The Wake Forest Nonhuman Primate Radiation Late Effects Cohort

Abstract

Wake Forest University School of Medicine (WFUSM) hosts the Radiation Late Effects Cohort (RLEC), a unique cohort of previously irradiated rhesus monkeys gathered from multiple institutions over twenty years. Supported by the National Institutes of Health’s National Institute of Allergy and Infectious Diseases (NIH/NIAID) Radiation/Nuclear program, it serves as a resource for the Centers for Medical Countermeasures against Radiation Consortium (CMCRC) and the wider biomedical research community. These animals act as a national resource for examining the long-term effects of radiation exposure. Since its establishment in 2007, the RLEC has studied 328 macaques, including 270 exposed to external beam ionizing radiation and 58 controls. Results to date reveal a multisystemic pattern of chronic illness, including metabolic disease and diabetes mellitus, hypertension, higher rates of cancers, long-term immune impairment, myocardial disease, cerebrovascular disease, low body weight, gonadal injury with reduced sex steroid output, cataracts, osteopenia, renal disease, compromised intestinal barrier function, and ongoing systemic inflammation. This summary highlights the essential aspects of the RLEC, the significant achievements of researchers using this resource, and the potential for further investigation of this unique animal population and its associated resources.

INTRODUCTION

Nonhuman Primates in Radiation Research

The value of nonhuman primates (NHP) in radiation research lies in their genetic, anatomic, and physiologic similarity to human primates. Rhesus macaques share approximately 93% (deoxyribonucleic acid) of their DNA sequence identity with humans (1), and they share with humans primate-specific neurologic, immunologic, reproductive, and other biological features that differ from those of rodents and other species. The landscape and outlook for NHP research in the U.S. have been the subject of a recent review by the National Academies of Science (2), highlighting the trend toward multidisciplinary collaborative long-term studies, transparency, and data sharing. The work described here aims to reflect the evolution of the field and to take advantage of a unique opportunity to study long-term effects of radiation.

Early work in the field of NHP radiation responses was focused on modeling the acute risks and consequences of nuclear weapon radiation exposures. These studies included the acute effects of high-dose exposure on functional abilities (3), the hematopoietic acute radiation syndrome (4), and morbidity/mortality from atomic bomb/fallout exposures (5). Several long-term follow-up studies were conducted, notably the United States Air Force 25-year study of proton exposures (6) and the long-term cohort study of mixed neutron and photon exposures in the Netherlands (7). These studies yielded a wealth of knowledge regarding the pathophysiology of radiation injury; however, they occurred prior to the modern era of molecular biology and genomics. Medical interventions developed during this period were largely preventative. They included small molecule radioprotectants such as the thiol analog amifostine (8) and the use of potassium iodide to reduce or displace radioactive iodine exposure of the thyroid gland (9).

More recent studies of radiation in NHPs were conducted in the post-9/11 era, following the creation of the NIH/NIAID Centers for Medical Countermeasures against Radiation and the development of other federal programs in support of U.S. national defense and preparedness. This second wave of studies was focused on post hoc mitigation, rather than protective strategies, and resulted in Food and Drug Administration (FDA) approvals of hematopoietic growth factor analogs filgrastim (10), pegfilgrastim (11), sargramostim (12) and others as reviewed by Singh and Seed (13).

With the increased number of NHP studies in the early 2000s came an opportunity to study the delayed effects of acute radiation exposure (DEARE). The major human population health burden of radiation exposure is chronic disease, including cancers, cardiac disease (14), and a variety of long-term non-cancer diseases leading to increased morbidity and mortality (15), but the interpretation of these findings is complicated by other risk factors such as smoking, uncertain radiation doses, comorbidities, and other uncontrolled sources of variation. The RLEC was established to investigate chronic disease outcomes in a controlled, well-characterized population of animals with high genetic and physiological similarity to humans.

The Radiation Late Effects Cohort

The WFUSM Radiation Late Effects Cohort (RLEC) was established in 2007. Irradiated rhesus macaques (Macaca mulatta) were donated to the RLEC after completing short-term acute-phase radiation studies. Sham-irradiated controls were obtained when available; however, controls from other sources were accepted (e.g., blood donor or breeding colony animals). The RLEC primarily includes rhesus macaques (Macaca mulatta), mostly males. Female animals have been increasingly added to the cohort to better address sex differences. Seventy-six percent of these animals are of Chinese genetic origin, 22% are of Indian genetic origin, and 2% are hybrid or of uncertain origin. Cynomolgus macaques (Macaca fascicularis) have recently been included in small numbers (n = 3), reflecting the growing use of this species in radiation research. Characteristics of the cohort are shown in Fig. 1 and Table 1; the overall census of the cohort is displayed in Fig. 2.

FIG. 1.

Graphical representation of the current census of living rhesus and cynomolgus macaques (*) in the RLEC. Deceased animals are shown in Schaaf et al.³ Each horizontal line represents one animal, shown on its own lifespan; the solid line shows the time under observation at WFUSM. Dotted lines show the time between irradiation and arrival at WFUSM.

TABLE 1.

Age and Sex Characteristics of Rhesus Macaques in the RLEC Cohort, Including Living and Deceased Animals

Radiation dose category	Sex	Age at irradiation avg ± SD median (range)	Proportion immature at irradiation (<4 years)	Age at present avg ± SD median (range)	Proportion currently aged (>20 years)
PBI w/5% BM sparing (9–10 Gy)	M	3.5 ± 0.5; 3.3 (3.0–4.3)	14/19	5.9 ± 0.9; 5.4 (5.24–7.5)	0/19
	F	4.7 ± 0.4; 4.7 (4.3–5.2)	0/9	7.3 ± 0.9; 7.6 (6.3–8.3)	0/9
>LD90 (>8 Gy)	M	3.7 ± 0.4; 3.7 (3.2–4.4)	10/13	11.9 ± 4.6; 12.1 (3.8–21.4)	1/13
	F	5.1 ± 0.0; 5.1 (5.1–5.1)	0/1	10.2 ± 0.0; 10.2 (10.2–10.2)	0/1
LD50-LD90 (6.75–8 Gy)	M	3.9 ± 0.9; 3.6 (2.4–7.0)	37/55	10.9 ± 3.6; 10.2 (2.8–19.2)	0/55
	F	4.4 ± 0.8; 4.2 (3.5–6.0)	10/29	10.0 ± 2.6; 8.5 (7.4–16.4)	0/29
LD10-LD50 (5.5–6.75 Gy)	M	5.9 ± 3.1; 4.7 (2.3–15.5)	20/63	12.1 ± 3.9; 11.3 (5.0–23.1);	4/63
	F	4.6 ± 1.1; 4.5 (3.1–8.4)	5/18	10.8 ± 1.9; 10.2 (7.6–14.6)	0/18
<LD10 (<5.5 Gy)	M	10.2 ± 2.1; 9.5 (7.2–15.4)	2/37	10.2 ± 2.1; 9.5 (7.2–15.4)	0/37
	F	5.1 ± 1.2; 5.1 (2.7–6.8)	3/23	11.0 ± 3.0; 10.1 (7.4–16.3)	0/23
	F*	7.6 ± 0.5; 7.3 (7.3–8.2)	0/3	8.8 ± 0.5; 8.7 (8.7–9.1)	0/3
Irradiated totals	M	4.8 ± 2.1; 4.2 (2.3–15.5)	83/187	10.7 ± 3.8; 10.1 (2.7–23.1)	5/187
	F	4.7 ± 1.0; 4.5 (2.7–8.4)	18/83	10.2 ± 2.7; 9.5 (3.6–16.4)	0/83
Controls	M	NA	NA	15.5 ± 4.6; 15.7 (9.0–25.4)	11/47
	F	NA	NA	20.4 ± 4.4; 19.6 (15.7–29.2)	4/11
Total = 328

^*Cynomolgus macaques (N = 3).

FIG. 2.

Cumulative census of NHPs in the RLEC, by year. The cohort is maintained at a maximum capacity of 200 animals.

All animals were in good health at the time of accession into the cohort. Eligibility for the RLEC required veterinary examinations at the donating institution and at WFUSM and review of medical records, past experimental history if any, disclosure of the irradiation protocol if irradiated, and mitigator type dose and route, and any supportive care or blood transfusions given. Irradiated animals were typically transferred at more than 60 days postirradiation to allow for full recovery from hematopoietic injury and underwent a 60–90 day quarantine at WFUSM. Animals were not required to be specific-pathogen free; rather, viral status was recorded based on serologic screens for simian retroviruses and macacine herpesvirus 1, vaccination history for measles or other pathogens, and any other relevant exposures.

Notably, 38% of animals in the cohort were exposed to irradiation before or near the age of sexual maturity (<4 years), and 90% were irradiated prior to the typical age of adult body size (<7 years). These cutoffs were derived from literature conventions for rhesus macaques, and validated by developmental features such as dentition (16), and body size (17). Thus, a de facto juvenile exposure sub-cohort exists, and most animals were irradiated as “young adults” or at younger ages.

The dose categories shown in Fig. 1 and Table 1 are derived from an extensive literature review conducted by Macvittie et al. (4), which yielded an LD₅₀ of 6.75 Gy for total-body irradiation (TBI) in rhesus macaques. In addition to TBI animals, the cohort includes animals exposed to partial-body irradiation (PBI) with 5% bone marrow sparing, achieved by shielding the animals from the knees down during irradiation.

Radiation Exposures

Radiation exposures are summarized in Tables 1 and 2. The RLEC includes NHPs exposed to photons from ⁶⁰Co and linear accelerator (Linac) sources at multiple institutions. The energy of exposure was 1.17 and 1.33 (mean, 1.25) MeV for ⁶⁰Co gamma rays based on the emission characteristic of the isotope, and for Linac exposures a spectrum of energies was delivered using 6 MV X rays (6 MeV X-ray maximum energy), having a mean monoenergetic energy of approximately 2 MeV (18). Dose rates were 0.6–0.8 Gy/min at the animal midline. The geometry of exposure was similar across sites, with the radiation beam perpendicular to the long axis of the body. Sites used parallel-opposed beams, either right and left lateral fields or anterior and posterior fields, with most sites using a split-dose strategy whereby half the dose was given to one side of the animal followed by repositioning of the animal or the source and delivery of the second half of the dose within a few min (typically less than 5 min). Dosimetry was validated with measurements prior to animal irradiations and some sites used in-field dosimeters at the time of irradiation. Radiation parameters have been published in the reports of acute radiation syndrome by most of the investigative teams who later donated animals to the RLEC (19–21). The information presented in Table 2 is taken from those reports or direct surveys of donor laboratories. The use of sedation/anesthesia or oxygen varied by site; however, blood hemoglobin oxygen saturation under awake, ketamine-sedated, and anesthetized conditions was typically >90%.

TABLE 2.

Radiation Exposures

Source: Co-60
Energy: 1.25 MeV
Dose rate: 0.6 Gy/min
Geometry	Sedation, gas	Sex	n	Total dose (Gy)	Age at irradiation (years)	Age (years)	Support
TBI, LR-RL simultaneous; Seated, 270 cm from source	Ketamine, room air	M	64	6.7 ± 1.0 (3.5–8.5)	5.7 ± 2.6 (3.1–14.2)	12.7 ± 3.5 (3.8–23.1)	SC, AB, BT
		F	24	5.7 ± 1.8 (1.1–7.2)	5.0 ± 0.8 (2.7–6.4)	13.3 ± 2.1 (8.7–16.4)	SC, AB, BT
TBI, AP-PA split; Standing, 172 cm from source	Awake, room air	M	27	6.0 ± 1.0 (4.0–6.8)	4.1 ± 1.0 (3.3–8.2)	9.0 ± 1.4(5.2–11.6)	SC
		F	36	6.0 ± 1.0 (4.0–6.8)	4.3 ± 1.0 (3.1–6.8)	9.2 ± 1.3 (7.4–13.0)	SC
PBIBM5, AP-PA split; Standing, 172 cm from source	Awake, room air	M	14	9.5 ± 0.4 (9.0–10.0)	3.2 ± 0.1 (3.0–3.4)	5.4 ± 0.1 (5.2–5.5)	SC
		F	9	9.8 ± 0.4 (9.0–10.0)	4.7 ± 0.4 (4.3–5.2)	7.3 ± 0.9 (6.3–8.3)	SC
Source: 6 MV LINAC
Energy: 2 MeV
Dose rate: 0.67–0.8 Gy/min
Geometry	Sedation, gas	Sex	n	Total dose (Gy)	Age at irradiation (years)	Age (years)	Support
TBI, LR-RL split; Supine, 120 cm from source	Ketamine & isoflurane, 100% oxygen	M	20	6.6 ± 0.1 (6.4–6.8)	3.2 ± 0.8 (2.3–4.6)	9.0 ± 2.4 (2.7–13.1)	SC, AB
TBI, PA-AP split; Supine, 153 cm from source	Awake, room air	M	21	6.1 ± 1.8 (4.0–8.4)	4.4 ± 0.9 (3.1–6.7)	10.6 ± 4.4 (5.7–21.4)	SC
		F	11	4.8 ± 1.5 (4.0–8.5)	5.4 ± 1.3 (4.4–8.4)	9.0 ± 1.9 (7.4–13.2)	SC
TBI, AP-PA split; Supine, cm from source not reported	Ketamine, room air	M	14	7.3 ± 0.5 (6.8–8.1)	3.9 ± 1.0 (3.0–7.0)	13.9 ± 2.9 (9.7–19.2)	SC, AB, BT
TBI, LR-RL split; Supine, 175 cm from source	Ketamine, room air	M	22	4.2 ± 0.6 (4.0–6.0)	6.2 ± 2.6 (4.6–15.5)	10.9 ± 3.5 (7.2–21.8)	SC
		F*	3	4.0 ± 0.0 (4.0–4.0)	7.6 ± 0.5 (7.3–8.2)	8.8 ± 0.5 (8.7–9.1)	SC

Notes. TBI = total-body irradiation; LR = left to right; RL = right to left; AP = anterior to posterior; PA = posterior to anterior; SC = supportive care (fluids, nutrition, warming); AB = antibiotic use; BT = blood transfusion, PBI BM5 = partial-body irradiation with 5% bone marrow sparing; source to mid-plane distance (cm) is included in the geometry column. Values for total dose and age are expressed as mean +/− standard deviation, followed by (range). Split doses typically require <5 min between doses.

^*Cynomolgus macaques.

In addition to the major radiation exposure prior to entry into the cohort, all NHPs in the RLEC received small doses of ionizing radiation each year during imaging procedures. High-resolution lung and whole-body computed tomography (CT) and quantitative dual X-ray absorptiometry (DEXA) scans were performed on all living animals in the cohort each year. The annual exposure amounts to a mean of 18 mGy/year total body exposure and 24 mGy/year to the chest (total and chest-only scans) between 2012 and 2018, and 8 mGy/year total body, 12 mGy/year to the chest from 2019 to present. For comparison, the average annual background radiation exposure in the United States is 6.2 mGy. The average radiation dose to a human undergoing a whole-body CT is 19.9 mGy (22), or about equal to the exposure to a NHP undergoing a whole-body CT on our scanner in operation between 2012 and 2018 and roughly twice the exposure to a NHP scanned on our newer scanner in operation since 2019.

Mitigators Used after Acute Exposure

Most animals in the cohort (65.7%) did not receive mitigator treatment during the acute radiation syndrome (ARS). Mitigators used in the remaining 34.3% of the animals include a variety of agents given 24 h postirradiation. The ARS mitigating effects for most of these agents are published. Mitigators include hematopoietic growth factors or analogs thereof e.g., granulocyte colony-stimulating factor (G-CSF), pegfilgrastim (Neulasta), romiplostim (Nplate), Nplate and Neulasta, thrombopoeitin, the thrombopoietin analog ALXN4100TPO, cytokines such as pegylated interleukin 11 (PEG-IL-11), toll-like receptor agonists fibroblast stimulating lipopeptide 1 (FSL-1) and entolimod (CBLB-502), antioxidants such as the thioredoxin analog ORP-100S, the endotoxin-binding protein rhBPI-21, the angiotensin converting enzyme inhibitor lisinopril, and the calmodulin kinase kinase 2 inhibitor STO-609 (Fig. 3).

FIG. 3.

Mitigating agents used during the acute radiation syndrome in RLEC cohort animals.

Housing and Monitoring Procedures

All animals in the RLEC were monitored twice daily for signs of illness, and 95% were socially housed in indoor-outdoor pens, with exceptions for animals in quarantine or under medical or behavioral management. Housing pens include high and low perches, mirrors, manipulanda, and visual barriers or structures to allow animals to seek either social interaction or privacy. Water was provided ad libitum. Animals were fed a nutritionally complete diet that met the National Academy of Sciences (NAS) requirements for NHPs (23) and also approximated the North American diet in macronutrient composition (Diet 5LOP; LabDiet, St. Louis, MO).

All animals were observed daily for signs of illness or social/behavioral difficulties, and appropriate veterinary care was provided. A full-time lab-independent behavioral management team monitored all animals on a rotating basis, and worked with RLEC staff to recommend best practices to assure the well-being of the animals. All animals received a routine schedule of healthcare and sample collection procedures, summarized in Table 3.

TABLE 3.

Recurring Examinations and Sample Collections Performed for the Animals in the RLEC

Assessment	Frequency
Health observations	Daily
Blood pressure	Annually
Hematology/Serum Chemistry	3×/year
Sample bank collections (serum, saliva, urine, feces, hair)	3×/year
Tuberculin test	3×/year
Veterinary physical examination, sedated	Annually
Abdominal ultrasound	Annually
Bronchoalveolar lavage	Annually
Immune profile (flow cytometry, thymopoiesis assay)	Annually
Ophthalmic examinations	Annually
Whole body and focused lung and bone CT scan	Annually
Bone marrow aspirate	Upon arrival and every 3rd year
Brain MRI	Upon arrival and every 3rd year
Cognitive testing	As needed
Dual-energy X-ray absorptiometry (DEXA) body composition	As needed
Echocardiography and Electrocardiography	As needed
GI endoscopy/biopsy	As needed
Glomerular filtration rate (iohexol)	As needed
Optical Coherence Tomography (retina)	As needed
PET scans	As needed
Pulse-wave velocity	As needed
Serum cortisol and sex steroids, LCMS	As needed

All procedures were conducted at WFUSM with approval by the Institutional Animal Care and Use Committee. WFUSM 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 U.S. Department of Health and Human Services. WFUSM maintains an Assurance on file in the Office for Protection from Research Risks, Office of the Director, National Institutes of Health, that accepts responsibility for the humane care and use of animals (OPRR #A-3391–01). The Laboratory Animal Care Program of WFUSM complies with the “Principles for Use of Animals,” the “Guide for the Care and Use of Laboratory Animals” (24), 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).

Major Patterns of Disease

Prior published reports of disease patterns from the RLEC are summarized in Table 4. A more detailed and current description of in-life findings and diagnostic criteria for major chronic diseases is outlined by Quillen et al.,² including the first report in this population of some expected radiation-associated pathologies such as cataracts. Schaaf et al.³ present an extensive analysis of postmortem findings in this issue. As we have reported previously (25, 26), these disorders co-occurred as part of a multi-morbidity aging-like disease phenotype, with some animals having more than 10 co-morbid conditions.

TABLE 4.

Published Reports of Radiation-associated Disease Patterns in the RLEC

Organ system	Disease process	Refs.
Any site	Cancers	(25–27)
Bone	Osteopenia	(26, 28)
Brain	Vascular injury, inflammation, white matter injury, cognitive impairment	(29–33)
Cardiovascular	Hypertension, myocardial fibrosis, cerebrovascular injury	(30, 34, 35)
Cutaneous	Dermatitis, alopecia	(25)
Endocrine	Diabetes, insulin resistance	(25, 26, 36, 37)
Gastrointestinal	Loss of intestinal barrier, gastrointestinal disease	(35, 38)
Hematologic	Chronic inflammatory profile	(39–41)
Immune	Response repertoire deficits, long term immunophenotype changes	(41–43)
Metabolic	Low body weight	(25)
Musculoskeletal	Loss of microvasculature, insulin resistance, fibrosis	(45, 46)
Oral/dental	Reduced periodontal disease	(25)
Reproductive	Testicular atrophy	(46)
Urinary	Azotemia, renal fibrosis	(47)

DISCUSSION

Insights, Caveats, and Opportunities

Late effects are a multisystemic disease, with some features resembling normal aging (25, 26), but also with distinctive patterns of disease that represent the greater sensitivity to radiation of specific organ systems such as the gonads (46), lens, and microvasculature throughout the body (31, 44). Specific pathogenic processes characteristic of radiation injury are likely; for example, DNA damage almost certainly underlies the observed carcinogenesis at multiple sites (27). Chronic, systemic and multi-organ inflammation are part of the multi-morbidity pattern in these animals (39–41), and there are long-term deficiencies in both humoral and cellular elements of the immune system (41–43), which may lead to disproportionate adverse effects of infectious disease.

The radiation doses administered to animals in the RLEC were selected by consideration of both scientific priorities and practical considerations. All exposures involved photons, either from ⁶⁰Co or linear accelerator irradiators; there were no exposures to the complex mixed prompt and delayed spectrum of photon, proton, neutron, and isotopic exposures characteristic of nuclear weapons. (48). The dose rate was relatively low for all animals (0.6–0.8 Gy/min), with no high-dose-rate exposures as would occur from a weapon or criticality accident. Nor were there radiation exposures characteristic of spaceflight, which consists of low dose rate (<1 mGy/day) but high-Z particle galactic cosmic radiation (49). Fractionation of the dose (as in radiation therapy) was not used. Most animals received two half-doses within a few minutes, with the desired dose delivered to the midline of the animal; this provided high dose homogeneity but differs from the likely unilateral and potentially nonuniform exposure after a blast.

Sex differences are difficult to interpret at present in the cohort; although the cohort is now approaching equal numbers of male and female subjects, earlier studies consisted primarily of males. The full spectrum of long-term disease may not yet be evident in females. Age is also a confounding variable, as discussed in previous publications (25, 26); controls and animals that received the highest doses are generally older than those that received mid-range doses. Furthermore, as new study cohorts enter the RLEC after treatment with a given mitigator, care is required in the analysis of outcomes to prevent premature attribution of mitigator benefits when comparing younger, healthier, newly arrived mitigator-treated animals with older, untreated animals already in the cohort.

The primary opportunity presented by this resource is in the long-term, longitudinal assessment of a complex multi-morbidity disease pattern in a primate species with close genetic and physiologic similarity to human subjects. Most of the late effects observed in the RLEC occurred with a latency of 4–5 years, highlighting the value of a long-term approach and continued follow-up of this irreplaceable resource. Newer accessions to the cohort, particularly partial-body exposures, will undoubtedly add invaluable new data to augment our understanding of radiation late effects. It could be argued that these partial-body irradiated animals are the closest to modeling a real-world accidental or malicious human exposure, which would not be a uniform total-body exposure.

In addition to the single high-dose exposures of animals in this cohort, they are exposed to milligray doses annually as part of their annual surveillance CT scans. Recent projections estimate that human CT doses in the mGy range may increase cancer incidence by 5% on a population basis (50). This represents an opportunity for studying low-dose exposures, either in previously unexposed control animals or in previously irradiated animals. Such studies could be conducted acutely, immediately before and after CT exposures, or over more extended periods to investigate the cumulative effects of repeated annual CT scans.

CONCLUSIONS

The RLEC is a unique preclinical model for studying radiation injury and chronic disease mitigators, including comprehensive in-life disease phenotyping and postmortem evaluations. The resource provides expert veterinary care, clinical imaging, detailed pathological assessments, and a platform to integrate experimental data to guide future research.