Acute Radiation-induced Lung Injury in the Non-human Primate: A Review and Comparison of Mortality and Co-morbidities Using Models of Partial-body Irradiation with Marginal Bone Marrow Sparing and Whole Thorax Lung Irradiation.

The nonhuman primate, rhesus macaque, is a relevant animal model that has been used to determine the efficacy of medical countermeasures to mitigate major signs of morbidity and mortality of radiation-induced lung injury. Herein, a literature review of published studies showing the evolution of lethal lung injury characteristic of the delayed effects of acute radiation exposure between the two significantly different exposure protocols, whole thorax lung irradiation and partial-body irradiation with bone marrow sparing in the nonhuman primate, is provided. The selection of published data was made from the open literature. The primary studies conducted at two research sites benefitted from the similarity of major variables; namely, both sites used rhesus macaques of approximate age and body weight and radiation exposure by LINAC-derived 6 MV photons at dose rates of 0.80 Gy min and 1.00 Gy min delivered to the midline tissue via bilateral, anterior/posterior, posterior/anterior geometry. An advantage relative to sex difference resulted from the use of male and female macaques by the Maryland and the Washington sites, respectively. Subject-based medical management was used for all macaques. The primary studies (6) provided adequate data to establish dose response relationships within 180 d for the radiation-induced lung injury consequent to whole thorax lung irradiation (male vs. female) and partial-body irradiation with bone marrow sparing exposure protocols (male). The dose response relationships established by probit analyses vs. linear dose relationships were characterized by two main parameters or dependent variables, a slope and LD50/180. Respective LD50/180 values for the primary studies that used whole thorax lung irradiation for respective male and female nonhuman primates were 10.24 Gy [9.87, 10.52] (n = 76, male) and 10.28 Gy [9.68, 10.92] (n = 40, female) at two different research sites. The respective slopes were steep at 1.73 [0.841, 2.604] and 1.15 [0.65, 1.65] probits per linear dose. The LD50/180 value and slope derived from the dose response relationships for the partial-body irradiation with bone marrow sparing exposure was 9.94 Gy [9.35, 10.29] (n = 87) and 1.21 [0.70, 1.73] probits per linear dose. A secondary study (1) provided data on limited control cohort of nonhuman primates exposed to whole thorax lung irradiation. The data supported the incidence of clinical, radiographic, and histological indices of the dose-dependent lung injury in the nonhuman primates. Tertiary studies (6) provided data derived from collaboration with the noted primary and secondary studies on control cohorts of nonhuman primates exposed to whole thorax lung irradiation and partial-body irradiation with bone marrow sparing exposure. These studies provided a summary of histological evidence of fibrosis, inflammation and reactive/proliferative changes in pneumonocytes characteristic of lung injury and data on biomarkers for radiation-induced lung injury based on matrix-assisted laser desorption ionization-mass spectrometry imaging and gene expression approaches. The available database in young rhesus macaques exposed to whole thorax lung irradiation or partial-body irradiation with bone marrow sparing using 6 MV LINAC-derived radiation with medical management showed that the dose response relationships were equivalent relative to the primary endpoint all-cause mortality. Additionally, the latency, incidence, severity, and progression of the clinical, radiographic, and histological indices of lung injury were comparable. However, the differences between the exposure protocols are remarkable relative to the demonstrated time course between the multiple organ injury of the acute radiation syndrome and that of the delayed effects of acute radiation exposure, respectively.

Efficacy of Neulasta or Neupogen on H-ARS and GI-ARS Mortality and Hematopoietic Recovery in Nonhuman Primates After 10-Gy Irradiation With 2.5% Bone Marrow Sparing.

A nonhuman primate model of acute, partial-body, high-dose irradiation with minimal (2.5%) bone marrow sparing was used to assess endogenous gastrointestinal and hematopoietic recovery and the ability of Neulasta (pegylated granulocyte colony-stimulating factor) or Neupogen (granulocyte colony-stimulating factor) to enhance recovery from myelosuppression when administered at an increased interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neulasta or Neupogen on mortality and morbidity due to the hematopoietic acute radiation syndrome and concomitant gastrointestinal acute radiation syndrome. Nonhuman primates were exposed to 10.0 Gy, 6 MV, linear accelerator-derived photons delivered at 0.80 Gy min. All nonhuman primates received subject-based medical management. Nonhuman primates were dosed daily with control article (5% dextrose in water), initiated on day 1 postexposure; Neulasta (300 µg kg), administered on days 1, 8, and 15 or days 3, 10, and 17 postexposure; or Neupogen (10 µg kg), administered daily postexposure following its initiation on day 1 or day 3 until neutrophil recovery (absolute neutrophil count ≥1,000 cells µL for 3 consecutive days). Mortality in the irradiated cohorts suggested that administration of Neulasta or Neupogen on either schedule did not affect mortality due to gastrointestinal acute radiation syndrome or mitigate mortality due to hematopoietic acute radiation syndrome (plus gastrointestinal damage). Following 10.0 Gy partial-body irradiation with 2.5% bone marrow sparing, the mean duration of neutropenia (absolute neutrophil count <500 cells µL) was 22.4 d in the control cohort vs. 13.0 and 15.3 d in the Neulasta day 1, 8, 15 and day 3, 10, 17 cohorts, relative to 16.2 and 17.4 d in the Neupogen cohorts initiated on day 1 and day 3, respectively. The absolute neutrophil count nadirs were 48 cells µL in the controls; 117 cells µL and 40 cells µL in the Neulasta days 1, 8, and 15 or days 3, 10, and 17 cohorts, respectively; and 75 cells µL and 37 cells µL in the Neupogen day 1 and day 3 cohorts, respectively. Therefore, the earlier administration of Neulasta or Neupogen was more effective in this model of marginal 2.5% bone marrow sparing. The approximate 2.5% bone marrow sparing may approach the threshold for efficacy of the lineage-specific medical countermeasure. The partial-body irradiation with 2.5% bone marrow sparing model can be used to assess medical countermeasure efficacy in the context of the concomitant gastrointestinal and hematopoietic acute radiation syndrome sequelae.

Acute and Chronic Kidney Injury in a Non-Human Primate Model of Partial-Body Irradiation with Bone Marrow Sparing.

The development of medical countermeasures against acute and delayed multi-organ injury requires animal models predictive of the human response to radiation and its treatment. Late chronic injury is a well-known feature of radiation nephropathy, but acute kidney injury has not been reported in an appropriate animal model. We have established a single-fraction partial-body irradiation model with minimal marrow sparing in non-human primates. Subject-based medical management was used including parenteral fluids according to prospective morbidity criteria. We show herein that 10 or 11 Gy exposures caused both acute and chronic kidney injury. Acute and chronic kidney injury appear to be dose-independent between 10 and 11 Gy. Acute kidney injury was identified during the first 50 days postirradiation and appeared to resolve before the occurrence of chronic kidney injury, which was progressively more severe up to 180 days postirradiation, which was the end of the study. These findings show that mitigation of the acute radiation syndrome by medical management will unmask delayed late effects that occur months after partial-body irradiation. They further emphasize that both acute and chronic changes in kidney function must be taken into account in the use and timing of mitigators and medical management for acute radiation syndrome and delayed effects of acute radiation exposure (DEARE).

A non-human primate model of radiation-induced cachexia.

Cachexia, or muscle wasting, is a serious health threat to victims of radiological accidents or patients receiving radiotherapy. Here, we propose a non-human primate (NHP) radiation-induced cachexia model based on clinical and molecular pathology findings. NHP exposed to potentially lethal partial-body irradiation developed symptoms of cachexia such as body weight loss in a time- and dose-dependent manner. Severe body weight loss as high as 20-25% was observed which was refractory to nutritional intervention. Radiographic imaging indicated that cachectic NHP lost as much as 50% of skeletal muscle. Histological analysis of muscle tissues showed abnormalities such as presence of central nuclei, inflammation, fatty replacement of skeletal muscle, and muscle fiber degeneration. Biochemical parameters such as hemoglobin and albumin levels decreased after radiation exposure. Levels of FBXO32 (Atrogin-1), ActRIIB and myostatin were significantly changed in the irradiated cachectic NHP compared to the non-irradiated NHP. Our data suggest NHP that have been exposed to high dose radiation manifest cachexia-like symptoms in a time- and dose-dependent manner. This model provides a unique opportunity to study the mechanism of radiation-induced cachexia and will aid in efficacy studies of mitigators of this disease.

The Effect of Radiation Dose and Variation in Neupogen® Initiation Schedule on the Mitigation of Myelosuppression during the Concomitant GI-ARS and H-ARS in a Nonhuman Primate Model of High-dose Exposure with Marrow Sparing.

A nonhuman primate (NHP) model of acute high-dose, partial-body irradiation with 5% bone marrow (PBI/BM5) sparing was used to assess the effect of Neupogen® [granulocyte colony stimulating factor (G-CSF)] to mitigate the associated myelosuppression when administered at an increasing interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neupogen® on the mortality or morbidity of the hematopoietic (H)- acute radiation syndrome (ARS) and concurrent acute gastrointestinal radiation syndrome (GI-ARS). NHP were exposed to 10.0 or 11.0 Gy with 6 MV LINAC-derived photons at approximately 0.80 Gy min. All NHP received medical management. NHP were dosed daily with control article (5% dextrose in water) initiated on day 1 post-exposure or Neupogen® (10 µg kg) initiated on day 1, day 3, or day 5 until recovery [absolute neutrophil count (ANC) ≥ 1,000 cells µL for three consecutive days]. Mortality in both the 10.0 Gy and 11.0 Gy cohorts suggested that early administration of Neupogen® at day 1 post exposure may affect acute GI-ARS mortality, while Neupogen® appeared to mitigate mortality due to the H-ARS. However, the study was not powered to detect statistically significant differences in survival. The ability of Neupogen® to stimulate granulopoiesis was assessed by evaluating key parameters for ANC recovery: the depth of nadir, duration of neutropenia (ANC < 500 cells µL) and recovery time to ANC ≥ 1,000 cells µL. Following 10.0 Gy PBI/BM5, the mean duration of neutropenia was 11.6 d in the control cohort vs. 3.5 d and 4.6 d in the day 1 and day 3 Neupogen® cohorts, respectively. The respective ANC nadirs were 94 cells µL, 220 cells µL, and 243 cells µL for the control and day 1 and day 3 Neupogen® cohorts. Following 11.0 Gy PBI/BM5, the duration of neutropenia was 10.9 d in the control cohort vs. 2.8 d, 3.8 d, and 4.5 d in the day 1, day 3, and day 5 Neupogen® cohorts, respectively. The respective ANC nadirs for the control and day 1, day 3, and day 5 Neupogen® cohorts were 131 cells µL, 292 cells µL, 236 cells µL, and 217 cells µL, respectively. Therefore, the acceleration of granulopoiesis by Neupogen® in this model is independent of the time interval between radiation exposure and treatment initiation up to 5 d post-exposure. The PBI/BM5 model can be used to assess medical countermeasure efficacy in the context of the concurrent GI- and H-ARS.

Immune cell reconstitution after exposure to potentially lethal doses of radiation in the nonhuman primate.

Delayed immune reconstitution remains a major cause of morbidity associated with myelosuppression induced by cytotoxic therapy or myeloablative conditioning for stem cell transplant, as well as potentially lethal doses of total- or partial-body irradiation. Restoration of a functional immune cell repertoire requires hematopoietic stem cell reconstitution for all immune cells and effective thymopoiesis for T cell recovery. There are no medical countermeasures available to mitigate damage consequent to high-dose, potentially lethal irradiation, and there are no well characterized large animal models of prolonged immunosuppression to assess efficacy of potential countermeasures. Herein, the authors describe a model of T and B cell reconstitution following lethal doses of partial-body irradiation with 5% bone marrow sparing that includes full exposure of the thymus. Rhesus macaques (n = 31 male, 5.5-11.3 kg body weight) were exposed to midline tissue doses of 9.0-12.0 Gy using 6 MV LINAC-derived photons at a dose rate of 0.80 Gy min, sparing approximately 5% of bone marrow (tibiae, ankles, and feet). All animals received medical management and were monitored for myeloid and lymphoid suppression and recovery through 180 d post-exposure. Myeloid recovery was assessed by neutrophil and platelet-related hematological parameters. Reconstitution of B and T cell subsets was assessed by flow cytometric immunophenotyping, and recent thymic emigrants were identified by RT-PCR of T cell receptor excision circles. Mortality was recorded through 180 d post-exposure. Acute myelo-suppression was characterized by severe neutropenia and thrombocytopenia, followed by recovery 30-60 d post-exposure. Total T (CD3+) and B (CD20+) cells were reduced significantly following exposure and exhibited differential recovery patterns post-exposure. Both CD4+ and CD8+ subsets of naïve T cells and total CD4+ T cell counts remained significantly lower than baseline through 180 d post-exposure. The failure of recent thymic emigrants and naïve T cell subsets to recover to normal baseline values reflects the severe radiation effects on the recovery of marrow-derived stem and early thymic progenitor cells, their mobilization and seeding of receptive thymic niches, and slow endogenous thymic regeneration.