Biomarkers to Predict Lethal Radiation Injury to the Rat Lung.

Currently, there are no biomarkers to predict lethal lung injury by radiation. Since it is not ethical to irradiate humans, animal models must be used to identify biomarkers. Injury to the female WAG/RijCmcr rat has been well-characterized after exposure to eight doses of whole thorax irradiation: 0-, 5-, 10-, 11-, 12-, 13-, 14- and 15-Gy. End points such as SPECT imaging of the lung using molecular probes, measurement of circulating blood cells and specific miRNA have been shown to change after radiation. Our goal was to use these changes to predict lethal lung injury in the rat model, 2 weeks post-irradiation, before any symptoms manifest and after which a countermeasure can be given to enhance survival. SPECT imaging with 99mTc-MAA identified a decrease in perfusion in the lung after irradiation. A decrease in circulating white blood cells and an increase in five specific miRNAs in whole blood were also tested. Univariate analyses were then conducted on the combined dataset. The results indicated that a combination of percent change in lymphocytes and monocytes, as well as pulmonary perfusion volume could predict survival from radiation to the lungs with 88.5% accuracy (95% confidence intervals of 77.8, 95.3) with a p-value of < 0.0001 versus no information rate. This study is one of the first to report a set of minimally invasive endpoints to predict lethal radiation injury in female rats. Lung-specific injury can be visualized by 99mTc-MAA as early as 2 weeks after radiation.

Wound Trauma Exacerbates Acute, but not Delayed, Effects of Radiation in Rats: Mitigation by Lisinopril.

The goal of this study is to understand and mitigate the effects of wounds on acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), for preparedness against a radiological attack or accident. Combined injuries from concomitant trauma and radiation are likely in these scenarios. Either exacerbation or mitigation of radiation damage by wound trauma has been previously reported in preclinical studies. Female WAG/RijCmcr rats received 13 Gy X-rays, with partial-body shielding of one leg. Within 2 h, irradiated rats and non-irradiated controls were given full-thickness skin wounds with or without lisinopril, started orally 7 days after irradiation. Morbidity, skin wound area, breathing interval and blood urea nitrogen were measured up to 160 days post-irradiation to independently evaluate wound trauma and DEARE. Wounding exacerbated morbidity in irradiated rats between 5 and 14 days post-irradiation (during the ARS phase), and irradiation delayed wound healing. Wounding did not alter delayed morbidities from radiation pneumonitis or nephropathy after 30 days post-irradiation. Lisinopril did not mitigate wound healing, but markedly decreased morbidity during DEARE from 31 through 160 days. The results derived from this unique model of combined injuries suggest different molecular mechanisms of injury and healing of ARS and DEARE after radiation exposure.

Mapping genetic modifiers of radiation-induced cardiotoxicity to rat chromosome 3.

Radiation therapy is used in ~50% of cancer patients to reduce the risk of recurrence and in some cases improve survival. Despite these benefits, doses can be limited by toxicity in multiple organs, including the heart. The underlying causes and biomarkers of radiation-induced cardiotoxicity are currently unknown, prompting the need for experimental models with inherent differences in sensitivity and resistance to the development of radiation-induced cardiotoxicity. We have identified the parental SS (Dahl salt-sensitive/Mcwi) rat strain to be a highly-sensitized model of radiation-induced cardiotoxicity. In comparison, substitution of rat chromosome 3 from the resistant BN (Brown Norway) rat strain onto the SS background (SS-3BN consomic) significantly attenuated radiation-induced cardiotoxicity. SS-3BN rats had less radiation-induced cardiotoxicity than SS rats, as measured by survival, pleural and pericardial effusions, echocardiogram parameters, and histological damage. Mast cells, previously shown to have predominantly protective roles in radiation-induced cardiotoxicity, were increased in the more resistant SS-3BN hearts postradiation. RNA sequencing from SS and SS-3BN hearts at 1 wk postradiation revealed 5,098 differentially expressed candidate genes across the transcriptome and 350 differentially expressed genes on rat chromosome 3, which coincided with enrichment of multiple pathways, including mitochondrial dysfunction, sirtuin signaling, and ubiquitination. Upstream regulators of enriched pathways included the oxidative stress modulating transcription factor, Nrf2, which is located on rat chromosome 3. Nrf2 target genes were also differentially expressed in the SS vs. SS-3BN consomic hearts postradiation. Collectively, these data confirm the existence of heritable modifiers in radiation-induced cardiotoxicity and provide multiple biomarkers, pathways, and candidate genes for future analyses. NEW & NOTEWORTHY This novel study reveals that heritable genetic factors have the potential to modify normal tissue sensitivity to radiation. Gene variant(s) on rat chromosome 3 can contribute to enhanced cardiotoxicity displayed in the SS rats vs. the BN and SS-3BN consomic rats. Identifying genes that lead to understanding the mechanisms of radiation-induced cardiotoxicity represents a novel method to personalize radiation treatment, as well as predict the development of radiation-induced cardiotoxicity.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Delayed renal injury in survivors of hematologic acute radiation syndrome.

PURPOSE: A mass casualty disaster involving radiological or nuclear agents continues to be a public health concern which requires consideration of both acute and late tissue toxicities in exposed victims. With the advent of advanced treatment options for the mitigation of hematological injuries, there are likely to be survivors of total body irradiation (TBI) exposures as high as 8-10 Gy. These survivors are at risk for a range of delayed multi-organ morbidities including progressive renal failure. MATERIAL AND METHODS: Here, we established the WAG/RijCmcr rat as an effective model for the evaluation of medical countermeasures (MCM) for acute hematologic radiation syndrome (H-ARS). The LD50/30 dose for adult and pediatric WAG/RijCmcr rats was determined for both sexes. We then confirmed the FDA-approved MCM pegfilgrastim (peg-GCSF, Neulasta®) mitigates H-ARS in adult male and female rats. Finally, we evaluated survival and renal dysfunction up to 300 d post-TBI in male and female adult rats. RESULTS: In the WAG/RijCmcr rat model, 87.5% and 100% of adult rats succumb to lethal hematopoietic acute radiation syndrome (H-ARS) at TBI doses of 8 and 8.5 Gy, respectively. A single dose of the hematopoietic growth factor peg-GCSF administered at 24 h post-TBI improved survival during H-ARS. Peg-GCSF treatment improved 30 d survival from 12.5% to 83% at 8 Gy and from 0% to 63% at 8.5 Gy. We then followed survivors of H-ARS through day 300. Rats exposed to TBI doses greater than 8 Gy had a 26% reduction in survival over days 30-300 compared to rats exposed to 7.75 Gy TBI. Concurrent with the reduction in long-term survival, a dose-dependent impairment of renal function as assessed by blood urea nitrogen (BUN) and urine protein to urine creatinine ratio (UP:UC) was observed. CONCLUSION: Together, these data show survivors of H-ARS are at risk for the development of delayed renal toxicity and emphasize the need for the development of medical countermeasures for delayed renal injury.

IPW-5371 mitigates the delayed effects of acute radiation exposure in WAG/RijCmcr rats when started 15 days after PBI with bone marrow sparing.

PURPOSE: To test IPW-5371 for the mitigation of the delayed effects of acute radiation exposure (DEARE). Survivors of acute radiation exposure are at risk for developing delayed multi-organ toxicities; however, there are no FDA-approved medical countermeasures (MCM) to mitigate DEARE. METHODS: WAG/RijCmcr female rat model of partial-body irradiation (PBI), by shielding part of one hind leg, was used to test IPW-5371 (7 and 20 mg kg-1 d-1) for mitigation of lung and kidney DEARE when started 15 d after PBI. Rats were fed known amounts of IPW-5371 using a syringe, instead of delivery by daily oral gavage, sparing exacerbation of esophageal injury by radiation. The primary endpoint, all-cause morbidity was assessed over 215 d. Secondary endpoints: body weight, breathing rate and blood urea nitrogen were also assessed. RESULTS: IPW-5371 enhanced survival (primary endpoint) as well as attenuated secondary endpoints of lung and kidney injuries by radiation. CONCLUSION: To provide a window for dosimetry and triage, as well as avoid oral delivery during the acute radiation syndrome (ARS), the drug regimen was started at 15 d after 13.5 Gy PBI. The experimental design to test mitigation of DEARE was customized for translation in humans, using an animal model of radiation that was designed to simulate a radiologic attack or accident. The results support advanced development of IPW-5371 to mitigate lethal lung and kidney injuries after irradiation of multiple organs.

Biological sex differences in renin angiotensin system enzymes ACE and ACE2 regulate normal tissue response to radiation injury.

Introduction: In experimental animal models, biological sex-differences in the manifestation and severity of normal tissue radiation injury have been well-documented. Previously we demonstrated male and female rats have differential and highly reproducible responses to high-dose partial body irradiation (PBI) with male rats having greater susceptibility to both gastrointestinal acute radiation syndrome (GI-ARS) and radiation pneumonitis than female rats. Methods: In the current study, we have investigated whether differential expression of the renin-angiotensin system (RAS) enzymes angiotensin converting enzyme (ACE) and ACE2 contribute to the observed sex-related differences in radiation response. Results: During the period of symptomatic pneumonitis, the relative ratio of ACE to ACE2 (ACE/ACE2) protein in the whole lung was significantly increased by radiation in male rats alone. Systemic treatment with small molecule ACE2 agonist diminazene aceturate (DIZE) increased lung ACE2 activity and reduced morbidity during radiation pneumonitis in both sexes. Notably DIZE treatment also abrogated morbidity in male rats during GI-ARS. We then evaluated the contribution of the irradiated bone marrow (BM) compartment on lung immune cell infiltration and ACE imbalance during pneumonitis. Transplantation of bone marrow from irradiated donors increased both ACE-expressing myeloid cell infiltration and immune ACE activity in the lung during pneumonitis compared to non-irradiated donors. Discussion: Together, these data demonstrate radiation induces a sex-dependent imbalance in the renin-angiotensin system enzymes ACE and ACE2. Additionally, these data suggest a role for ACE-expressing myeloid cells in the pathogenesis of radiation pneumonitis. Finally, the observed sex-differences underscore the need for consideration of sex as a biological variable in the development of medical countermeasures for radiation exposure.

Differences in radiation-induced heart dysfunction in male versus female rats.

PURPOSE: Radiation therapy remains part of the standard of care for breast, lung, and esophageal cancers. While radiotherapy improves local control and survival, radiation-induced heart dysfunction is a common side effect of thoracic radiotherapy. Cardiovascular dysfunction can also result from non-therapeutic total body radiation exposures. Numerous studies have evaluated the relationship between radiation dose to the heart and cardiotoxicity, but relatively little is known about whether there are differences based on biological sex in radiation-induced heart dysfunction (RIHD). MATERIALS AND METHODS: We evaluated whether male and female inbred Dahl SS rats display differences in RIHD following delivery of 24 Gy in a single fraction to the whole heart using a 1.5 cm beam size (collimater). We also compared the 2.0 cm vs. 1.5 cm collimator in males. Pleural and pericardial effusions and normalized heart weights were measured, and echocardiograms were performed. RESULTS: Female SS rats displayed more severe RIHD relative to age-matched SS male rats. Normalized heart weight was significantly increased in females, but not in males. A total of 94% (15/16) of males and 55% (6/11) of females survived 5 months after completion of radiotherapy (p < .01). Among surviving rats, 100% of females and 14% of males developed moderate-to-severe pericardial effusions at 5 months. Females demonstrated increased pleural effusions, with the mean normalized pleural fluid volume for females and males being 56.6 mL/kg ± 12.1 and 10.96 mL/kg ± 6.4 in males (p = .001), respectively. Echocardiogram findings showed evidence of heart failure, which was more pronounced in females. Because age-matched female rats have smaller lungs, a higher percentage of the total lung was treated with radiation in females than males using the same beam size. After using a larger 2 cm beam in males which results in higher lung exposure, there was not a significant difference between males and females in terms of the development of moderate-to-severe pericardial effusions or pleural effusions. Treatment of males with a 2 cm beam resulted in comparable increases in LV mass and reductions in stroke volume to female rats treated with a 1.5 cm beam. CONCLUSION: Together, these results illustrate that there are differences in radiation-induced cardiotoxicity between male and female SS rats and add to the data that lung radiation doses, in addition to other factors, may play an important role in cardiac dysfunction following heart radiation exposure. These factors may be important to factor into future mitigation studies of radiation-induced cardiotoxicity.

Mitigation of Multi-Organ Radiation Injury with ACE2 Agonist Diminazene Aceturate.

The renin-angiotensin system (RAS) is known to regulate the pathogenesis of radiation-induced injury as inhibitors of the RAS enzyme angiotensin converting enzyme (ACE) have established function as mitigators of multi-organ radiation injury. To further elucidate the role of RAS signaling during both the acute and delayed syndromes of radiation exposure, we have evaluated whether pharmacologic modulation of alternate RAS enzyme angiotensin converting enzyme 2 (ACE2) reduces the pathogenesis of multi-organ radiation-induced injuries. Here, we demonstrate pharmacologic ACE2 activation with the small molecule ACE2 agonist diminazene aceturate (DIZE) improves survival in rat models of both hematologic acute radiation syndrome (H-ARS) and multi-organ delayed effects of acute radiation exposure (DEARE). In the H-ARS model, DIZE treatment increased 30-day survival by 30% compared to vehicle control rats after a LD50/30 total-body irradiation (TBI) dose of 7.75 Gy. In the mitigation of DEARE, ACE2 agonism with DIZE increased median survival by 30 days, reduced breathing rate, and reduced blood urea nitrogen (BUN) levels compared to control rats after partial-body irradiation (PBI) of 13.5 Gy. DIZE treatment was observed to have systemic effects which may explain the multi-organ benefits observed including mobilization of hematopoietic progenitors to the circulation and a reduction in plasma TGF-beta levels. These data suggest the ACE2 enzyme plays a critical role in the RAS-mediated pathogenesis of radiation injury and may be a potential therapeutic target for the development of medical countermeasures for acute radiation exposure.

Pharmacologic ACE-Inhibition Mitigates Radiation-Induced Pneumonitis by Suppressing ACE-Expressing Lung Myeloid Cells.

PURPOSE: Radiation-induced lung injury is a major dose-limiting toxicity for thoracic radiation therapy patients. In experimental models, treatment with angiotensin converting enzyme (ACE) inhibitors mitigates radiation pneumonitis; however, the mechanism of action is not well understood. Here, we evaluate the direct role of ACE inhibition on lung immune cells. METHODS AND MATERIALS: ACE expression and activity were determined in the lung immune cell compartment of irradiated adult rats after either high dose fractionated radiation therapy to the right lung (5 fractions × 9 Gy) or a single dose of 13.5 Gy partial body irradiation. Mitigation of radiation-induced pneumonitis with the ACE-inhibitor lisinopril was evaluated in the 13.5 Gy rat partial body irradiation model. During pneumonitis, we characterized inflammation and immune cell content in the lungs and bronchoalveolar lavage fluid. In vitro mechanistic studies were performed using primary human monocytes and the human monocytic THP-1 cell line. RESULTS: In both the partial body irradiation and fractionated radiation therapy models, radiation increased ACE activity in lung immune cells. Treatment with lisinopril improved survival during radiation pneumonitis (P = .0004). Lisinopril abrogated radiation-induced increases in bronchoalveolar lavage fluid monocyte chemoattractant protein 1 (chemokine ligand 2) and MIP-1a cytokine levels (P < .0001). Treatment with lisinopril reduced both ACE expression (P = .006) and frequency of CD45+ CD11b+ lung myeloid cells (P = .004). In vitro, radiation injury acutely increased ACE activity (P = .045) and reactive oxygen species (ROS) generation (P = .004) in human monocytes, whereas treatment with lisinopril blocked radiation-induced increases in both ACE and ROS. Radiation-induced ROS generation was blocked by pharmacologic inhibition of either NADPH oxidase 2 (P = .012) or the type 1 angiotensin receptor (P = .013). CONCLUSIONS: These data demonstrate radiation-induced ACE activation within the immune compartment promotes the pathogenesis of radiation pneumonitis, while ACE inhibition suppresses activation of proinflammatory immune cell subsets. Mechanistically, our in vitro data demonstrate radiation directly activates the ACE/type 1 angiotensin receptor pathway in immune cells and promotes generation of ROS via NADPH oxidase 2.

Lisinopril Mitigates Radiation-Induced Mitochondrial Defects in Rat Heart and Blood Cells.

The genetic bases and disparate responses to radiotherapy are poorly understood, especially for cardiotoxicity resulting from treatment of thoracic tumors. Preclinical animal models such as the Dahl salt-sensitive (SS) rat can serve as a surrogate model for salt-sensitive low renin hypertension, common to African Americans, where aldosterone contributes to hypertension-related alterations of peripheral vascular and renal vascular function. Brown Norway (BN) rats, in comparison, are a normotensive control group, while consomic SSBN6 with substitution of rat chromosome 6 (homologous to human chromosome 14) on an SS background manifests cardioprotection and mitochondrial preservation to SS rats after injury. In this study, 2 groups from each of the 3 rat strains had their hearts irradiated (8 Gy X 5 fractions). One irradiated group was treated with the ACE-inhibitor lisinopril, and a separate group in each strain served as nonirradiated controls. Radiation reduced cardiac end diastolic volume by 9-11% and increased thickness of the interventricular septum (11-16%) and left ventricular posterior wall (14-15%) in all 3 strains (5-10 rats/group) after 120 days. Lisinopril mitigated the increase in posterior wall thickness. Mitochondrial function was measured by the Seahorse Cell Mitochondrial Stress test in peripheral blood mononuclear cells (PBMC) at 90 days. Radiation did not alter mitochondrial respiration in PBMC from BN or SSBN6. However, maximal mitochondrial respiration and spare capacity were reduced by radiation in PBMC from SS rats (p=0.016 and 0.002 respectively, 9-10 rats/group) and this effect was mitigated by lisinopril (p=0.04 and 0.023 respectively, 9-10 rats/group). Taken together, these results indicate injury to the heart by radiation in all 3 strains of rats, although the SS rats had greater susceptibility for mitochondrial dysfunction. Lisinopril mitigated injury independent of genetic background.

Radiation Increases Bioavailability of Lisinopril, a Mitigator of Radiation-Induced Toxicities.

There are no FDA-approved drugs to mitigate the delayed effects of radiation exposure that may occur after a radiological attack or nuclear accident. To date, angiotensin-converting enzyme inhibitors are one of the most successful candidates for mitigation of hematopoietic, lung, kidney, and brain injuries in rodent models and may mitigate delayed radiation injuries after radiotherapy. Rat models of partial body irradiation sparing part of one hind leg (leg-out PBI) have been developed to simultaneously expose multiple organs to high doses of ionizing radiation and avoid lethal hematological toxicity to study the late effects of radiation. Exposures between 9 and 14 Gy damage the gut and bone marrow (acute radiation syndrome), followed by delayed injuries to the lung, heart, and kidney. The goal of the current study is to compare the pharmacokinetics (PK) of a lead angiotensin converting enzyme (ACE) inhibitor, lisinopril, in irradiated vs. nonirradiated rats, as a step toward licensure by the FDA. Methods: Female WAG/RijCmcr rats were irradiated with 12.5-13 Gy leg-out PBI. At day 35 after irradiation, during a latent period for injury, irradiated and nonirradiated siblings received a single gavage (0.3 mg, 0.6 mg) or intravenous injection (0.06 mg) of lisinopril. Plasma, urine, lung, liver and kidney levels of lisinopril were measured at different times. PK modeling (R package) was performed to track distribution of lisinopril in different compartments. Results: A two-compartment (central plasma and periphery) PK model best fit lisinopril measurements, with two additional components, the gavage and urine. The absorption and renal clearance rates were similar between nonirradiated and irradiated animals (respectively: ratios 0.883, p = 0.527; 0.943, p = 0.605). Inter-compartmental clearance (from plasma to periphery) for the irradiated rats was lower than for the nonirradiated rats (ratio 0.615, p = 0.003), while the bioavailability of the drug was 33% higher (ratio = 1.326, p < 0.001). Interpretation: Since receptors for lisinopril are present in endothelial cells lining blood vessels, and radiation induces vascular regression, it is possible that less lisinopril remains bound in irradiated rats, increasing circulating levels of the drug. However, this study cannot rule out changes in total amount of lisinopril absorbed or excreted long-term, after irradiation in rats.

Brain-derived neurotrophic factor promotes immune reconstitution following radiation injury via activation of bone marrow mesenchymal stem cells.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family which has been extensively studied for its roles in neural development, long-term memory, brain injury, and neurodegenerative diseases. BDNF signaling through tropomyosin receptor kinase B (TrkB) stimulates neuronal cell survival. For this reason, small molecule TrkB agonists are under pre-clinical develoment for the treatment of a range of neurodegenerative diseases and injuries. Our laboratory recently reported BDNF is secreted by pro-regenerative endothelial progenitor cells (EPCs) which support hematopoietic reconstitution following total body irradiation (TBI). Here we report BDNF-TrkB signaling plays a novel regenerative role in bone marrow and thymic regeneration following radiation injury. Exogenous administration of BDNF or TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following myelosuppressive radiation injury promoted faster recovery of mature blood cells and hematopoietic stem cells capable of multi-lineage reconstitution. BDNF promotes hematopoietic regeneration via activation of PDGFRα+ bone marrow mesenchymal stem cells (MSCs) which increase secretion of hematopoietic cytokines interleukin 6 (IL-6) and leukemia inhibitory factor (LIF) in response to TrkB activation. These data suggest pharmacologic activation of the BDNF pathway with either BDNF or 7,8-DHF may be beneficial for treatment of radiation or chemotherapy induced myelosuppression.

Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

There is a need for countermeasures to mitigate lethal acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE). In WAG/RijCmcr rats, ARS occurs by 30-days following total body irradiation (TBI), and manifests as potentially lethal gastrointestinal (GI) and hematopoietic (H-ARS) toxicities after >12.5 and >7 Gy, respectively. DEARE, which includes potentially lethal lung and kidney injuries, is observed after partial body irradiation >12.5 Gy, with one hind limb shielded (leg-out PBI). The goal of this study is to enhance survival from ARS and DEARE by polypharmacy, since no monotherapy has demonstrated efficacy to mitigate both sets of injuries. For mitigation of ARS following 7.5 Gy TBI, a combination of three hematopoietic growth factors (polyethylene glycol (PEG) human granulocyte colony-stimulating factor (hG-CSF), PEG murine granulocyte-macrophage-CSF (mGM-CSF), and PEG human Interleukin (hIL)-11), which have shown survival efficacy in murine models of H-ARS were tested. This triple combination (TC) enhanced survival by 30-days from ∼25% to >60%. The TC was then combined with proven medical countermeasures for GI-ARS and DEARE, namely enrofloxacin, saline and the angiotensin converting enzyme inhibitor, lisinopril. This combination of ARS and DEARE mitigators improved survival from GI-ARS, H-ARS, and DEARE after 7.5 Gy TBI or 13 Gy PBI. Circulating blood cell recovery as well as lung and kidney function were also improved by TC + lisinopril. Taken together these results demonstrate an efficacious polypharmacy to mitigate radiation-induced ARS and DEARE in rats.

Corrigendum: Polypharmacy to Mitigate Acute and Delayed Radiation Syndromes.

[This corrects the article DOI: 10.3389/fphar.2021.634477.].

Rat Models of Partial-body Irradiation with Bone Marrow-sparing (Leg-out PBI) Designed for FDA Approval of Countermeasures for Mitigation of Acute and Delayed Injuries by Radiation.

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.

Acquired Immunity Is Not Essential for Radiation-Induced Heart Dysfunction but Exerts a Complex Impact on Injury.

While radiation therapy (RT) can improve cancer outcomes, it can lead to radiation-induced heart dysfunction (RIHD) in patients with thoracic tumors. This study examines the role of adaptive immune cells in RIHD. In Salt-Sensitive (SS) rats, image-guided whole-heart RT increased cardiac T-cell infiltration. We analyzed the functional requirement for these cells in RIHD using a genetic model of T- and B-cell deficiency (interleukin-2 receptor gamma chain knockout (IL2RG-/-)) and observed a complex role for these cells. Surprisingly, while IL2RG deficiency conferred protection from cardiac hypertrophy, it worsened heart function via echocardiogram three months after a large single RT dose, including increased end-systolic volume (ESV) and reduced ejection fraction (EF) and fractional shortening (FS) (p < 0.05). Fractionated RT, however, did not yield similarly increased injury. Our results indicate that T cells are not uniformly required for RIHD in this model, nor do they account for our previously reported differences in cardiac RT sensitivity between SS and SS.BN3 rats. The increasing use of immunotherapies in conjunction with traditional cancer treatments demands better models to study the interactions between immunity and RT for effective therapy. We present a model that reveals complex roles for adaptive immune cells in cardiac injury that vary depending on clinically relevant factors, including RT dose/fractionation, sex, and genetic background.

Delayed Effects of Acute Radiation Exposure (Deare) in Juvenile and Old Rats: Mitigation by Lisinopril.

Our goal is to develop lisinopril as a mitigator of delayed effects of acute radiation exposure in the National Institute of Allergy and Infectious Diseases program for radiation countermeasures. Published studies demonstrated mitigation of delayed effects of acute radiation exposure by lisinopril in adult rats. However, juvenile or old rats beyond their reproductive lifespans have never been tested. Since no preclinical models of delayed effects of acute radiation exposure were available in these special populations, appropriate rat models were developed to test lisinopril after irradiation. Juvenile (42-d-old, prepubertal) female and male WAG/RijCmcr (Wistar) rats were given 13-Gy partial-body irradiation with only part of one hind limb shielded. Lethality from lung injury between 39-58 d and radiation nephropathy between 106-114 d were recorded. All irradiated-only juvenile rats were morbid from delayed effects of acute radiation exposure by 114 d, while lisinopril (24 mg m d) started 7 d after irradiation and continued improved survival to 88% (p = 0.0015, n ≥ 8/group). Old rats (>483-d-old, reproductively senescent) were irradiated with 13-Gy partial-body irradiation keeping part of one leg shielded and additionally shielding the head in some animals. Irradiated old females developed lethal nephropathy, and all became morbid by 170 d after irradiation, though no rats displayed lethal radiation pneumonitis. Similar results were observed for irradiated geriatric males, though 33% of rats remained alive at 180 d after irradiation. Lisinopril mitigated radiation nephropathy in old rats of both sexes. Finally, comparison of delayed effects of acute radiation exposure between irradiated juvenile, adult, and old rats showed younger rats were more sensitive to delayed effects of acute radiation exposure with earlier manifestation of injuries to some organs.

Cardiac Remodeling and Reversible Pulmonary Hypertension During Pneumonitis in Rats after 13-Gy Partial-Body Irradiation with Minimal Bone Marrow Sparing: Effect of Lisinopril.

Total-body irradiation causes acute and delayed toxicity to hematopoietic, pulmonary, cardiac, gastrointestinal, renal, and other organ systems. Angiotensin-converting enzyme inhibitors mitigate many of the delayed injuries to these systems. The purpose of this study was to define echocardiographic features in rats at two times after irradiation, the first before lethal radiation pneumonitis (50 d) and the second after recovery from pneumonitis but before lethal radiation nephropathy (100 d), and to determine the actions of the angiotensin-converting enzyme inhibitor lisinopril. Four groups of female WAG/RijCmcr rats at 11-12 wk of age were studied: nonirradiated, nonirradiated plus lisinopril, 13-Gy partial-body irradiation sparing one hind leg (leg-out partial-body irradiation), and 13-Gy leg-out partial-body irradiation plus lisinopril. Lisinopril was started 7 d after radiation. Echocardiograms were obtained at 50 and 100 d, and cardiac histology was assessed after 100 d. Irradiation without lisinopril demonstrated echocardiographic transient pulmonary hypertension by 50 d which was largely resolved by 100 d in survivors. Irradiated rats given lisinopril showed no increase in pulmonary artery pressures at 50 d but exhibited left ventricular remodeling. By 100 d these rats showed some signs of pulmonary hypertension. Lisinopril alone had no impact on echocardiographic end points at either time point in nonirradiated rats. Mild increases in mast cells and fibrosis in the heart were observed after 100 d following 13-Gy leg-out partial-body irradiation. These data demonstrate irradiation-induced pulmonary hypertension which was reversed in survivors of pneumonitis. Lisinopril modified cardiovascular remodeling to enhance survival in this model from 41% to 86% (p = 0.0013).