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

Abstract

The Wake Forest Radiation Late Effects Cohort (RLEC), formerly known as the Radiation Survivors Cohort, of rhesus (Macaca mulatta) non-human primates (NHPs) is a unique colony of long-term survivors of total-body irradiation (TBI). The cohort includes 212 live animals, with 17% being unirradiated controls, and 104 deceased animals, including 15% controls. This cohort has been monitored for over 16 years, with an average observation period of 5 years. Irradiated NHPs were exposed to single TBI doses ranging from 1.14 to 8.5 Gy (average = 6.1 Gy). One animal received 10 Gy partial-body irradiation with approximately 5% bone marrow sparing. In this paper, we present the postmortem findings from 104 deceased members of the RLEC. Animals underwent a comprehensive, standardized necropsy, which included a complete gross and histopathologic examination of 36 organs and tissues. For this study, necropsy reports of 104 deceased animals (87 irradiated and 17 controls) were reviewed by two board-certified veterinary pathologists (GWS and JMC), and all diagnoses were cataloged. A total of 2,790 diagnoses were recorded across all organ systems and analyzed for statistical differences between irradiated and control animals using Fisher’s exact test. Deceased control animals ranged in age from 9.9 to 21.4 years (mean = 16 years), whereas irradiated animals were younger, with ages from 2.7 to 23.1 years (mean = 11.6 years, P = 0.0001). The time from irradiation to death ranged from 0.3 to 14.4 years (average = 6.4 years). Radiation doses for these deceased animals ranged from 3.5 to 8.5 Gy (average = 6.6 Gy). The prevalence of most lesions was not statistically different from controls. Common findings among the irradiated animals included multi-organ fibrosis and chronic inflammation. Additionally, there was an increased occurrence of neoplasia in the irradiated animals. These data represent comprehensive, systemic, long-term pathology assessments conducted on a large group of NHPs years after total-body irradiation in the molecular era. They provide a solid foundation for molecular and translational studies of radiation late effects. The fact that many of the same lesions appeared in both the irradiated and unirradiated control animals, despite the significant age difference, suggests an accelerated-aging phenotype in the survivors.

INTRODUCTION

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.

METHODS

Animal Population

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.

FIG. 1.

Age at death plotted with mean and standard deviation. The age at death was significantly younger for irradiated animals when compared to controls using an unpaired t test, P = 0.0001. Age at death for both irradiated and control animals were normally distributed using both the D’Agostino & Pearson and Shapiro-Wilk tests for normality.

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.

FIG. 2.

Cohort demographics. Radiation dose (Gy) and lethality dose groups are shown on the Y-axis and age on the X-axis. Each line represents an individual animal’s lifespan. The hashed line begins when the animal was irradiated and the solid line begins when the animal was transferred to Wake Forest University School of Medicine. The lines end at the age of death. Note that one animal received 10 Gy with 5% bone marrow sparing partial body irradiation.

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).

Clinical Assessment

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).

Euthanasia and Necropsy

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.

TABLE 1.

Criteria for Clinical Diagnoses and Euthanasia in the Radiation Late Effects Cohort

Organ/disease	Criteria for clinical diagnosis	Criteria for euthanasia or removal
Diabetes	Hemoglobin A1c (HbA1c) >6.5%, 3 fasting blood glucose measurements > 100 mg/dL, any non-fasted blood glucose > 200 mg/dL	Long term insulin treatment
Kidney	BUN > 30 mg/dl or Cr > 1.1 mg/dl, Loss of renal volume >50%	Clinical renal failure/uremia
Lung	Pulmonary consolidation on CT scan or emphysema >25%, SPO2 < 80%; Respiratory rate >80 bpm	Respiratory distress
GI	Any lesion on endoscopy, diarrhea (severity code >2 for >5 days)	Chronic diarrhea with weight loss
Skin	Persistent dermatitis, alopecia or loss of pigmentation	Severe extensive dermatitis
Heart/CV	Murmur detected on auscultation or echocardiography; MAP >120, adult stroke volume <5 mls and CO <0.5 L/min at .7 years	Congestive heart failure or substantial dysfunction
Brain	Y/N MRI lesions visible on SWI, neurologic abnormality on clinical exam	Severe neurologic abnormality, infarct or edema on MRI
Behavior	Any behavioral abnormality requiring individual management plan	Unmanageable self-injurious behavior or depression
Cancer	Any neoplastic disease (imaging or biopsy)	Malignancy
Bone	Adult bone mineral content (BMC) < 274g or density (BMD) < 0.373 g/cc	Fracture
Obese	Waist circumference > 45 cm or DEXA Body Fat > 30%	Unintended weight loss >25% in non-obese animal
Underweight	Waist circumference < 25 cm or DEXA adult body fat < 12.3%

TABLE 2.

Specific Tissues and Anatomic Locations that were Examined Histologically by Veterinary Pathologists

Organ system	Tissue	Anatomic location (if applicable)
Nervous system	Brain	Prefrontal cortex, anterior striatum, frontal cortex, hippocampus, midbrain, occipital cortex, brainstem, cerebellum, meninges
	Spinal cord Sciatic nerve	Cervical spine
Cardiovascular	Heart	Right ventricle, left ventricle, interventricular septum
Respiratory	Lung	One section from all six lung lobes
Lymphatic	Spleen
	Lymph nodes Thymus Tonsil	Axillary, inguinal, mesenteric, tracheobronchial
Gastrointestinal/Digestive	Stomach	Greater curvature
	Intestine Liver Gallbladder Pancreas	Duodenum, jejunum, ileum, ascending colon, descending colon
Endocrine	Adrenal glands Thyroid glands Pituitary gland
Hematopoietic	Bone marrow	Sternum, humoral head
Renal/Urinary	Kidney Urinary bladder	Pelvis, medulla and cortex from both kidneys
Reproductive	Testes or ovaries
	Epididymides or oviducts	Head of epididymides
	Uterus or prostate Cervix	Cranial and caudal lobes of prostate
	Penis or vagina
Integument	Skin	Lateral thigh, mammary gland
Skeletomuscular	Skeletal muscle	Biceps femoris
	Bone	Sternal vertebra, humerus
Adipose	Subcutaneous adipose
	Visceral adipose
Special senses	Eyes	Entire globe, additional sections of retina to include macula, lenses

Note. Tissues were collected and examined identically for both the irradiated and control animals.

Necropsy Report Review

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.

Statistical Analysis

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.

Table 22.

Whole Body Lesions

Whole body lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Emaciation	0	0	2 (15%)	4 (5%)	0.586
Obesity	1 (100%)	6 (38%)	1 (8%)	4 (5%)	0.00045

RESULTS

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.

TABLE 3.

Cardiovascular Lesions

Cardiovascular lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Arteriosclerosis	0	7 (44%)	5 (38%)	16 (22%)	0.2295
Degeneration, myocardial	0	6 (38%)	1 (8%)	13 (18%)	0.0906
Adipocyte infiltration, myocardium	0	3 (19%)	1 (8%)	4 (5%)	0.1208
Edema	0	0	0	1 (1%)	1
Fibrosis, myocardial	0	12 (75%)	4 (30%)	32 (43%)	0.0344
Hyperplasia, arterial smooth muscle	0	1 (6%)	0	1 (1%)	0.3015
Hypertrophy, cardiomegaly	0	1 (6%)	0	5 (7%)	1
Inflammation	0	1 (6%)	2 (15%)	15 (20%)	0.2939
Mineralization	0	1 (6%)	0	3 (4%)	0.5159
Necrosis	0	1 (6%)	0	1 (1%)	0.3015
Thrombosis	0	0	0	4 (5%)	1

Table 13.

Neoplasia

Neoplasia Type and diagnosis	Control		Irradiated		(Adapted from Sills et al, IJROBP, 2021)
	Female (n= 1)	Male (n = 16)	Female (n = 13)	Male (n= 74)	Site
Sarcoma
Malignant nerve sheath tumor	0	0	0	5 (7%)	Liver, kidney, skin
Endothelial malignancies	0	0	0	5 (7%)	Liver, lung, skin, spleen
Malignant glomus tumor	0	0	0	3 (4%)	Kidney, seminal vesicles, skin
Osteosarcoma	0	0	1 (8%)	1 (1%)	Bone
Fibrosarcoma	0	0	0	1 (1%)	Skin
Leiomyosarcoma	0	0	0	1 (1%)	Small intestine
Myxosarcoma	0	0	0	1 (1%)	Skin
Gastrointestinal stromal tumor	0	0	0	1 (1%)	Small intestine
Carcinoma
Renal carcinoma	0	0	0	4 (5%)	Kidney
Hepatocellular carcinoma	0	1 (6%)	0	3 (4%)	Liver
Biliary carcinoma	0	0	0	1 (1%)	Liver
Mammary carcinoma	0	0	0	1 (1%)	Mammary gland
Basal cell carcinoma	0	0	0	1 (1%)	Skin
Trophoblastic tumor	0	0	1 (8%)	0	Uterus
Benign mesenchymal
Lipoma	0	1 (6%)	0	4 (5%)	Skin, stomach
Leiomyoma	0	0	3 (23%)	1 (1%)	Uterus, liver
Acrochordon	0	1 (6%)	0	2 (3%)	Skin
Cutaneous fibroma	0	1 (6%)	0	2 (3%)	Skin
Hemangioma	0	0	0	2 (3%)	Skin, spleen
Neurofibroma	0	0	0	2 (3%)	Skin
Chondrolipoma	0	0	0	1 (1%)	Skin
Collagenous hamartoma	0	0	0	1 (1%)	Skin
Complex odontoma	0	1 (6%)	0	0	Oral cavity
Osteochondroma	0	0	0	1 (1%)	Bone
Osteoma	0	0	0	1 (1%)	Bone
Benign epithelial
Renal adenoma	0	0	0	4 (5%)	Kidney
Pituitary adenoma	0	1 (6%)	0	1 (1%)	Pituitary
Hepatocellular adenoma	0	0	0	2 (3%)	Liver
Biliary cystadenoma	0	0	0	1 (1%)	Liver
Islet cell adenoma	0	0	0	1 (1%)	Pancreas
Keratinizing acanthoma	0	0	0	1 (1%)	Skin
Papilloma	0	0	0	1 (1%)	Skin
Prostatic adenoma	0	0	0	1 (1%)	Prostate
Trichoblastoma	0	0	0	1 (1%)	Skin
Other (malignant)
Acute myeloid leukemia	0	0	0	2 (3%)	Marrow
Lymphoma	0	0	1 (8%)	1 (1%)	Lymph node
Chemoreceptor tumor	0	0	0	1 (1%)	Heart
Glioblastoma	0	0	0	1 (1%)	Brain
Leydig cell tumor	0	0	0	1 (1%)	Testis
Melanocytoma	0	0	0	1 (1%)	Skin
Meningioma	0	0	1 (8%)	0	Brain

Note. Statistical tests for benign and malignant tumor prevalence is shown in Table 14.

Table 14.

Neoplasia (Benign vs. Malignant)

Total neoplastic lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n =1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Malignant neoplasia	0	1 (6%)	4 (31%)	35 (47%)	0.0108
Benign neoplasia	0	5 (31%)	3 (23%)	37 (50%)	0.2863

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).

Table 6.

Gastrointestinal Lesions

GI lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Amyloidosis, intestines	0	0	0	2 (3%)	1
Atrophy, small intestine	0	1 (6%)	0	3 (4%)	0.5159
Atrophy, exocrine pancreas	0	0	1 (8%)	3 (4%)	1
Diverticula, large intestine	0	7 (44%)	0	3 (4%)	0.0000825
Adipose infiltration, exocrine pancreas	0	0	0	2 (3%)	1
Edema, large intestine	0	0	0	1 (1%)	1
Fibrosis, intestines	0	0	1 (8%)	5 (7%)	0.586
Fibrosis, exocrine pancreas	0	0	0	1 (1%)	1
Hyperplasia, small intestine, goblet cells	0	1 (6%)	0	2 (3%)	0.4179
Hyperplasia, large intestine	0	0	0	1 (1%)	1
Hyperplasia, pancreatic ducts	0	0	0	3 (4%)	1
Inflammation, stomach	0	8 (50%)	6 (46%)	26 (35%)	0.4294
Inflammation, small intestine	0	1 (6%)	1 (8%)	15 (20%)	0.2944
Inflammation, large intestine	0	2 (13%)	3 (23%)	18 (24%)	0.3497
Metaplasia, Paneth cell, large intestine	0	0	0	2 (3%)	1
Trichobezoar, stomach	0	0	0	1 (1%)	1
Impaction, large intestine	0	0	0	1 (1%)	1
Necrosis, exocrine pancreas	0	0	0	1 (1%)	1
Perforation, intestines	0	0	0	4 (5%)	1
Villus fusion, small intestine	0	0	0	1 (1%)	1

FIG. 3.

Selected examples of cardiovascular lesions. Panel A: Extensive myocardial fibrosis in the interventricular septum of a 17.2-year-old unirradiated (control) male rhesus macaque. Panel B: Arteriosclerosis in a coronary artery from the same control animal. Panel C: Severe arterial fibrosis in the testis of a 10.4-year-old male rhesus macaque who received 8.05 Gy total-body irradiation six years earlier. Panel D: Severe histiocytic eosinophilic vasculitis in a pulmonary artery of a 10.2-year-old male rhesus macaque who received 7.2 Gy total-body irradiation five years earlier. The high-magnification inset shows mural infiltration of eosinophils and histiocytes.

Table 15.

Ocular Lesions

Ocular lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Cataracts*	0	5 (31%)	6 (46%)	39 (53%)	0.115
Cystoid degeneration, retina	0	2 (13%)	1 (8%)	2 (3%)	0.1867
Degeneration, retina	0	0	0	3 (4%)	1
Inflammation, eye (other than retina)	0	0	1 (8%)	2 (3%)	1
Ulcer, cornea	0	0	0	1 (1%)	1
Vascular redundancy	0	1 (6%)	0	0	0.1635

^*Denotes in-life ophthalmic diagnosis. Due to artifacts from tissue fixation, cataracts were the only lesion in this study more reliably diagnosed in living animals.

Table 19.

Male Reproductive Lesions

Male reproductive lesions Pathologic process	Control	Irradiated	Fisher’s exact test
	Male (n = 16)	Male (n = 74)	P value
Atrophy, prostate	0	6 (8%)	0.586
Atrophy, testis	4 (25%)	59 (80%)	0.000052
Degeneration, prostate	0	1 (1%)	1
Fibrosis, prostate	0	4 (5%)	1
Hyperplasia, prostate	0	4 (5%)	1
Hyperplasia, seminal vesicle	0	1 (1%)	1
Inflammation, epididymis	0	2 (3%)	1
Inflammation, prostate	5 (31%)	18 (24%)	0.5433
Inflammation, seminal vesicle	0	2 (3%)	1
Inflammation, testis	0	1 (1%)	1
Mineralization, prostate	1 (6%)	4 (5%)	1
Mineralization, seminal vesicle	0	4 (5%)	1

Table 20.

Respiratory Lesions

Respiratory lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Anthracosis, lung	0	10 (63%)	3 (23%)	32 (43%)	0.1867
Atelectasis, lung	0	1 (6%)	1 (8%)	1 (1%)	0.4179
Edema, lung	0	0	0	2 (3%)	1
Emphysema, lung	1 (100%)	6 (38%)	7 (54%)	15 (20%)	0.2368
Fibrosis/smooth muscle hyperplasia, lung	1 (100%)	5 (31%)	6 (46%)	51 (79%)	0.029
Histiocytosis, lung	0	2 (13%)	0	2 (3%)	0.124
Hyperplasia, epithelial, lung	1 (100%)	1 (6%)	1 (8%)	16 (22%)	0.732
Inflammation, lung/pleura	0	0	3 (23%)	24 (32%)	0.0053
Inflammation, trachea	0	2 (13%)	0	2 (3%)	0.124
Osseous metaplasia, trachea	1 (100%)	3 (19%)	0	9 (12%)	0.2198
Pneumothorax	0	0	0	2 (3%)	1
Telangiectasis, lung	0	1 (6%)	0	1 (1%)	0.3015

FIG. 4.

Selected examples of respiratory lesions. Panel A: Severe smooth muscle hyperplasia and fibrosis in the lung of a 6.9-year-old male rhesus macaque who received 7.55 Gy total-body irradiation four years earlier. Panel B: Masson’s trichrome staining from the same animal showing mild interstitial fibrosis. Panel C: Chronic histiocytic pneumonia in the lung of a 7.5-year-old male rhesus macaque who received 6.5 Gy total-body irradiation 4.5 years earlier. Panel D: Mild lymphohistiocytic pneumonia in a 7.6-year-old male rhesus macaque who received 6.6 Gy total-body irradiation five years earlier.

TABLE 8.

Hepatobiliary Lesions

Hepatobiliary lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Amyloidosis, liver	0	0	0	2 (3%)	1
Atrophy/degeneration, hepatocellular	0	2 (13%)	0	6 (8%)	0.6143
Cyst, liver	0	2 (13%)	2 (15%)	18 (24%)	0.516
Fibrosis, liver	0	1 (6%)	0	9 (12%)	1
Glycogenesis, hepatocellular	0	0	0	1 (1%)	1
Hepatomegaly	0	1 (6%)	0	0	0.1635
Hyperplasia, liver, stellate cell	0	1 (6%)	0	2 (3%)	0.4179
Inflammation, liver	0	4 (25%)	4 (30%)	21 (28%)	0.7742
Lipidosis, liver	0	4 (25%)	2 (15%)	16 (22%)	0.7535
Necrosis, hepatocellular	0	1 (6%)	0	4 (5%)	1

Table 17.

Renal Lesions

Renal lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Atrophy, glomerular	0	0	1 (8%)	0	1
Atrophy, tubular			1 (8%)	0	1
Amyloidosis	0	0	0	4 (5%)	1
Cysts, cortical or medullary	0	1 (6%)	0	19 (26%)	0.1835
Degeneration/Necrosis	0	0	0	3 (4%)	1
Dysplasia, tubular	0	0	0	1 (1%)	1
Fibrosis	1 (100%)	4 (25%)	2 (15%)	22 (30%)	1
Inflammation	1 (100%)	1 (6%)	3 (23%)	22 (30%)	0.3556
Membranoproliferative glomerulonephropathy	1 (100%)	6 (38%)	1 (8%)	26 (35%)	0.4124
Proteinosis, tubular	0	0	0	1 (1%)	1

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).

FIG. 5.

Examples of renal neoplasia. Panel A: Renal sarcoma in a 5.2-year-old male rhesus macaque who received 6.8 Gy total-body irradiation one year earlier. The high-magnification inset shows streams and whorls of malignant spindle-shaped mesenchymal cells. Panel B: Renal carcinoma in a 11.3-year-old male rhesus maaque who received 7.2 Gy total-body irradiation 8 years earlier. The high-magnification inset shows haphazardly arranged malignant epithelial cells on a fibrous stroma infiltrated by scattered lymphocytes. Panel C: Renal adenoma in a 17-year-old male rhesus macaque who received 8.05 Gy total-body irradiation 13.7 years earlier. The high-magnification inset shows neoplastic epithelial cells arranged in tubules and trabeculae on a fibrous stroma, with small, scattered areas of hemorrhage and necrosis. Panel D: Renal glomus tumor from a 7.5-year-old male rhesus macaque who received 6.5 Gy total-body irradiation 4.5 years earlier, demonstrating strong membranous and cytoplasmic reactivity for a-smooth muscle actin immunohistochemistry.

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).

DISCUSSION

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).

TABLE 5.

Endocrine Lesions

Endocrine lesions	CONTROL		IRRADIATED		Fisher’s exact test
Pathologic Process	Female (n=1)	Male (n=16)	Female (n=13)	Male (n=74)	p value
Amyloidosis, thyroid and adrenal	0	0	0	1 (1%)	1
Amyloidosis, endocrine pancreas	0	2 (13%)	1 (8%)	13 (18%)	1
Atrophy, thyroid	0	0	0	4 (5%)	1
Atrophy, adrenocortical	0	0	0	1 (1%)	1
Cyst, pituitary (Rathke’s pouch)	1 (100%)	3 (19%)	1 (8%)	6 (8%)	0.0786
Cyst, thyroid and adrenal	0	0	0	1 (1%)	1
Degeneration/Mineralization, endocrine pancreas	0	0	0	2 (3%)	1
Degeneration, adrenal vacuolation	0	0	0	3 (4%)	1
Hyperplasia, thyroid, follicular	0	0	0	3 (4%)	1
Hyperplasia, thyroid, C cell	0	1 (6%)	0	2 (3%)	0.4179
Hyperplasia, pituitary	0	1 (6%)	0	2 (3%)	0.4179
Hyperplasia, endocrine pancreas	0	2 (13%)	1 (8%)	7 (9%)	0.6659
Hyperplasia, adrenomedullary	0	1 (6%)	0	0	0.1635
Hyperplasia, adrenocortical	0	3 (19%)	1 (8%)	12 (16%)	0.7228
Inflammation, thyroid	0	1 (6%)	1 (8%)	1 (1%)	0.4179
Metaplasia, adrenal, adipose	0	0	0	2 (3%)	1
Mineralization, adrenal	1 (100%)	3 (19%)	1 (8%)	6 (8%)	0.0786

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.

CONCLUSIONS

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.

TABLE 4.

Central Nervous System Lesions

CNS lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Atrophy	0	1 (6%)	0	1 (1%)	0.3015
Degeneration/necrosis, brain, vascular	0	0	2 (15%)	13 (18%)	0.1231
Degeneration, spinal cord/nerve	0	1 (6%)	0	1 (1%)	0.3015
Edema	0	1 (6%)	0	3 (4%)	0.5159
Fibrosis, meningeal	0	1 (6%)	1 (8%)	7 (9%)	1
Vascular malformation	0	1 (6%)	0	7 (9%)	1
Inflammation	0	1 (6%)	4 (30%)	5 (7%)	1
Metaplasia, spinal cord hamartoma (white matter)	0	1 (6%)	0	0	0.1635
Ventricular dilatation	1 (100%)	0	0	2 (3%)	0.4179
Pigmentation, hemosiderosis	0	0	0	6 (8%)	0.586
Pigmentation, lipofuscin and/or melanin	0	2 (13%)	3 (23%)	8 (11%)	1
Perivascular clear space	0	0	2 (15%)	3 (4%)	0.5885

TABLE 7.

Hematopoietic Lesions

Hematopoietic lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n =1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Atrophy, bone marrow	0	0	0	4 (5%)	1
Extramedullary hematopoiesis, any site	0	0	1 (8%)	4 (5%)	0.5885
Dysplasia, bone marrow	0	0	0	1 (1%)	1
Fibrosis, bone marrow	0	0	1 (8%)	1 (1%)	1
Hyperplasia, bone marrow	0	2 (13%)	1 (8%)	13 (18%)	1
Hemosiderosis, bone marrow	0	0	1 (8%)	1 (1%)	1

TABLE 9.

Integumentary Lesions

Integumentary lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Edema, subcutaneous	0	1 (6%)	0	1 (1%)	0.3015
Fibrosis, skin	0	1 (6%)	0	1 (1%)	0.3015
Hyperplasia/hyperkeratosis, skin	0	3 (19%)	2 (15%)	6 (8%)	0.3823
Inflammation, skin	0	3 (19%)	3 (23%)	15 (20%)	1
Adnexal gland abnormality, skin	0	0	0	3 (4%)	1
Melanosis, skin	0	1 (6%)	0	1 (1%)	0.3015
Wound, skin	0	2 (13%)	0	4 (5%)	0.2532

TABLE 10.

Lymphoid Tissue Lesions

Lymphoid tissue lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Amyloidosis, spleen	0	0	0	2 (3%)	1
Amyloidosis, lymph node	0	2 (13%)	0	4 (5%)	0.2532
Anthracosis, lymph node	0	1 (6%)	3 (23%)	14 (19%)	0.2939
Atrophy, thymus	0	8 (50%)	5 (38%)	35 (47%)	1
Atrophy, lymphoid depletion, any site	0	2 (13%)	0	13 (18%)	1
Dysplasia, spleen, ectopy, any site	0	0	0	1 (1%)	1
Dysplasia, spleen, adipocyte infiltration	0	0	1 (8%)	0	1
Ectopic tissue, spleen, any site	0	0	0	1 (1%)	1
Eosinophilia, lymph node	0	0	0	1 (1%)	1
Fibrosis, lymph node	0	0	0	2 (3%)	1
Fibrosis, spleen	0	1 (6%)	0	1 (1%)	0.3042
Hemosiderosis, spleen	0	0	0	2 (3%)	1
Histiocytosis, lymph node	0	1 (6%)	1 (8%)	18 (24%)	0.1835
Histiocytosis, spleen	0	0	0	1 (1%)	1
Inflammation, lymph node	0	1 (6%)	0	0	0.1635
Inflammation, tonsil	0	1 (6%)	0	9 (12%)	1
Lithaisis/mineralization, tonsil	0	0	0	2 (3%)	1
Lymphangiectasia, any site	0	1 (6%)	1 (8%)	1 (1%)	0.4179
Lymphoid depletion, any site	0	2 (13%)	0	13 (18%)	1
Lymphoid hyperplasia, any site	1 (100%)	5 (32%)	3 (23%)	29 (39%)	1
Plasmacytosis, spleen	0	0	0	1 (1%)	1
Proteinosis, spleen	0	0	0	1 (1%)	1

Table 11.

Mammary Lesions

Mammary lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Hyperplasia, mammary gland	0	0	0	1 (1%)	1
Secretory change, mammary gland	0	0	0	1 (1%)	1
Inflammation, mammary gland	0	2 (13%)	0	1 (1%)	0.0687

Table 12.

Musculoskeletal Lesions

Musculoskeletal lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n =1)	Male (n = 16)	Female (n =13)	Male (n = 74)	P value
Arthritis	1 (100%)	6 (38%)	3 (23%	12 (16%)	0.0467
Atrophy/growth arrest, bone	0	0	0	2 (3%)	1
Atrophy, muscle	0	1 (6%)	0	2 (3%)	0.4179
Hamartoma, bone	0	1 (6%)	0	0	0.1635
Hypertrophy, bone	0	0	1 (8%)	1 (1%)	1
Inflammation, muscle	0	0	0	5 (7%)	1
Kyphosis	0	1 (6%)	0	2 (3%)	0.4179
Necrosis, muscle, any site	0	2 (13%)	0	3 (4%)	0.1867
Spondylosis	0	4 (25%)	0	6 (8%)	0.0559

Table 16.

Oral Lesions

Oral lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n = 1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Atrophy, salivary gland	0	0	0	1 (1%)	1
Cyst, salivary gland	0	0	0	1 (1%)	1
Dental disease	0	6 (38%)	4 (31%)	17 (23%)	0.3704
Inflammation, pharynx	0	0	0	3 (4%)	1
Inflammation, salivary gland	1 (100%)	0	0	4 (5%)	1
Inflammation, tongue	0	1 (6%)	0	4 (5%)	1
Necrosis, tongue	1 (100%)	0	0	1	0.3015

Table 18.

Female Reproductive Lesions

Female reproductive lesions Pathologic process	Control	Irradiated	Fisher’s exact test
	Female (n = 1)	Female (n = 13)	P value
Cyst, ovary	0	2 (15%)	1
Endometriosis	0	4 (31%)	1
Hemorrhage, cervix	1 (100%)	1 (8%)	0.1429
Hyperplasia, endometrium	0	1 (8%)	1
Inflammation, cervix	0	1 (8%)	1
Polyp, oviduct	0	1 (8%)	1
Squamous metaplasia, cervix	0	1 (8%)	1

Table 21.

Urinary Tract Lesions (excluding renal)

Urinary lesions Pathologic process	Control		Irradiated		Fisher’s exact test
	Female (n =1)	Male (n = 16)	Female (n = 13)	Male (n = 74)	P value
Amyloidosis, urinary bladder	0	0	0	1 (1%)	1
Fibrosis, urinary bladder	0	0	0	3 (4%)	1
Inflammation, urinary bladder	0	1 (6%)	1 (8%)	2 (3%)	0.5159