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1.  Intrahepatic lipid, not visceral or muscle fat, is correlated with insulin resistance in older, female Rhesus macaques 
Obesity (Silver Spring, Md.)  2013;21(10):2021-2028.
Little is known of the effect of body composition on glucose metabolism in the aging female non-human primate. We studied these variables in older female Rhesus macaques.
Design and Methods
Female Rhesus macaques (Macacamulatta, n = 19, age range 23–30 yrs) underwent magnetic resonance imaging and 1H spectroscopy to quantify total abdominal fat, visceral fat (VF), subcutaneous fat (SF) area, extramyocellular lipid (EMCL), intramyocellular lipid (IMCL) and intrahepatic lipid (IHL) content, and DEXA scan for whole body composition. A subgroup (n=12) underwent a fasting blood draw and intravenous glucose tolerance test.
SF correlated with homeostatic model assessment of insulin resistance (HOMAIR) and quantitative insulin sensitivity check index (QUICKI), but not after adjustment for fat mass. IHL demonstrated the strongest correlation with HOMAIR, QUICKI and calculated insulin sensitivity index (CSI), and remained significant after adjustment for fat mass. VF, IMCL, and EMCL did not correlate with any of our measures of insulin sensitivity.
Despite a greater amount of VF compared to SF, VF was not associated with markers of insulin resistance (IR) in the older female monkey. Instead, IHL is a marker for IR in the fasting and post-prandial state in these animals.
PMCID: PMC3661746  PMID: 23408675
Body Composition; Insulin Resistance; Magnetic Resonance; Fat Distribution; Obesity
2.  Testosterone Increases Circulating Dehydroepiandrosterone Sulfate Levels in the Male Rhesus Macaque 
The adrenal steroid dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are two of the most abundant hormones in the human circulation. Furthermore, they are released in a circadian pattern and show a marked age-associated decline. Adult levels of DHEA and DHEAS are significantly higher in males than in females, but the reason for this sexual dimorphism is unclear. In the present study, we administered supplementary androgens [DHEA, testosterone and 5α-dihydrotestosterone (DHT)] to aged male rhesus macaques (Macaca mulatta). While this paradigm increased circulating DHEAS immediately after DHEA administration, an increase was also observed following either testosterone or DHT administration, resulting in hormonal profiles resembling levels observed in young males in terms of both amplitude and circadian pattern. This stimulatory effect was limited to DHEAS, as an increase in circulating cortisol was not observed. Taken together, these data demonstrate an influence of the hypothalamo-pituitary–testicular axis on adrenal function in males, possibly by sensitizing the zona reticularis to the stimulating action of adrenocorticopic hormone. This represents a plausible mechanism to explain sex differences in circulating DHEA and DHEAS levels, and may have important implications in the development of hormone therapies designed for elderly men and women.
PMCID: PMC4070064  PMID: 25009533
adrenal gland; aging; androgen; dehydroepiandrosterone; non-human primate; testosterone
3.  Changes in spontaneous activity assessed by accelerometry correlate with extent of cerebral ischemia-reperfusion injury in the nonhuman primate 
Translational stroke research  2012;3(4):442-451.
The use of accelerometry to monitor activity in human stroke patients has revealed strong correlations between objective activity measurements and subjective neurological findings. The goal of our study was to assess the applicability of accelerometry-based measurements in experimental animals undergoing surgically-induced cerebral ischemia. Using a nonhuman primate cortical stroke model, we demonstrate for the first time that monitoring locomotor activity prior to and following cerebrovascular ischemic injury using an accelerometer is feasible in adult male rhesus macaques and that the measured activity outcomes significantly correlate with severity of brain injury. The use of accelerometry as an unobtrusive, objective preclinical efficacy determinant could complement standard practices involving subjective neurological scoring and magnetic resonance imaging in nonhuman primates. Similar activity monitoring devices to those employed in this study are currently in use in human clinical studies, underscoring the feasibility of this approach for assessing the clinical potential of novel treatments for cerebral ischemia.
PMCID: PMC3619447  PMID: 23580904
accelerometer; actigraphy; ischemia; nonhuman primate; rhesus macaque; stroke
4.  Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline 
Age  2011;34(5):1093-1110.
The cognitive decline associated with normal aging was long believed to be due primarily to decreased synaptic density and neuron loss. Recent studies in both humans and non-human primates have challenged this idea, pointing instead to disturbances in white matter (WM) including myelin damage. Here, we review both cross-sectional and longitudinal studies in humans and non-human primates that collectively support the hypothesis that WM disturbances increase with age starting at middle age in humans, that these disturbances contribute to age-related cognitive decline, and that age-related WM changes may occur as a result of free radical damage, degenerative changes in cells in the oligodendrocyte lineage, and changes in microenvironments within WM.
PMCID: PMC3448998  PMID: 22203458
Aging; White matter; Myelination
5.  Perimenopausal regulation of steroidogenesis in the nonhuman primate 
Neurobiology of Aging  2011;33(7):1487.e1-1487.e13.
Human aging is characterized by a marked decrease in circulating levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate (DHEAS), hormonal changes associated with cognitive decline. Despite beneficial effects of DHEA supplementation in rodents, studies in elderly humans have generally failed to show cognitive improvement after treatment. In the present study we evaluate the effects of age and estradiol supplementation on expression of genes involved in the de novo synthesis of DHEA and its conversion to estradiol in the rhesus macaque hippocampus. Using RT-PCR we demonstrate the expression of genes associated with this synthesis in several areas of the rhesus brain. Furthermore, real-time PCR reveals an age-related attenuation of hippocampal expression level of the genes CYP17A1, STS, and 3BHSD1/2. Additionally, short-term administration of estradiol is associated with decreased expression of CYP17A1, STS, SULT2B1, and AROMATASE, consistent with a downregulation not only of estrogen synthesis from circulating DHEA, but also of de novo DHEA synthesis within the hippocampus. These findings suggest a decline in neurosteroidogenesis may account for the inefficacy of DHEA supplementation in elderly humans, and that central steroidogenesis may be a function of circulating hormones and menopausal status.
PMCID: PMC3196783  PMID: 21683476
Aging; Dehydroepiandrosterone; Hormone replacement; Menopause; Neurosteroidogenesis
6.  Association of microtubule associated protein-2, synaptophysin, and apolipoprotein E mRNA and protein levels with cognition and anxiety levels in aged female Rhesus macaques 
Behavioural Brain Research  2012;232(1):1-6.
The dendritic protein microtubule associated protein 2 (MAP-2), the presynaptic marker synaptophysin (SYN), and apolipoprotein E (apoE), a protein which plays a role in lipid transport and metabolism and affects synaptic activity show changes with age. We analyzed post-mortem tissue from aged female Rhesus macaques cognitively tested in a spatial maze and classified as good spatial performers (GSP) or poor spatial performers (PSP) and behaviorally tested in a playroom and classified as bold or reserved animals. MAP2, SYN, and APOE mRNA and protein levels in the prefrontal cortex (PFC), hippocampus, and amygdala, were assessed using qRT-PCR and western blot. In the amygdala, bold monkeys had higher levels of MAP2 and SYN mRNA than reserved monkeys. MAP2 mRNA correlated positively with amygdala size on the right, left, and combined left and right sides, while SYN mRNA levels correlated positively with the size of the right amygdala. In the hippocampus, SYN and apoE protein levels were higher in GSP than PSP animals. Thus, in aged nonhuman primates, classification of measures of anxiety is associated with differences in selected mRNA, but not protein, levels. In contrast, classification of cognitive performance is associated with differences in selected protein, but not mRNA, levels.
PMCID: PMC3361595  PMID: 22475553
MAP-2; SYN; APOE; cognitive function
7.  Astrocytes in aged non-human primate brain gray matter synthesize excess hyaluronan 
Neurobiology of Aging  2011;33(4):830.e13-830.e24.
The glycosaminoglycan hyaluronan (HA) accumulates in central nervous system lesions where it limits astrogliosis but also inhibits oligodendrocyte progenitor cell (OPC) maturation. The role of hyaluronan in normative brain aging has not been previously investigated. Here, we tested the hypothesis that HA accumulates in the aging non-human primate brain. We found that HA levels significantly increase with age in the gray matter of rhesus macaques. HA accumulation was linked to age-related increases in the transcription of HA Synthase-1 (HAS1) expressed by reactive astrocytes but not changes in the expression of other HAS genes or hyaluronidases. HA accumulation was accompanied by increased expression of CD44, a transmembrane HA receptor. Areas of gray matter with elevated HA in older animals demonstrated increased numbers of olig2+ OPCs, consistent with the notion that HA may influence OPC expansion or maturation. Collectively, these data indicate that HAS1 and CD44 are transcriptionally upregulated in astrocytes during normative aging and are linked to HA accumulation in gray matter.
PMCID: PMC3227765  PMID: 21872361
hyaluronan; non-human primate; astrocytes; aging; oligodendrocyte progenitor cells
8.  Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: implications for aging 
Biochimica et biophysica acta  2011;1822(2):111-119.
The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.
PMCID: PMC3249524  PMID: 22056405
9.  Hippocampal M1 receptor function associated with spatial learning and memory in aged female rhesus macaques 
Age  2010;33(3):309-320.
Of the acetylcholine muscarinic receptors, the type 1 (M1) and type 2 (M2) receptors are expressed at the highest levels in the prefrontal cortex (PFC) and hippocampus, brain regions important for cognition. As equivocal findings of age-related changes of M1 and M2 in the nonhuman primate brain have been reported, we first assessed age-related changes in M1 and M2 in the PFC and hippocampus using saturation binding assays. Maximum M1 receptor binding, but not affinity of M1 receptor binding, decreased with age. In contrast, the affinity of M2 receptor binding, but not maximum M2 receptor binding, increased with age. To determine if in the elderly cognitive performance is associated with M1 or M2 function, we assessed muscarinic function in elderly female rhesus macaques in vivo using a scopolamine challenge pharmacological magnetic resonance imaging and in vitro using saturation binding assays. Based on their performance in a spatial maze, the animals were classified as good spatial performers (GSP) or poor spatial performers (PSP). In the hippocampus, but not PFC, the GSP group showed a greater change in T2*-weighted signal intensity after scopolamine challenge than the PSP group. The maximum M1 receptor binding and receptor binding affinity was greater in the GSP than the PSP group, but no group difference was found in M2 receptor binding. Parameters of circadian activity positively correlated with the difference in T2*-weighted signal intensity before and after the challenge, the maximum M1 receptor binding, and the M1 receptor binding affinity. Thus, while in rhesus macaques, there are age-related decreases in M1 and M2 receptor binding, in aged females, hippocampal M1, but not M2, receptor function is associated with spatial learning and memory and circadian activity.
PMCID: PMC3168603  PMID: 20890730
M1 receptor; Scopolamine phMRI; Spatial maze
10.  Accelerated immune senescence and reduced response to vaccination in ovariectomized female rhesus macaques 
Age  2010;33(3):275-289.
Aging is associated with a general dysregulation in immune function, commonly referred to as “immune senescence”. Several studies have shown that female sex steroids can modulate the immune response. However, the impact of menopause-associated loss of estrogen and progestins on immune senescence remains poorly understood. To help answer this question, we examined the effect of ovariectomy on T-cell homeostasis and function in adult and aged female rhesus macaques. Our data show that in adult female rhesus macaques, ovariectomy increased the frequency of naïve CD4 T cells. In contrast, ovariectomized (ovx) aged female rhesus macaques had increased frequency of terminally differentiated CD4 effector memory T cells and inflammatory cytokine-secreting memory T cells. Moreover, ovariectomy reduced the immune response (T-cell cytokine and IgG production) following vaccination with modified vaccinia ankara in both adult and aged female rhesus macaques compared to ovary-intact age-matched controls. Interestingly, hormone therapy (estradiol alone or in conjunction with progesterone) partially improved the T-cell response to vaccination in aged ovariectomized female rhesus macaques. These data suggest that the loss of ovarian steroids, notably estradiol and progesterone, may contribute to reduced immune function in post-menopausal women and that hormone therapy may improve immune response to vaccination in this growing segment of the population.
PMCID: PMC3168610  PMID: 20814751
Aging; Immune senescence; Ovariectomy; T cells; Vaccine; Estrogen; Progestin
11.  Japanese macaque encephalomyelitis: a spontaneous multiple sclerosis-like disease in a nonhuman primate 
Annals of neurology  2011;70(3):362-373.
To describe Japanese macaque encephalomyelitis (JME), a spontaneous inflammatory demyelinating disease occurring in the Oregon National Primate Research Center’s (ONPRC) colony of Japanese macaques (JM, Macaca fuscata).
JM with neurologic impairment were removed from the colony, evaluated and treated with supportive care. Animals were humanely euthanized and their central nervous system (CNS) examined.
ONPRC’s JM colony was established in 1965 and no cases of JME occurred until 1986. Since 1986, 56 JM spontaneously developed a disease characterized clinically by paresis of one or more limbs, ataxia or ocular motor paresis. Most animals were humanely euthanized during their initial episode. Three recovered, later relapsed and were then euthanized. There was no gender predilection and the median age for disease was 4 years. Magnetic resonance imaging of eight cases of JME revealed multiple gadolinium enhancing T1-weighted hyperintensities in the white matter of the cerebral hemispheres, brainstem, cerebellum and cervical spinal cord. The CNS of monkeys with JME contained multifocal plaque-like demyelinated lesions of varying ages, including acute and chronic, active demyelinating lesions with macrophages and lymphocytic periventricular infiltrates, and chronic, inactive demyelinated lesions. A previously undescribed gamma-herpesvirus was cultured from acute JME white matter lesions. Cases of JME continue to affect 1–3% of the ONPRC colony per year.
JME is a unique spontaneous disease in a nonhuman primate that has similarities with multiple sclerosis (MS) and is associated with a novel simian herpesvirus. Elucidating the pathogenesis of JME may shed new light on MS and other human demyelinating diseases.
PMCID: PMC3170450  PMID: 21674589
12.  Proof of concept: pharmacological preconditioning with a Toll-like receptor agonist protects against cerebrovascular injury in a primate model of stroke 
Cerebral ischemic injury is a significant portion of the burden of disease in developed countries; rates of mortality are high and the costs associated with morbidity are enormous. Recent therapeutic approaches have aimed at mitigating the extent of damage and/or promoting repair once injury has occurred. Often, patients at high risk of ischemic injury can be identified in advance and targeted for antecedent neuroprotective therapy. Agents that stimulate the innate pattern recognition receptor, Toll-like receptor 9, have been shown to induce tolerance (precondition) to ischemic brain injury in a mouse model of stroke. Here, we demonstrate for the first time that pharmacological preconditioning against cerebrovascular ischemic injury is also possible in a nonhuman primate model of stroke in the rhesus macaque. The model of stroke used is a minimally invasive transient vascular occlusion, resulting in brain damage that is primarily localized to the cortex and as such, represents a model with substantial clinical relevance. Finally, K-type (also referred to as B-type) cytosine-guanine-rich DNA oligonucleotides, the class of agents employed in this study, are currently in use in human clinical trials, underscoring the feasibility of this treatment in patients at risk of cerebral ischemia.
PMCID: PMC3099644  PMID: 21285967
CpG ODN; ischemia; neuroprotection; nonhuman primate; stroke; Toll-like receptors
13.  Spatial Memory Performance Associated with Measures of Immune Function in Elderly Female Rhesus Macaques 
European geriatric medicine  2011;2(2):117-121.
We recently reported that in aged female rhesus macaques, spatial learning and memory correlates with circadian sleep-wake measures and hippocampal muscarinic type 1 (M1) receptor binding. To investigate if spatial memory also correlates with measures of immune function, we now assessed the magnitude of the adaptive immune response to vaccination in the same old female rhesus macaques. Cognitively characterized animals were classified as good spatial performers (GSP) or poor spatial performers (PSP) based on performance in the Spatial Foodport maze. The GSP group had higher frequency of CD8, but not CD4, interferon-γ (IFN-γ) producing cells following vaccination compared to the PSP group, suggesting a stronger CD8 T cell response in the GSP group. In addition, the number of CD-8 IFN-γ positive cells correlated with measures of sleep quality. Interestingly, the PSP group had a significantly higher antibody titer compared to the GSP group, and antibody titer negatively correlated with day-time activity. Thus, in aged female rhesus macaques, superior cognitive performance is correlated with a more robust CD8 T cell response but a reduced antibody response to vaccination.
PMCID: PMC3097089  PMID: 21603071
spatial learning and memory; immune senescence; circadian activity
14.  Age-related decreases in SYN levels associated with increases in MAP-2, apoE, and GFAP levels in the rhesus macaque prefrontal cortex and hippocampus 
Age  2010;32(3):283-296.
Loss of synaptic integrity in the hippocampus and prefrontal cortex (PFC) may play an integral role in age-related cognitive decline. Previously, we showed age-related increases in the dendritic marker microtubule associated protein 2 (MAP-2) and the synaptic marker synaptophysin (SYN) in mice. Similarly, apolipoprotein E (apoE), involved in lipid transport and metabolism, and glial fibrillary acidic protein (GFAP), a glia specific marker, increase with age in rodents. In this study, we assessed whether these four proteins show similar age-related changes in a nonhuman primate, the rhesus macaque. Free-floating sections from the PFC and hippocampus from adult, middle-aged, and aged rhesus macaques were immunohistochemically labeled for MAP-2, SYN, apoE, and GFAP. Protein levels were measured as area occupied by fluorescence using confocal microscopy as well as by Western blot. In the PFC and hippocampus of adult and middle-aged animals, the levels of SYN, apoE, and GFAP immunoreactivity were comparable but there was a trend towards higher MAP-2 levels in middle-aged than adult animals. There was significantly less SYN and more MAP-2, apoE, and GFAP immunoreactivity in the PFC and hippocampus of aged animals compared to adult or middle-aged animals. Thus, the age-related changes in MAP-2, apoE, and GFAP levels were similar to those previously observed in rodents. On the other hand, the age-related changes in SYN levels were not, but were similar to those previously observed in the aging human brain. Taken together, these data emphasize the value of the rhesus macaque as a pragmatic translational model for human brain aging.
PMCID: PMC2926858  PMID: 20640549
Aging; Nonhuman primate; MAP-2; Synaptophysin; apoE
Experimental gerontology  2009;45(3):208-216.
Apyrimidinic/apurinic endonuclease (APE) is a key protein involved in the base excision DNA repair (BER) pathway of oxidative DNA lesions. Using a novel oligonucleotide substrate, we demonstrate that APE activity in the frontal/parietal cortex (F/PCTX), cerebellum, brainstem, midbrain and hypothalamus declined with age in rats on an ad libitum (AL) diet. In contrast, APE activity for these brain regions was ~1.5-3 times higher in young, caloric restricted (CR) rats. Despite continuous CR treatment in all animals since six weeks of age, APE activity in the CR group started to decline by middle-age and continued into old age. However, CR maintained APE activity at a level that was significantly higher than that in AL rats across age and in the brain regions examined. Because Western analysis of APE, DNA polymerase β and DNA ligase III levels in the F/PCTX of both CR and AL rats remained unchanged with age, this suggests that the increased APE activity in CR rats is the result of differential post-translational modification of APE.
PMCID: PMC2826610  PMID: 20005284
Exonuclease; apyrimidinic/apurinic endonuclease (APE); DNA polymerase β; DNA ligase III; frontal/parietal cortex; 8-oxodeoxyguanosine
16.  Influence of 17β-estradiol and progesterone on GABAergic gene expression in the arcuate nucleus, amygdala and hippocampus of the rhesus macaque 
Brain research  2009;1307:28-42.
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and the responsiveness of neurons to GABA can be modulated by sex steroids. To better understand how ovarian steroids influence GABAergic system in the primate brain, we evaluated the expression of genes encoding GABA receptor subunits, glutamic acid decarboxylase (GAD) and a GABA transporter in the brains of female rhesus macaques. Ovariectomized adults were subjected to a hormone replacement paradigm involving either 17β-estradiol (E), or E plus progesterone (E+P). Untreated animals served as controls. Using GeneChip® microarray analysis and real-time RT-PCR (qPCR), we examined gene expression differences within and between the amygdala (AMD), hippocampus (HPC) and arcuate nuclei of the medial basal hypothalamus (MBH). The results from PCR corresponded with results from representative GeneChip® probesets, and showed similar effects of sex steroids on GABA receptor subunit gene expression in the AMD and HPC, and a more pronounced expression than in the MBH. Exposure to E+P attenuated GAD1, GAD2 and SLC32A1 gene expression in the AMD and HPC, but not in the MBH. GABA receptor subunit gene expression was generally higher in the AMD and HPC than in the MBH, with the exception of receptor subunits ε and γ2. Taken together, the data demonstrate differential regulation of GABA receptor subunits and GABAergic system components in the MBH compared to the AMD and HPC of rhesus macaques. Elevated ε and reduced δ subunit expression in the MBH supports the hypothesis that the hypothalamic GABAergic system is resistant to the modulatory effects of sex steroids.
PMCID: PMC2787920  PMID: 19833106
gamma-aminobutyric acid; GABA receptor; Macaca mulatta; microarray
17.  In vivo manipulation of gene expression in non-human primates using lentiviral vectors as delivery vehicles 
Methods (San Diego, Calif.)  2009;49(1):70-77.
Nonhuman primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.
PMCID: PMC2732747  PMID: 19559089
Lentiviral Vectors; Gene Regulation; Non-Human Primates
18.  Gene expression profiling in the rhesus macaque: experimental design considerations 
Methods (San Diego, Calif.)  2009;49(1):26-31.
The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-hour expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.
PMCID: PMC2734384  PMID: 19467336
Circadian rhythms; Macaca Mulatta; Menstrual cycle; Microarray; Sex steroids; Rhesus Macaque Genome Array
19.  Gene expression profiling in the rhesus macaque: methodology, annotation and data interpretation 
Methods (San Diego, Calif.)  2009;49(1):42-49.
Gene microarray analyses represent potentially effective means for high-throughput gene expression profiling in nonhuman primates. In the companion article we emphasize effective experimental design based on the in vivo physiology of the rhesus macaque, whereas this article emphasizes considerations for gene annotation and data interpretation using gene microarray platforms from Affymetrix®. Initial annotation of the rhesus genome array was based on Affymetrix® human GeneChips®. However, annotation revisions improve the precision with which rhesus transcripts are identified. Annotation of the rhesus GeneChip® is under continuous revision with large percentages of probesets under multiple annotation systems having undergone multiple reassignments between March 2007 and November 2008. It is also important to consider that quantitation and comparison of gene expression levels across multiple chips requires appropriate normalization. External corroboration of microarray results using PCR-based methodology also requires validation of appropriate internal reference genes for normalization of expression values. Many tools are now freely available to aid investigators with microarray normalization and selection of internal reference genes to be used for independent corroboration of microarray results.
PMCID: PMC2739830  PMID: 19467334
Macaca Mulatta; Microarray; GeneChip® Rhesus Macaque Genome Array
20.  Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain 
BMC Molecular Biology  2010;11:47.
Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods.
We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix® GeneChip® rhesus Macaque Genome Array.
Having shown that qRT-PCR and Affymetrix® GeneChip® data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages.
Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip® facilitated more targeted analysis than could be accomplished using the rhesus GeneChip®. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed.
Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.
PMCID: PMC2914640  PMID: 20565976
21.  A Population-Average MRI-Based Atlas Collection of the Rhesus Macaque 
NeuroImage  2008;45(1):52-59.
Magnetic resonance imaging (MRI) studies of non-human primates are becoming increasingly common; however, the well-developed voxel-based methodologies used in human studies are not readily applied to non-human primates. In the present study, we create a population-average MRI-based atlas collection for the rhesus macaque (Macaca mulatta) that can be used with common brain mapping packages such as SPM or FSL. In addition to creating a publicly available T1-weighted atlas (, probabilistic tissue classification maps and T2-weighted atlases were also created. Theses atlases are aligned to the MRI volume from the Saleem-Logothetis (2006) atlas providing an explicit link to histological sections. Additionally, we have created a transform to integrate these atlases with the F99 surface-based atlas in CARET. It is anticipated that these tools will help facilitate voxel-based imaging methodologies in non-human primate species, which in turn may increase our understanding of brain function, development, and evolution.
PMCID: PMC2659879  PMID: 19059346
rhesus macaque; MRI; atlas; Macaca mulatta; priors; brain mapping; probabilistic
22.  A New Model of Cortical Stroke in the Rhesus Macaque 
Primate models are essential tools for translational research in stroke, but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (MCA) (distal to the orbitofrontal branch), and both anterior cerebral arteries (ACA) were occluded with vascular clips. Bilateral occlusion of the ACA was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia were induced for 45, 60, or 90 minutes (n=2/timepoint) and infarct volume and neurological outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T2-weighted MRI. Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride (TTC) and cresyl violet staining corroborated MRI results. Neurological deficit scores worsened gradually with longer occlusion times. A subset of animals (n=5) underwent 60 minutes of ischemia resulting in consistent infarct volumes primarily located to the cortex which correlated well with neurological deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.
PMCID: PMC2828874  PMID: 19384334
Cerebral ischemia; MRI; Neurological deficit; Non-human primate; Rhesus macaque; Stroke
23.  Free radical damage to cerebral cortex in Alzheimer's Disease, Microvascular Brain Injury, and Smoking 
Annals of neurology  2009;65(2):226-229.
Evidence supports a pathogenic role for free radical injury to brain regions in Alzheimer's disease (AD); however, clinical trial results are only mildly encouraging. Examining brains from The Adult Changes in Thought study offers a unique perspective. Selectively increased free radical damage to cerebral cortex was associated with AD, microvascular brain injury (μ-VBI), and current smoking, but not with antioxidant supplement usage. Our results support suppression of free radical injury to brain as a therapeutic target for AD and μ-VBI; however, future clinical trials should consider other antioxidants or doses than those identified in our study.
PMCID: PMC2729331  PMID: 19259965
24.  Glutamate receptor subunit expression in the rhesus macaque locus coeruleus 
Brain research  2007;1173:53-65.
The locus coeruleus (LC) is a major noradrenergic brain nucleus that regulates states of arousal, optimizes task-oriented decision-making, and may also play an important role in modulating the activity of the reproductive neuroendocrine axis. Rodent studies have shown that the LC is responsive to glutamate receptor agonists, and that it expresses various glutamate receptor subunits. However, glutamate receptor subunit expression has not been extensively examined in the primate LC. We previously demonstrated expression of the NR1 NMDA glutamate receptor subunit in the rhesus macaque LC, and now extend this work by also examining the expression of non-NMDA (AMPA and kainate) ionotropic glutamate receptor subunits. Using in situ hybridization histochemistry and immunohistochemistry, we confirmed the presence of the obligatory NR1 subunit in the LC. In addition, we demonstrated expression of the AMPA glutamate receptor subunits GluR1, GluR2 and GluR3. More extensive receptor profiling, using rhesus monkey gene microarrays (Affymetrix GeneChip®), further corroborated the histological findings and showed expression of mRNA encoding ionotropic glutamate receptor subunits NR2A, NR2D, GluR4, and GluR6, as well as the metabotropic glutamate receptor subunits mGluR1, mGluR3, mGluR4, mGluR5 and mGluR7. These data provide a foundation for future examination of how changes in glutamate receptor composition contribute to the control of primate physiology.
PMCID: PMC2067256  PMID: 17765206
Locus coeruleus; Microarray; Gene array; Rhesus macaque; Glutamate receptor; in situ hybridization

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