and GIMAP family members. Fluid intake per gram body weight decreased with age in males, and decreased after irradiation in all mice. Geriatric mice of substrain C57BL/6JN sourced from the National Institute on Aging were significantly more radiosensitive than C57BL/6J mice from Jackson Labs aged at our institution, indicating mouse source and substrain should be considered in geriatric radiation studies. This work highlights the importance of sex, vendor, and other considerations in studies relating to hematopoiesis and aging, identifies novel sex-specific functional and molecular changes in aging hematopoietic cells at steady state and after irradiation, and presents well-characterized aging mouse models poised for MCM efficacy testing for treatment of acute radiation effects in the elderly.
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Síndrome Aguda da Radiação , Animais , Modelos Animais de Doenças , Feminino , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tolerância a RadiaçãoRESUMO
ABSTRACT: Near total body exposure to high-dose ionizing radiation results in organ-specific sequelae, including acute radiation syndromes and delayed effects of acute radiation exposure. Among these sequelae are acute kidney injury and chronic kidney injury. Reports that neither oxidative stress nor inflammation are dominant mechanisms defining radiation nephropathy inspired an unbiased, discovery-based proteomic interrogation in order to identify mechanistic pathways of injury. We quantitatively profiled the proteome of kidney from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Kidney was analyzed by liquid chromatography-tandem mass spectrometry. Out of the 3,432 unique proteins that were identified, we found that 265 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 230 proteins showed strong upregulation while 35 proteins showed downregulation. Bioinformatics analysis revealed significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. These data will be useful for a greater understanding of the molecular mechanisms of injury in well-characterized animal models of partial body irradiation with minimal bone marrow sparing. These data may be potentially useful in the future development of medical countermeasures.
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Síndrome Aguda da Radiação , Lesões Experimentais por Radiação , Síndrome Aguda da Radiação/diagnóstico , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/metabolismo , Animais , Medula Óssea/efeitos da radiação , Rim/efeitos da radiação , Macaca mulatta , Proteômica , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismoRESUMO
ABSTRACT: To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model, Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies. Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3), and adhesion molecules (fibronectin, integrin ß6, ICAM-1) were further studied using real-time PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration. Cytokine/chemokine-induced excessive innate immune response after thoracic irradiation plays an important role in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine-induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.
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Pulmão , Tórax , Animais , Imunidade Inata , Pulmão/efeitos da radiação , Macaca mulatta , Metaloporfirinas , Tórax/efeitos da radiaçãoRESUMO
ABSTRACT: Exposure to ionizing radiation following a nuclear or radiological incident results in potential acute radiation syndromes causing sequelae of multi-organ injury in a dose- and time-dependent manner. Currently, medical countermeasures against radiation injury are limited, and no biomarkers have been approved by regulatory authorities. Identification of circulating plasma biomarkers indicative of radiation injury can be useful for early triage and injury assessment and in the development of novel therapies (medical countermeasures). Aims of this study are to (1) identify metabolites and lipids with consensus signatures that can inform on mechanism of injury in radiation-induced multi-organ injury and (2) identify plasma biomarkers in non-human primate (NHP) that correlate with tissues (kidney, liver, lung, left and right heart, jejunum) indicative of radiation injury, assessing samples collected over 3 wk post-exposure to 12 Gy partial body irradiation with 2.5% bone marrow sparing. About 180 plasma and tissue metabolites and lipids were quantified through Biocrates AbsoluteIDQ p180 kit using liquid chromatography and mass spectrometry. System-wide perturbations of specific metabolites and lipid levels and pathway alterations were identified. Citrulline, Serotonin, PC ae 38:2, PC ae 36:2, and sum of branched chain amino acids were identified as potential biomarkers of radiation injury. Pathway analysis revealed consistent changes in fatty acid oxidation and metabolism and perturbations in multiple other pathways.
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Síndrome Aguda da Radiação , Síndrome Aguda da Radiação/diagnóstico , Síndrome Aguda da Radiação/etiologia , Animais , Biomarcadores , Macaca mulatta , Metabolômica , Doses de Radiação , Radiação IonizanteRESUMO
ABSTRACT: The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimated LD50/60 from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalent LD50/60 exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed the LD50/60 (10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60-d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.
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Síndrome Aguda da Radiação , Síndrome Aguda da Radiação/diagnóstico , Síndrome Aguda da Radiação/etiologia , Animais , Medula Óssea/efeitos da radiação , Relação Dose-Resposta à Radiação , Macaca mulatta , Irradiação Corporal Total/efeitos adversosRESUMO
ABSTRACT: Medical countermeasure development under the US Food and Drug Administration animal rule requires validated animal models of acute radiation effects. The key large animal model is the non-human primate, rhesus macaque. To date, only the rhesus macaque has been used for both critical supportive data and pivotal efficacy trials seeking US Food and Drug Administration approval. The potential for use of the rhesus for other high priority studies such as vaccine development underscores the need to identify another non-human primate model to account for the current lack of rhesus for medical countermeasure development. The cynomolgus macaque, Macaca fascicularis, has an existing database of medical countermeasure development against the hematopoietic acute radiation syndrome, as well as the use of radiation exposure protocols that mimic the likely nonuniform and heterogenous exposure consequent to a nuclear terrorist event. The review herein describes published studies of adult male cynomolgus macaques that used two exposure protocols-unilateral, nonuniform total-body irradiation and partial-body irradiation with bone marrow sparing-with the administration of subject-based medical management to assess mitigation against the hematopoietic acute radiation syndrome. These studies assessed the efficacy of cytokine combinations and cell-based therapy to mitigate acute radiation-induced myelosuppression. Both therapeutics were shown to mitigate the myelosuppression of the hematopoietic acute radiation syndrome. Additional studies being presented herein further defined the dose-dependent hematopoietic acute radiation syndrome of cynomolgus and rhesus macaques and a differential dose-dependent effect with young male and female cynomolgus macaques. The database supports the investigation of the cynomolgus macaque as a comparable non-human primate for efficacy testing under the US Food and Drug Administration animal rule. Critical gaps in knowledge required to validate the models and exposure protocols are also identified.
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Síndrome Aguda da Radiação , Contramedidas Médicas , Exposição à Radiação , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/prevenção & controle , Animais , Feminino , Macaca fascicularis , Macaca mulatta , Masculino , Exposição à Radiação/efeitos adversosRESUMO
ABSTRACT: Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a non-human primate model of partial body irradiation with minimal bone marrow sparing, lung was analyzed from animals irradiated with 12 Gy at timepoints every 4 d up to 21 d after irradiation and compared to non-irradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry followed by pathway analysis. Out of the 3,101 unique proteins that were identified, we found that 252 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 215 proteins showed strong up-regulation while 37 proteins showed down-regulation. Canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, fibrosis, and retinoic acid signaling were identified. The proteomic profiling of lung conducted here represents an untargeted systems biology approach to identify acute molecular events in the non-human primate lung that could potentially be initiating events for radiation-induced lung injury.
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Lesões Experimentais por Radiação , Pneumonite por Radiação , Animais , Medula Óssea/efeitos da radiação , Pulmão/metabolismo , Primatas , Proteômica , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismoRESUMO
ABSTRACT: Radiation sequelae is complex and characterized by multiple pathologies, which occur over time and nonuniformly throughout different organs. The study of the mesenteric lymph node (MLN) due to its importance in the gastrointestinal system is of particular interest. Other studies have shown an immediate post-irradiation reduction in cellularity due to the known effects of irradiation on lymphoid cell populations, but the molecular and functional mechanisms that lead to these cellular alterations remain limited. In this work, we show the use of lipidomic, proteomic, and mass spectrometry imaging in the characterization of the effects of acute radiation exposure on the MLN at different time points after ionizing radiation (IR) from 4 d to 21 d after 12 Gy partial body irradiation with 2.5% bone marrow sparing. The combined analyses showed a dysregulation of the lipid and protein composition in the MLN after IR. Protein expression was affected in numerous pathways, including pathways regulating lipids such as LXR/RXR activation and acute phase response. Lipid distribution and abundance was also affected by IR in the MLN, including an accumulation of triacylglycerides, a decrease in polyunsaturated glycerophospholipids, and changes in polyunsaturated fatty acids. Those changes were observed as early as 4 d after IR and were more pronounced for lipids with a higher concentration in the nodules and the medulla of the MLN. These results provide molecular insight into the MLN that can inform on injury mechanism in a non-human primate model of the acute radiation syndrome of the gastrointestinal tract. Those findings may contribute to the identification of therapeutic targets and the development of new medical countermeasures.
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Medula Óssea , Lesões Experimentais por Radiação , Animais , Medula Óssea/efeitos da radiação , Lipidômica , Linfonodos/patologia , Macaca mulatta , Espectrometria de Massas , Proteômica , Lesões Experimentais por Radiação/patologiaRESUMO
ABSTRACT: High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. An active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the non-human primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.
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Medula Óssea , Retinoides , Animais , Medula Óssea/efeitos da radiação , Homeostase , Nutrientes , Primatas/metabolismo , Retinoides/metabolismo , Tretinoína/farmacologiaRESUMO
ABSTRACT: High-dose radiation exposure results in hematopoietic and gastrointestinal acute radiation syndromes followed by delayed effects of acute radiation exposure, which encompasses multiple organs, including heart, kidney, and lung. Here we sought to further characterize the natural history of radiation-induced heart injury via determination of differential protein and metabolite expression in the heart. We quantitatively profiled the proteome and metabolome of left and right ventricle from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of 3 wk. Global proteome profiling identified more than 2,200 unique proteins, with 220 and 286 in the left and right ventricles, respectively, showing significant responses across at least three time points compared to baseline levels. High-throughput targeted metabolomics analyzed a total of 229 metabolites and metabolite combinations, with 18 and 22 in the left and right ventricles, respectively, showing significant responses compared to baseline levels. Bioinformatic analysis performed on metabolomic and proteomic data revealed pathways related to inflammation, energy metabolism, and myocardial remodeling were dysregulated. Additionally, we observed dysregulation of the retinoid homeostasis pathway, including significant post-radiation decreases in retinoic acid, an active metabolite of vitamin A. Significant differences between left and right ventricles in the pathology of radiation-induced injury were identified. This multi-omic study characterizes the natural history and molecular mechanisms of radiation-induced heart injury in NHP exposed to PBI with minimal bone marrow sparing.
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Síndrome Aguda da Radiação , Medula Óssea , Primatas , Proteômica , Lesões por Radiação , Síndrome Aguda da Radiação/patologia , Animais , Medula Óssea/efeitos da radiação , Doses de Radiação , Lesões por Radiação/metabolismoRESUMO
ABSTRACT: The goal of this study was to develop rat models of partial body irradiation with bone-marrow sparing (leg-out PBI) to test medical countermeasures (MCM) of both acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE) under the FDA animal rule. The leg-out PBI models were developed in female and male WAG/RijCmcr rats at doses of 12.5-14.5 Gy. Rats received supportive care consisting of fluids and antibiotics. Gastrointestinal ARS (GI-ARS) was assessed by lethality to d 7 and diarrhea scoring to d 10. Differential blood counts were analyzed between d 1-42 for the natural history of hematopoietic ARS (H-ARS). Lethality and breathing intervals (BI) were measured between d 28-110 to assess delayed injury to the lung (L-DEARE). Kidney injury (K-DEARE) was evaluated by measuring elevation of blood urea nitrogen (BUN) between d 90-180. The LD50/30, including both lethality from GI-ARS and H-ARS, for female and male rats are 14.0 Gy and 13.5 Gy, respectively, while the LD50/7 for only GI-ARS are 14.3 Gy and 13.6 Gy, respectively. The all-cause mortalities, including ARS and L-DEARE, through 120 d (LD50/120) are 13.5 Gy and 12.9 Gy, respectively. Secondary end points confirmed occurrence of four distinct sequelae representing GI, hematopoietic, lung, and kidney toxicities after leg-out PBI. Adult rat models of leg-out PBI showed the acute and long-term sequelae of radiation damage that has been reported in human radiation exposure case studies. Sex-specific differences were observed in the DRR between females and males. These rat models are among the most useful for the development and approval of countermeasures for mitigation of radiation injuries under the FDA animal rule.
Assuntos
Síndrome Aguda da Radiação , Sistema Hematopoético , Contramedidas Médicas , Exposição à Radiação , Lesões Experimentais por Radiação , Síndrome Aguda da Radiação/tratamento farmacológico , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/prevenção & controle , Animais , Medula Óssea/efeitos da radiação , Feminino , Masculino , Lesões Experimentais por Radiação/complicações , Lesões Experimentais por Radiação/prevenção & controle , RatosRESUMO
Medical countermeasures (MCMs) for hematopoietic acute radiation syndrome (H-ARS) should be evaluated in well-characterized animal models, with consideration of at-risk populations such as pediatrics. We have developed pediatric mouse models of H-ARS and delayed effects of acute radiation exposure (DEARE) for efficacy testing of MCMs against radiation. Male and female C57BL/6J mice aged 3, 4, 5, 6, 7 and 8 weeks old (±1 day) were characterized for baseline hematopoietic and gastrointestinal parameters, radiation response, efficacy of a known MCM, and DEARE at six and 12 months after total-body irradiation (TBI). Weanlings (age 3 weeks) were the most radiosensitive age group with an estimated LD50/30 of 712 cGy, while mice aged 4 to 8 weeks were more radioresistant with an estimated LD50/30 of 767-787 cGy. Female weanlings were more radiosensitive than males at 3 and 4 weeks old but became significantly more radioresistant after the pubertal age of 5 weeks. The most dramatic increase in body weight, RBC counts and intestinal circumference length occurred from 3 to 5 weeks of age. The established radiomitigator Neulasta® (pegfilgrastim) significantly increased 30-day survival in all age groups, validating these models for MCM efficacy testing. Analyses of DEARE among pediatric survivors revealed depressed weight gain in males six months post-TBI, and increased blood urea nitrogen at 12 months post-TBI which was more severe in females. Hematopoietic DEARE at six months post-TBI appeared to be less severe in survivors from the 3- and 4-week-old groups but was equally severe in all age groups by 12 months of age. Similar to our other acute radiation mouse models, there was no appreciable effect of Neulasta used as an H-ARS MCM on the severity of DEARE. In summary, these data characterize a pediatric mouse model useful for assessing the efficacy of MCMs against ARS and DEARE in children.
Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Filgrastim/farmacologia , Sistema Hematopoético/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/fisiopatologia , Animais , Modelos Animais de Doenças , Sistema Hematopoético/fisiopatologia , Sistema Hematopoético/efeitos da radiação , Humanos , Camundongos , Pediatria , Tolerância a Radiação/efeitos da radiação , Irradiação Corporal Total/efeitos adversosRESUMO
Studies performed decades ago in the canine and nonhuman primate established the dose response relationships for the hematopoietic acute radiation syndrome in response to mixed neutron/gamma, x-radiation, and Co gamma radiation. There were no published studies that determined the dose response relationships for the gastrointestinal acute radiation syndrome in response to either noted radiation quality. This analysis of a retrospective, unpublished study provided the dose response relationships in a canine model for the acute gastrointestinal syndrome relative to the acute hematopoietic syndrome due to mixed neutron/gamma radiation. Canines were exposed to total-body, steady state, bilateral, 0.40 Gy min, mixed neutron/gamma (5.4:1) radiation from a TRIGA reactor. The average neutron/gamma energy (MeV) was 0.85/0.9, and exposure was reported as midline tissue dose. Medical management was not administered. The mixed neutron/gamma exposure resulted in an estimated LD50/6 of 2.83 Gy [2.76, 2.94] and LD50/30 of 2.16 Gy [2.01, 2.24] for the GI- and H-ARS respectively. The mean survival times for decedents after mixed neutron/gamma exposure approximate to the LD50/6 were 8.5 d, 10.5 d, and 4 d for 2.75 Gy, 2.80 Gy, 3.00, and 3.12 Gy exposures, respectively. The mean survival times for decedents for mixed neutron/gamma exposure approximate to the LD50/30 were 21.3 d and 15.6 d for 2.00 Gy and 2.25 Gy, respectively. Furthermore, the dose response relationships for the acute hematopoietic syndrome due to mixed neutron/gamma exposure (0.85/0.9 MeV; 5.4:1) resulted in an estimated relative biological effectiveness of 1.2 as compared with reference Co gamma radiation.