Non-Human Primates Receiving High-Dose Total-Body Irradiation are at Risk of Developing Cerebrovascular Injury Years Postirradiation.

Nuclear accidents and acts of terrorism have the potential to expose thousands of people to high-dose total-body iradiation (TBI). Those who survive the acute radiation syndrome are at risk of developing chronic, degenerative radiation-induced injuries [delayed effects of acute radiation (DEARE)] that may negatively affect quality of life. A growing body of literature suggests that the brain may be vulnerable to radiation injury at survivable doses, yet the long-term consequences of high-dose TBI on the adult brain are unclear. Herein we report the occurrence of lesions consistent with cerebrovascular injury, detected by susceptibility-weighted magnetic resonance imaging (MRI), in a cohort of non-human primate [(NHP); rhesus macaque, Macaca mulatta] long-term survivors of high-dose TBI (1.1-8.5 Gy). Animals were monitored longitudinally with brain MRI (approximately once every three years). Susceptibility-weighted images (SWI) were reviewed for hypointensities (cerebral microbleeds and/or focal necrosis). SWI hypointensities were noted in 13% of irradiated NHP; lesions were not observed in control animals. A prior history of exposure was correlated with an increased risk of developing a lesion detectable by MRI (P = 0.003). Twelve of 16 animals had at least one brain lesion present at the time of the first MRI evaluation; a subset of animals (n = 7) developed new lesions during the surveillance period (3.7-11.3 years postirradiation). Lesions occurred with a predilection for white matter and the gray-white matter junction. The majority of animals with lesions had one to three SWI hypointensities, but some animals had multifocal disease (n = 2). Histopathologic evaluation of deceased animals within the cohort (n = 3) revealed malformation of the cerebral vasculature and remodeling of the blood vessel walls. There was no association between comorbid diabetes mellitus or hypertension with SWI lesion status. These data suggest that long-term TBI survivors may be at risk of developing cerebrovascular injury years after irradiation.

White Matter is the Predilection Site of Late-Delayed Radiation-Induced Brain Injury in Non-Human Primates.

Fractionated whole-brain irradiation for the treatment of intracranial neoplasia causes progressive neurodegeneration and neuroinflammation. The long-term consequences of single-fraction high-dose irradiation to the brain are unknown. To assess the late effects of brain irradiation we compared transcriptomic gene expression profiles from nonhuman primates (NHP; rhesus macaques Macaca mulatta) receiving single-fraction total-body irradiation (TBI; n = 5, 6.75-8.05 Gy, 6-9 years prior to necropsy) to those receiving fractionated whole-brain irradiation (fWBI; n = 5, 40 Gy, 8 × 5 Gy fractions; 12 months prior to necropsy) and control comparators (n = 5). Gene expression profiles from the dorsolateral prefrontal cortex (DLPFC), hippocampus (HC) and deep white matter (WM; centrum semiovale) were compared. Stratified analyses by treatment and region revealed that radiation-induced transcriptomic alterations were most prominent in animals receiving fWBI, and primarily affected white matter in both TBI and fWBI groups. Unsupervised canonical and ontologic analysis revealed that TBI or fWBI animals demonstrated shared patterns of injury, including white matter neuroinflammation, increased expression of complement factors and T-cell activation. Both irradiated groups also showed evidence of impaired glutamatergic neurotransmission and signal transduction within white matter, but not within the dorsolateral prefrontal cortex or hippocampus. Signaling pathways and structural elements involved in extracellular matrix (ECM) deposition and remodeling were noted within the white matter of animals receiving fWBI, but not of those receiving TBI. These findings indicate that those animals receiving TBI are susceptible to neurological injury similar to that observed after fWBI, and these changes persist for years postirradiation. Transcriptomic profiling reaffirmed that macrophage/microglial-mediated neuroinflammation is present in radiation-induced brain injury (RIBI), and our data provide novel evidence that the complement system may contribute to the pathogenesis of RIBI. Finally, these data challenge the assumption that the hippocampus is the predilection site of injury in RIBI, and indicate that impaired glutamatergic neurotransmission may occur in white matter injury.

Cerebrovascular Remodeling and Neuroinflammation is a Late Effect of Radiation-Induced Brain Injury in Non-Human Primates.

Fractionated whole-brain irradiation (fWBI) is a mainstay of treatment for patients with intracranial neoplasia; however late-delayed radiation-induced normal tissue injury remains a major adverse consequence of treatment, with deleterious effects on quality of life for affected patients. We hypothesize that cerebrovascular injury and remodeling after fWBI results in ischemic injury to dependent white matter, which contributes to the observed cognitive dysfunction. To evaluate molecular effectors of radiation-induced brain injury (RIBI), real-time quantitative polymerase chain reaction (RT-qPCR) was performed on the dorsolateral prefrontal cortex (DLPFC, Brodmann area 46), hippocampus and temporal white matter of 4 male Rhesus macaques (age 6-11 years), which had received 40 Gray (Gy) fWBI (8 fractions of 5 Gy each, twice per week), and 3 control comparators. All fWBI animals developed neurologic impairment; humane euthanasia was elected at a median of 6 months. Radiation-induced brain injury was confirmed histopathologically in all animals, characterized by white matter degeneration and necrosis, and multifocal cerebrovascular injury consisting of perivascular edema, abnormal angiogenesis and perivascular extracellular matrix deposition. Herein we demonstrate that RIBI is associated with white matter-specific up-regulation of hypoxia-associated lactate dehydrogenase A (LDHA) and that increased gene expression of fibronectin 1 (FN1), SERPINE1 and matrix metalloprotease 2 (MMP2) may contribute to cerebrovascular remodeling in late-delayed RIBI. Additionally, vascular stability and maturation associated tumor necrosis super family member 15 (TNFSF15) and vascular endothelial growth factor beta (VEGFB) mRNAs were increased within temporal white matter. We also demonstrate that radiation-induced brain injury is associated with decreases in white matter-specific expression of neurotransmitter receptors SYP, GRIN2A and GRIA4. We additionally provide evidence that macrophage/microglial mediated neuroinflammation may contribute to RIBI through increased gene expression of the macrophage chemoattractant CCL2 and macrophage/microglia associated CD68. Global patterns in cerebral gene expression varied significantly between regions examined (P < 0.0001, Friedman's test), with effects most prominent within cerebral white matter.

Long-Term Cognitive Functioning in Single-Dose Total-Body Gamma-Irradiated Rhesus Monkeys ( Macaca mulatta ).

In this study, the effects of a potentially lethal radiation exposure on the brain for long-term cognitive sequelae were investigated using Rhesus macaques ( Macaca mulatta ) adopted from other facilities after analysis of acute radiation response via the Centers for Medical Countermeasures against Radiation (CMCR) network. Fifty-nine animals were given the opportunity to participate in cognitive cage-side testing. The animals that received single-dose gamma irradiation were significantly less likely to engage in cognitive testing than the controls, suggesting that irradiated animals may have differences in cognitive ability. Five irradiated (6.75-8.05 Gy) and three naïve control animals self-selected, were extensively trained and administered a simple visual discrimination with reversal (SVD+R) task 2-3 times per week for 11-18 months. Each session consisted of 30 trials in which the animals were required to choose the correct visual stimulus for a food reward. After the initial presentation, the stimulus that signaled the presence of food was twice reversed once the animal reached criterion (90% accuracy across four consecutive sessions). While the limited sample size precluded definitive statistical analysis, irradiated animals took longer to reach the criterion subsequent to reversal than did control animals, suggesting a relative deficiency in cognitive flexibility. These results provide preliminary data supporting the potential use of a nonhuman primate model to study radiation-induced, late-delayed cognitive deficits.

Late Effects of Total-Body Gamma Irradiation on Cardiac Structure and Function in Male Rhesus Macaques.

Heart disease is an increasingly recognized, serious late effect of radiation exposure, most notably among breast cancer and Hodgkin's disease survivors, as well as the Hiroshima and Nagasaki atomic bomb survivors. The purpose of this study was to evaluate the late effects of total-body irradiation (TBI) on cardiac morphology, function and selected circulating biomarkers in a well-established nonhuman primate model. For this study we used male rhesus macaques that were exposed to a single total-body dose of ionizing gamma radiation (6.5-8.4 Gy) 5.6-9.7 years earlier at ages ranging from ∼3-10 years old and a cohort of nonirradiated controls. Transthoracic echocardiography was performed annually for 3 years on 20 irradiated and 11 control animals. Myocardium was examined grossly and histologically, and myocardial fibrosis/collagen was assessed microscopically and by morphometric analysis of Masson's trichrome-stained sections. Serum/plasma from 27 irradiated and 13 control animals was evaluated for circulating biomarkers of cardiac damage [N-terminal pro B-type natriuretic protein (nt-proBNP) and troponin-I], inflammation (CRP, IL-6, MCP-1, sICAM) and microbial translocation [LPS-binding protein (LBP) and sCD14]. A higher prevalence of histological myocardial fibrosis was observed in the hearts obtained from the irradiated animals (9/14) relative to controls (0/3) (P = 0.04, χ(2)). Echocardiographically determined left ventricular end diastolic and systolic diameters were significantly smaller in irradiated animals (repeated measures ANOVA, P < 0.001 and P < 0.008, respectively). Histomorphometric analysis of trichrome-stained sections of heart tissue demonstrated ∼14.9 ± 1.4% (mean ± SEM) of myocardial area staining for collagen in irradiated animals compared to 9.1 ± 0.9 % in control animals. Circulating levels of MCP-1 and LBP were significantly higher in irradiated animals (P < 0.05). A high incidence of diabetes in the irradiated animals was associated with higher plasma triglyceride and lower HDLc but did not appear to be associated with cardiovascular phenotypes. These results demonstrate that single total-body doses of 6.5-8.4 Gy produced long-term effects including a high incidence of myocardial fibrosis, reduced left ventricular diameter and elevated systemic inflammation. Additional prospective studies are required to define the time course and mechanisms underlying radiation-induced heart disease in this model.

Pathology of fractionated whole-brain irradiation in rhesus monkeys ( Macaca mulatta ).

Fractionated whole-brain irradiation (fWBI), used to treat brain metastases, often leads to neurologic injury and cognitive impairment. The cognitive effects of irradiation in nonhuman primates (NHP) have been previously published; this report focuses on corresponding neuropathologic changes that could have served as the basis for those effects in the same study. Four rhesus monkeys were exposed to 40 Gy of fWBI [5 Gy × 8 fraction (fx), 2 fx/week for four weeks] and received anatomical MRI prior to, and 14 months after fWBI. Neurologic and histologic sequelae were studied posthumously. Three of the NHPs underwent cognitive assessments, and each exhibited radiation-induced impairment associated with various degrees of vascular and inflammatory neuropathology. Two NHPs had severe multifocal necrosis of the forebrain, midbrain and brainstem. Histologic and MRI findings were in agreement, and the severity of cognitive decrement previously reported corresponded to the degree of observed pathology in two of the animals. In response to fWBI, the NHPs showed pathology similar to humans exposed to radiation and show comparable cognitive decline. These results provide a basis for implementing NHPs to examine and treat adverse cognitive and neurophysiologic sequelae of radiation exposure in humans.

Assessment of Multiplate platelet aggregometry using citrate, heparin or hirudin in Rhesus macaques.

Electrical impedance aggregometry (EIA) has gained popularity in clinical and research applications. Nonhuman primates are used to study disease and drug-related mechanisms that affect hemostasis, therefore establishing normal EIA parameters are necessary. The anticoagulants sodium heparin, hirudin and sodium citrate and three agonists, ADP, ASPI, and collagen were evaluated. Whole blood from 12 adult male rhesus macaques was collected to evaluate anticoagulants, sodium heparin, hirudin and sodium citrate using three agonists (ADP, ASPI and collagen), on the Multiplate® 5.0 Analyzer. Platelet function was reported for three parameters: Area under the curve (AUC), aggregation, and aggregation velocity. There was a significant difference in mean AUC between citrate and heparin samples, and citrate and hirudin samples regardless of the agonist used. There was no difference in AUC between heparin and hirudin. ADP-activated samples showed an increase in impedance with hirudin samples compared to citrate. Furthermore, heparin and hirudin out-perform citrate as the anticoagulant for EIA in the macaque. Finally, this study demonstrates the utility of the Multiplate® system in this model and provides important insight into anticoagulant choice when using EIA.

Handedness and lateralised tympanic membrane temperature in relation to approach-avoidance behaviour in Garnett's bushbaby (Otolemur garnettii).

Studies of handedness suggest a relationship between hemispheric specialisation and emotional processing. Recently measures of lateralised tympanic membrane temperature (TMT) have identified similar relationships (i.e., the left hemisphere is involved in approach behaviour and the right hemisphere avoidance behaviour). In the present study we examined lateralised changes in TMT in response to social interaction in 10 Garnett's bushbabies. Additionally, we examined whether handedness could be used as a predictor of approach-avoidance tendencies. We found a positive association between temperature change and both allogrooming and affiliative approach. Social behaviour did not differ between right- and left-handed bushbabies. These findings are discussed in terms of existing theories of asymmetric emotional processing. Overall, the data suggest that there is a left hemisphere specialisation for processing approach-related behaviours, which is consistent with existing models of lateralised emotional processing. Our data also indicate that TMT is a reliable, cost-effective measure of cerebral activation that is less invasive and more practical than alternative measures such as EEG, PET, and fMRI.

Age-related decline in lateralised prey capture success in Garnett's bushbaby (Otolemur garnettii).

We examined differences in prey capture success when reaching for moving prey with the preferred and non-preferred hand (as determined previously using stationary food items) in 12 Garnett's bushbabies (Otolemur garnettii). Hand preference was determined by a test of simple reaching for stationary food items. We assessed both the frequency of hand use and success rates for each hand in capturing live mealworms. We also examined the effect of age on overall prey capture success. Subjects were individually presented with live mealworms in a cup partially filled with a cornmeal medium. The preferred hand was used significantly more often than the non-preferred hand to obtain the moving prey; however, no differences were found in the frequency of usage of the left vs the right hand. Furthermore, there were no differences in the success rates of the left vs the right hand, nor the preferred vs the non-preferred hand. There was a significant negative correlation between age and prey capture success. These data suggest that age, rather than preferred hand, may be the most relevant factor in the bushbabies' prey capture success.

Multiple measures of laterality in Garnett's bushbaby (Otolemur garnettii).

Behavioral laterality, a common measure of hemispheric specialization of the brain, has been examined in multiple tasks across several species of prosimian primates; however, there is inconsistency among findings between and within species that leaves many questions about laterality unanswered. Most studies have employed few measures of laterality, most commonly handedness. This study examined multiple measures of laterality within subjects in 17 captive-born Garnett's bushbabies (Otolemur garnettii) to assess the consistency of lateralized behaviors and to examine possible influences such as age, posture, novelty, and arousal to elucidate the relations between direction and strength of laterality. We measured reaching, turning bias, scent marking, tail wrapping, leading foot, side-of-mouth preference, and hand use in prey capture. Because autonomic arousal has been invoked as a determinant of strength of lateralization, we included multiple tasks that would allow us to test this hypothesis. All subjects were significantly lateralized on simple reaching tasks (P<0.01) and tail wrapping (P<0.01). Moreover, the number of animals lateralized on turning (P<0.01), leading limb (P<0.05), mouth use (P<0.01), and prey capture (P<0.01) was greater than would be expected by chance alone. There was consistency in the strength and direction of hand biases across different postures. Tasks requiring hand use were more strongly lateralized than tasks not involving hand use (P<0.001). The data do not support the assumption that arousal (as subjectively categorized) or novelty strengthens lateralized responding. The results of this study are discussed in terms of the effects of arousal, posture, and age on lateralized behavior.

Rating vs. coding in animal personality research.

Animal personality research has become increasingly popular over the past few decades. The two main methods used to examine individual differences in animals are rating and coding. The rating method involves human scoring of an animal's behavioral tendencies along various behavioral dimensions, such ratings are typically based on the human rater's experience with the animal. The coding method also requires humans to score an animal's behavior, but differs in that the scoring is based on the animal's immediate behavior in a specific context. This brief report describes the use of both the rating and coding methods to examine personality within a group of 10 Garnett's bushbabies (Otolemur garnettii). The results indicated that individual personalities do exist in bushbabies, but also suggested that the rating method is heavily influenced by the rater's experience with an animal. Consequently, it is important that the nature of the rater's interactions with the target animals be considered when using the rating method to assess animal personality.

L-tryptophan and correlates of self-injurious behavior in small-eared bushbabies (Otolemur garnettii).

Self-injurious behavior (SIB) among captive primates is a recurring problem for those who manage such facilities. Its prevalence highlights the need for research evaluating the effectiveness of potential treatment approaches. In the present study, 4 wk of dietary supplementation with L-tryptophan (100 mg daily) was evaluated for the treatment of self-inflicted wounds in 22 small-eared bushbabies, a prosimian primate, with a history of SIB. The treatment significantly reduced stereotypy and was associated with a reduction in wound area and severity. In terms of physiologic measures, preexisting high levels of cortisol were reduced in bushbabies with SIB, whereas serotonin concentrations were increased after 4 wk of treatment. Results indicate that L-tryptophan as a dietary supplement may be a viable adjunct to standard husbandry procedures for animals exhibiting maladaptive behaviors such as stereotypy and SIB.

Efficacy of auditory enrichment in a prosimian primate (Otolemur garnettii).

Research suggests that auditory environmental enrichment might reduce abnormal behavior in certain primate species. The authors evaluated the behavioral effects of exposure to music in a prosimian primate (Garnett's bushbaby; Otolemur garnettii). They exposed bushbabies to a Mozart concerto for 15 min per day for 20 d (5 h exposure total), video-recorded them and subsequently analyzed the frequency of subjects' grooming and stereotypic behaviors. The authors compared the data with baseline behavioral data that had been recorded over a 20-d period before the experimental treatment. Neither stereotypy nor grooming behavior varied as a result of exposure to music. These results do not support the hypothesis that auditory enrichment in the form of exposure to music is an effective means of reducing stereotypic behavior in O. garnettii.

Cortisol response to relocation stress in Garnett's bushbaby (Otolemur garnettii).

Relocation of research animals, either within a facility or from one facility to another, is assumed to be stressful. Development of appropriate research methodologies may be facilitated by understanding the extent and duration of the physiological response to relocation stress and whether the stress can be buffered by environmental or social factors, such as the presence of a cagemate. To characterize the response to relocation stress in Garnett's bushbaby, we assessed cortisol concentrations in nine female and six male bushbabies during relocation to a different facility; six of the animals were pair-housed at the time of the move and were moved with their respective cagemates. Fecal cortisol was assessed at three time points: 1) baseline (1 day prior to moving); 2) relocation (the day the animals were relocated); and 3) post-relocation (7 days after relocation). Cortisol concentrations were higher at the relocation time point than at baseline and post-relocation, which did not differ. Cortisol concentration did not differ as a function of having a cagemate during relocation. Although relocation resulted in a significant (P < 0.05) increase in cortisol excretion concentration, the levels returned to baseline within 7 days after the stressor.