Postmortem Findings from the Wake Forest University Radiation Late Effects Cohort of Rhesus Monkeys (Macaca mulatta).

This study focused on deceased rhesus monkeys that were part of a long-term survival cohort monitored for systemic effects of radiation. Most of these animals were of Chinese origin, with nine irradiated males from India. These animals were irradiated at various institutions primarily to investigate the acute effects of radiation and then transferred to the Wake Forest School of Medicine (WFUSM), where they joined the Radiation Late Effects Cohort (RLEC) (see Fig. 1 for demographics). The deceased controls included 16 males and 1 female, while the deceased irradiated group comprised 76 males and 13 females. The controls and irradiated animals used for this study were obtained from WFUSM, the Armed Forces Radiobiology Research Institute (Bethesda, MD), Lovelace Respiratory Research Institute (Albuquerque, NM), the University of Maryland School of Medicine (Baltimore, MD), University of Illinois Chicago (Chicago, IL), Citox Labs (Laval, Quebec, Canada), and Primate Products (Immokalee, FL). Irradiated animals received TBI under IACUC oversight at their previous institution or WFUSM, using one of two strategies: 1. Linear accelerator (LINAC)-derived photon irradiation at a nominal mean energy of 2 MeV, delivered at 80 cGy/min as a split dose, with half delivered anterior-posterior and half posterior-anterior; or 2. Cobalt 60-derived gamma radiation, delivered either as a split-dose (Citox) or simultaneously, bilaterally (AFRRI) at 60 cGy/min. One female animal received 10 Gy partial-body irradiation with approximately 5% bone marrow sparing. These were potentially lethal doses in the absence of supportive care; the LD 10/30 for rhesus macaques is ~5.5 Gy, the LD 50/30 is ~6.7 Gy, and the LD 90/30 is 8 Gy ( 17 ). Surviving animals were subsequently transferred to WFUSM for long-term study after irradiation. The LD 50/15 for the one female rhesus that received 10 Gy partial-body irradiation with ~5% bone marrow sparing is ~12 Gy. At these relatively high doses, the lethality dose groups did not vary significantly between LINAC and cobalt-60 sources ( 17 ). Deceased control animals were 9.9 to 21.4 years old (mean = 16 years), whereas irradiated animals were younger at 2.7 to 23.1 years (mean = 11.6 years, P = 0.0001) (see Fig. 1 ). Among the irradiated animals, the age at death between male and females was not significant (P = 0.3936); the median age for males was 11.2 years, and 10.3 for females. The time since irradiation to death varied between 0.3 and 14.4 years (mean = 6.4 years), and the age at irradiation ranged from 2.3 to 15.5 years (mean = 4.8 years, with a standard deviation of 2.3 years). TBI doses for these deceased animals ranged from 3.5 to 8.5 Gy (mean = 6.6 Gy). One animal received 10 Gy partial-body irradiation with ~5% bone marrow sparing. A graphical representation of demographics is shown in Fig. 2 . In this cohort, there was no significant correlation between age at irradiation and postirradiation survival time. Animals were fed a Western diet (Typical American Diet; Purina LabDiet 5L0P) supplemented with fresh fruits and vegetables and provided ad libitum water. They were housed socially but sexually segregated in indoor-outdoor pens whenever possible or in group cages if necessary for safe handling or medical care. Care was taken to ensure the animals in the groups were socially compatible. Environmental enrichment, including fruits/vegetables, toys, puzzles, climbing, and hiding environments, was provided continuously on a rotating basis. Behavioral well-being was also monitored and recommendations made as needed by an independent behavioral management team. Sampling was scheduled so that the animals were sedated the minimum number of times required for data collection. All procedures were conducted with approval by the WFUSM Institutional Animal Care and Use Committee. Wake Forest University is committed to providing a high-quality program of animal care in compliance with state and federal Animal Welfare Acts and the standards and policies of the United States Department of Health and Human Services. WFUSM has an Assurance on file in the Office for Protection from Research Risks, Office of the Director, National Institutes of Health, which accepts responsibility for the humane care and use of animals (OPRR #A-3391-01). The Laboratory Animal Care Program of WFUSM complies with the Guide for the Care and Use of Laboratory Animals and all provisions of the Animal Welfare Act and has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) since April 8, 1966 (AAALAC File #8). All animals were assessed twice daily for food consumption, defecation, urination, evidence of discomfort or illness. Animals were routinely screened three times a year with clinical exams, complete blood counts, and serum chemistry panels. Signs of hematologic failure or infection were monitored using the methods of Uckun, as modified from the Children’s Cancer Group Clinical Toxicity Criteria ( 18 ). Animals were euthanized and taken for necropsy when they met predefined clinical criteria ( Table 1 ), or in rare cases, died unexpectedly. A complete necropsy was performed, followed by systematic histologic evaluation of all major organ systems and tissue types. The specific tissues examined histologically are detailed in Table 2 . Necropsy reports were generated for each animal by veterinary pathology residents and fellows, and reviewed by faculty veterinary pathologists. These reports used standardized morphologic diagnoses to ensure consistency over time. Two veterinary pathologists (GWS and JMC) reviewed one hundred and four necropsy reports for all animals in the RLEC that were either euthanized or died between 2008 and 2022. Diagnoses from these reports were compiled and standardized: for instance, if one report listed “hemangiosarcoma” and another “angiosarcoma,” both were unified as angiosarcomas to align with current diagnostic terminology. Diagnoses with a known cause unrelated to irradiation, such as conspecific trauma or healing surgical wounds, were excluded. In total, there were 2,790 diagnoses, representing 284 unique diagnoses. Fisher’s exact tests were used to compare the prevalence of individual diagnoses between control and irradiated animals, and statistical significance was defined as a P value ≤ 0.05. Fisher’s exact test was chosen due to small n of many diagnoses, often occurring in less than five animals, and also as it is the de facto test used in toxicologic pathology for the categorical comparison of two groups ( 18 ). Radiation was treated as a categorical variable with two possible values: yes or no. Given that there were 284 unique diagnoses, further stratification by radiation dose as a continuous variable or by dose group was beyond the scope of this paper. Sex differences are shown in Tables 1 - 22 ; however, the historical bias of using male animals for radiation research and the small number of deceased females prevented statistical analysis of diagnoses between the sexes. Continuous variables such as age at death were analyzed using D’Agostino and Pearson and Shapiro-Wilk tests for normality, and unpaired t tests. All statistics were performed with GraphPad Prism, version 10.1.2.

Tables 3 - 22 show prevalence of individual histologic morphologic diagnoses by major organ system. Neoplasia was catalogued separately ( Tables 13 and 14 ), and organized by malignant or benign type. Relatively few lesion frequencies were significantly different between irradiated and control animals. This was likely due to the confounding age difference between deceased control and irradiated animals, as many lesions were consistent with aging, such as fibrosis and chronic inflammation, but occurred at younger ages in the irradiated animals. Certain lesions, such as myocardial fibrosis, diverticulosis, and obesity ( Tables 3 , 6 , and 22 , and Fig. 3 ), all strongly correlated with age, were more common in the older control animals ( p ≤ 0.05). Similarly, lesions typically linked to the delayed effects of acute radiation exposure—such as cataracts, testicular atrophy, pulmonary inflammation and fibrosis, and malignant neoplasia—were significantly more prevalent in the irradiated animals ( Tables 14 , 15 , 19 and 20 , and Fig. 4 ). Although not statistically significant, inflammation throughout the GI tract, liver, and kidneys was common in irradiated animals, usually lymphoplasmacytic in nature ( Tables 6 , 8 and 17 ). As mentioned above, malignant neoplasia was significantly more prevalent in the irradiated animals ( Table 14 ). Notable tumor types diagnosed included malignant peripheral nerve sheath tumors, angiosarcomas, osteosarcomas, acute myeloid leukemias, and glomus tumors (P = 0.0108; Table 13 ). Many of these tumors were not diagnosed until necropsy, which precluded an accurate assessment of the age of onset of neoplasia. A previous study, which included a subset of the subjects in this cohort, from Sills et al. ( 19 ), suggested that irradiation accelerated tumorigenesis. Of note, the prevalence and diversity of renal neoplasia was unexpectedly high in the irradiated animals, which included sarcomas, carcinomas, and a glomus tumor ( Fig. 5 ). In addition to neoplasia, other lesions commonly seen in the kidneys of irradiated animals included corticomedullary cysts, lymphoplasmacytic inflammation, and fibrosis, although none reached statistical significance. Membranoproliferative glomerulopathy was another common finding, and despite being a lesion associated with aging, it had nearly identical prevalence between control and irradiated animals, suggesting that irradiation reduced the age of onset for chronic inflammatory kidney disease ( Table 17 ).

Myocardial fibrosis was previously reported as more prevalent in irradiated animals, but that report was limited to an age-matched subset of this cohort, whereas this report contained a group of much older controls ( 20 ). We also note that myocardial fibrosis is a commonly reported lesion in aged rhesus macaques, and thus we believe that the higher necropsy prevalence in the present study is an effect of age ( 21 ). Similarly, the higher prevalence of arteriosclerosis in our data likely represents an age effect. The higher prevalence of cardiovascular disease in irradiated human populations is well known ( 22 , 23 ). The renal interstitial fibrosis and chronic glomerular injury observed in this cohort were like those described by Cohen et al. ( 9 ), in a high-dose (10–12 Gy) partial-body irradiation model of rhesus monkeys, some of which survived to 180 days postirradiation. However, in contrast to that study, which found minimal mononuclear cellular infiltrate/inflammation, low-grade inflammation was a relatively common finding among the irradiated and control animals in this study (30% vs. 25%, respectively), and may be an age-related change ( 9 , 24 ). The lack of clear radiation-associated morphological changes in gastrointestinal disease was interesting. Although none of the animals in this study received a high enough dose to develop acute GI syndrome, we have shown that diarrheal disease is more common in irradiated animals and that the GI mucosal barrier is compromised ( 20 , 25 ). Indeed, other groups have demonstrated chronic mucosal barrier injury in NHPs that received partial-body irradiation ( 26 ). At doses higher than this cohort received, GI injury is well-described in various animal models ( 27 , 28 ). A functional impairment may be present in this cohort of NHPs, but without overt pathology, or that the well-documented immune impairment in this cohort contributes to the compromised ability to respond to GI infection ( 29 - 31 ). Testicular atrophy was one of the most evident effects of irradiation, as we have previously documented ( 32 ). Importantly, we previously found that severely affected animals have both reduced spermatogenesis and decreased androgen production; a hypoandrogenic state can potentially impact many organ systems. The increased occurrence of pulmonary inflammation, fibrosis, and smooth muscle hyperplasia in irradiated animals within our population indicates that even at doses below the 10 Gy threshold for radiation pneumonitis, low-grade lung pathology is observed ( 11 , 14 ). The lower prevalence of obesity is presumably due to chronic GI disease (primarily diarrhea) but also impaired adipocyte function in irradiated animals, as demonstrated by Bacarella et al. ( 33 ). These damaged and dysfunctional adipocytes likely contribute to the development of insulin resistance and then type II diabetes years after irradiation, despite generally being underweight. The pre-diabetic and diabetic animals in this study showed consistent amyloid buildup in the endocrine pancreas ( Table 5 ), ( 34 ). Neoplasia is a well documented and expected outcome of radiation exposure. Survivors of the atomic bomb blasts in Hiroshima and Nagasaki exhibited an increased risk for leukemia and lymphoma earlier in postirradiation follow-up (<10 years). In comparison, the risk of other solid tumors steadily increased after 10 years postirradiation ( 2 ). The prevalence of leukemia and lymphoma in our cohort (4%), despite the comparatively small N, was relatively similar. However, these animals generally received much higher doses of radiation: A mean of 6.6 Gy compared to the Japanese survivors, most of whom were exposed to an estimated ≤ 2 Gy. The preponderance of soft tissue sarcomas was an unexpected finding and was discussed in depth in the context of this cohort by Sills et al. ( 19 ). Additionally the prevalence of glomus tumors was surprising (3 in this cohort of deceased animals, and 2 additional in currently living, irradiated members of the cohort at the time of publication), as these are extremely rare tumors of pericyte origin. In humans, these rare tumors most commonly arise from the glomus body, which senses temperature and blood pressure, and the tumors are almost exclusively found in the fingertips ( 35 - 38 ). In our population, glomus tumors were identified in the kidneys, subcutaneous tissues of the trunk, and the seminal vesicles. Of note, at the time of publication, there are two living, male, irradiated animals that also have glomus tumors (one in the liver and the other in the kidney). Brain injury in irradiated rhesus monkeys, either due to high doses of fractionated radiation or in the context of limited age-matched studies in this cohort, has demonstrated microvascular and white-matter injury ( 39 - 42 ). Despite the high prevalence of cerebrovascular degeneration and necrosis in the irradiated animals, none were statistically significant. Most prior studies of brain injury in this cohort relied on MRI analysis and were not restricted to deceased members of the cohort. Additionally, MRI provides high-resolution detail of the entire brain, identifying small lesions that may not be visible during gross or histopathologic exams. The overall numbers of females in this study were limited due to the misguided historical bias placed on males in animal models of radiation injury; however, it is worth noting that 4 of the 13 irradiated females (31%) developed endometriosis. Endometriosis was also a reported finding in the United States Air Force cohort of irradiated rhesus monkeys, which were observed for up to 25 years postirradiation ( 43 ). Overall, the findings can be roughly grouped into two categories: deterministic outcomes of radiation exposure, such as testicular injury, cataracts, and multiorgan fibrosis, and stochastic outcomes related to either age or radiation, or more likely, a combination of both, including multiorgan chronic inflammation and neoplasia. The fundamental mechanisms of radiation injury, ROS injury and DNA damage (genomic instability), are also key hallmarks of age-related injury, and may explain some of the pathologic overlap observed in this cohort ( 44 ). The age difference between irradiated and control animals, and the fact that irradiated animals were aging as they were observed has made differentiating age versus radiation effects difficult. Indeed, these findings further support the idea that the irradiated animals exhibit an “accelerated aging” phenotype ( 25 , 45 ). Many of the lesions described in the irradiated animals are similar to those described in colonies of geriatric rhesus monkeys, some of which were decades older ( 21 ). This work offers some of the most comprehensive pathologic descriptions in a large, aging cohort of irradiated NHPs observed for over 14 years and will serve as a solid foundation for future molecular and statistical studies.

With few notable exceptions, most findings in the irradiated animals were similar to those observed in aging control animals, despite the animals being significantly younger. These findings support the potential of this cohort, and radiation exposure in general, to deepen our knowledge of aging and resilience to systemic stressors.

The advent of technology enabling the splitting of atoms marked a dubious milestone in human history, giving rise to the medical phenomenon of injury from high doses of ionizing radiation. Following the nuclear attacks on Hiroshima and Nagasaki in 1945, hundreds of thousands of survivors grappled with the immediate and enduring consequences of radiation exposure, with some continuing to suffer its delayed effects even 78 years later. An increase in cases of cancer, thyroid disease, cardiovascular disease, intestinal disease and respiratory disease have all been reported among these survivors ( 1 , 2 ). Similarly, survivors of accidental radiation exposure often experience a higher rate of health problems such as neoplasia and cardiovascular disease ( 3 - 5 ). A need to better understand and characterize the delayed effects of acute radiation exposure led to the formation of the Wake Forest Radiation Late Effects Cohort of rhesus macaques (RLEC). Formerly known as the Radiation Survivors Cohort, this is a cohort of animals monitored after exposure to varying doses of total-body irradiation (TBI) and approximately 5% bone marrow-sparing partial-body irradiation. These animals were initially used to study acute radiation syndrome (ARS), and the survivors later joined the RLEC for lifelong observation. For a more comprehensive historical perspective and description of this cohort, see Olson et al. ( 6 ). Rhesus macaques ( Macaca mulatta ) share approximately 95 to 97.5% genetic similarity with humans and are one of the most well-characterized large animal models utilized in biomedical research ( 7 ). The acute response of the rhesus monkey to ionizing radiation is similar to that of humans and has been well documented up to 180 days after irradiation ( 8 - 15 ). The United States Air Force conducted studies that performed postmortem examinations up to 25 years postirradiation in the 1960s; however, detailed pathology findings from all major organ systems were never published ( 16 ). Herein, we systematically describe the histologic lesions in all major organ systems observed up to 14.4 years postirradiation.