Osteoarthritic changes in vervet monkey knees correlate with meniscus degradation and increased matrix metalloproteinase and cytokine secretion.

OBJECTIVE: Meniscus injury increases osteoarthritis risk but its pathobiology in osteoarthritis is unclear. We hypothesized that older adult vervet monkeys would exhibit knee osteoarthritic changes and the degenerative menisci from these animals would secrete matrix metalloproteinases (MMPs) and pro-inflammatory cytokines that contribute to the development of osteoarthritis. DESIGN: In a cross sectional analysis of healthy young adult (9-12 years) and old (19-26 years) adult female vervet monkeys, knees were evaluated in vivo with computed tomography (CT) imaging, and joint tissues were morphologically graded at necropsy. Meniscus explants were subsequently cultured to evaluate meniscal MMP and cytokine secretion. RESULTS: CT images revealed significant bony osteoarthritic changes in 80% of older monkeys which included increases in osteophyte number and meniscal calcification. Meniscus and cartilage degradation scores were greater in the older monkeys and were positively correlated (r > 0.7). Menisci from older animals exhibiting osteoarthritic changes secreted significantly more MMP-1, MMP-3, and MMP-8 than healthy menisci from younger monkeys. Older menisci without significant osteoarthritic changes secreted more IL-7 than healthy young menisci while older osteoarthritic menisci secreted more IL-7 and granulocyte-macrophage colony-stimulating factor than healthy older menisci. CONCLUSIONS: Aged vervets develop naturally occurring knee osteoarthritis that includes involvement of the meniscus. Degenerative menisci secreted markedly increased amounts of matrix-degrading enzymes and inflammatory cytokines. These factors would be expected to act on the meniscus tissue and local joint tissues and may ultimately promote osteoarthritis development. These finding also suggest vervet monkeys are a useful animal model for studying the progression of osteoarthritis.

Chronic stress, metabolism, and metabolic syndrome.

The prevalence of obesity has rapidly escalated and now represents a major public health concern. Although genetic associations with obesity and related metabolic disorders such as diabetes and cardiovascular disease have been identified, together they account for a small proportion of the incidence of disease. Environmental influences such as chronic stress, behavioral and metabolic disturbances, dietary deficiency, and infection have now emerged as contributors to the development of metabolic disease. Although epidemiological data suggest strong associations between chronic stress exposure and metabolic disease, the etiological mechanisms responsible remain unclear. Mechanistic studies of the influence of chronic social stress are now being conducted in both rodent and nonhuman primate models, and phenotypic results are consistent with those in humans. The advantage of these models is that potential neural mechanisms may be examined and interventions to treat or prevent disease may be developed and tested. Further, circadian disruption and metabolic conditions such as diabetes mellitus could increase susceptibility to other stressors or serve as a stressor itself. Here, we review data from leading investigators discussing the interrelationship between chronic stress and development of metabolic disorders.

Polycystic ovary syndrome with endometrial hyperplasia in a cynomolgus monkey (Macaca fascicularis).

A 13-year-old, obese, female cynomolgus monkey (Macaca fascicularis) was observed in a 5-year neurobehavioral study and was humanely euthanatized for experimental purposes. During this observational study, the monkey was noted to ovulate only rarely (0-3 times a year), with a prolonged menstrual cycle length (up to 161 days), hyperandrogenism (androstenedione area under the curve in response to adrenocorticotropic hormone up to 27.64 ng/ml), and hyperinsulinemia (fasting insulin up to 65.85 microIU/ml). This animal's body mass index was 65.46 kg/m(2), with central obesity. On postmortem examination, the uterus was moderately enlarged, with an eccentric lumen and a broad-based endometrial polyp that consisted of complex glandular hyperplasia with atypia. Both ovaries contained many 2- to 3-mm follicles, without any corpora lutea. A diagnosis of polycystic ovary syndrome was made based on the clinical history, endocrinology, and gross and histopathologic findings.

Triphasic oral contraceptive treatment alters the behavior and neurobiology of female cynomolgus monkeys.

Oral contraceptives (OCs) are the most widely prescribed and effective of the reversible contraceptive methods. In addition to inhibiting ovulation, OCs alter central nervous system function in women; however, methodological problems have prevented clear human studies. Thus, in this experiment we investigated the effects of OC treatment on behavior, hypothalamic- pituitary-adrenal axis function and the central nervous system in 75 adult female cynomolgus monkeys (Macaca fascicularis) housed in social groups of four to five monkeys per pen. Monkey social groups were randomly divided into either a control or an OC treatment group which was administered a clinically prescribed OC (Triphasil(R), levonorgestrel and ethinyl estradiol tablets) for 2 years. OC treatment increased the frequency of contact aggression received, time spent in locomotion, and sitting close to another animal, and decreased time spent fearfully scanning. OC treatment decreased heart rate, increased activity levels, and increased baseline cortisol concentrations and the cortisol response to adrenocorticotropin compared to control animals. OC treatment decreased the prolactin response to fenfluramine suggesting decreased serotonergic activity. These results suggest that this triphasic OC disrupts social behavior, hypothalamic-pituitary-adrenal axis regulation and the underlying central nervous system function.

Soy and social stress affect serotonin neurotransmission in primates.

Stress and sex steroidal milieu can each influence mood in women. The purpose of this study was to compare the effect of long-term conjugated equine estrogen (CEE), soy phytoestrogen (SPE), and social subordination stress on dorsal raphe serotonin neurotransmission of ovariectomized cynomolgus monkeys. Tryptophan hydroxylase (TPH) and serotonin reuptake transporter (SERT) protein content were determined, and the in vitro degradation of macaque SERT protein was examined in the presence and absence of protease inhibitors, serotonin (5-HT), and citalopram. Like CEE, SPE increased TPH protein levels. Social subordinates had markedly lower TPH protein levels than dominants regardless of hormone replacement. Therefore, these two variables had independent and additive effects. CEE and SPE increased SERT, and social status had no effect. Thus, the hormone-induced increase in SERT was accompanied by increased 5-HT synthesis and neuronal firing, which appears biologically reasonable as 5-HT prevented SERT degradation in vitro.

Are subordinates always stressed? A comparative analysis of rank differences in cortisol levels among primates.

Among primate species there is pronounced variation in the relationship between social status and measures of stress physiology. An informal meta-analysis was designed to investigate the basis of this diversity across different primate societies. Species were included only if a substantial amount of published information was available regarding both social behavior and rank-related differences in stress physiology. Four Old World and three New World species met these criteria, including societies varying from small-group, singular cooperative breeders (common marmoset and cotton top tamarin) to large-troop, multi-male, multi-female polygynous mating systems (rhesus, cynomolgus, talapoin, squirrel monkeys, and olive baboon). A questionnaire was formulated to obtain information necessary to characterize the stress milieu for individuals in particular primate societies. We standardized cortisol values within each species by calculating the ratio of basal cortisol concentrations of subordinates to those of dominants in stable dominance hierarchies and expressing the ratio as a percentage (relative cortisol levels). The meta-analysis identified two variables that significantly predicted relative cortisol levels: subordinates exhibited higher relative cortisol levels when they (1). were subjected to higher rates of stressors, and (2). experienced decreased opportunities for social (including close kin) support. These findings have important implications for understanding the different physiological consequences of dominant and subordinate social status across primate societies and how social rank may differ in its behavioral and physiological manifestations among primate societies.

Induction and maintenance of ethanol self-administration in cynomolgus monkeys (Macaca fascicularis): long-term characterization of sex and individual differences.

BACKGROUND: Investigations of oral ethanol self-administration in nonhuman primates have revealed important parallels with human alcohol use and abuse, yet many fundamental questions concerning the individual risk to, and the biological basis of, excessive ethanol consumption remain unanswered. Moreover, many conditions of access to ethanol in nonhuman primate research are largely unexplored. This set of experiments extends within- and across-session exposure to ethanol to more fully characterize individual differences in oral ethanol self-administration. METHODS: Eight male and eight female adult cynomolgus monkeys (Macaca fascicularis) were exposed to daily oral ethanol self-administration sessions for approximately 9 months. During the first 3 months, a fixed-time (FT) schedule of food delivery was used to induce the consumption of an allotted dose of ethanol in 16-hr sessions. Subsequently, the FT schedule was suspended, and ethanol was available ad libitum for 6 months in 16- or 22-hr sessions. RESULTS: Cynomolgus monkeys varied greatly in their propensity to self-administer ethanol, with sex and individual differences apparent within 10 days of ethanol exposure. Over the last 3 months of ethanol access, individual average ethanol intakes ranged from 0.6 to 4.0 g/kg/day, resulting in blood ethanol concentrations from 5 to 235 mg/dl. Males drank approximately 1.5-fold more than females. In addition, heavy-, moderate-, and light-drinking phenotypes were identified by using daily ethanol intake and the percentage of daily calories obtained from ethanol as criteria. CONCLUSIONS: Cynomolgus monkeys displayed a wide intersubject range of oral ethanol self-administration with a procedure that used a uniform and prolonged induction that restricted early exposure to ethanol and subsequently allowed unlimited access to ethanol. There were sex and stable individual differences in the propensity of monkeys to consume ethanol, indicating that this species will be important in characterizing risk factors associated with heavy-drinking phenotypes.

Steroid regulation of tryptophan hydroxylase protein in the dorsal raphe of macaques.

BACKGROUND: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme for the synthesis of serotonin, and serotonin is a pivotal neurotransmitter in the regulation of mood, affective behavior, pituitary hormone secretion, and numerous autonomic functions. We previously demonstrated that estradiol (E) and progesterone (P) increase TPH mRNA levels in the dorsal raphe of macaques. METHODS: This study employed western blotting and densitometric quantitation to determine whether the changes observed at the level of gene expression were manifested by changes in TPH protein expression and whether modified estrogens or progestins had actions similar to the native ligands. In addition, the effect of the antiestrogen tamoxifen was examined. Ovariectomized (ovx) rhesus and cynomolgus macaques were untreated or treated with E, P, E+P, equine estrogens (EE), medroxyprogesterone (MPA), EE+MPA, or tamoxifen. The dorsal raphe region was subjected to Western analysis. RESULTS: E treatment for 28 days increased TPH protein mass four to six fold over ovariectomized controls. Addition of P to the E regimen or treatment with P for 28 days after E priming did not alter TPH from E treatment alone. Treatment of ovx macaques with a low dose of P caused a two-fold increase in TPH protein. Treatment of ovariectomized macaques for 30 months with EE alone or MPA alone significantly increased TPH protein; however, unlike P, the addition of MPA to the EE regimen blocked the stimulatory effect of EE. Tamoxifen treatment significantly reduced TPH protein compared to EE and ovariectomized control animals. CONCLUSION: The stimulatory effect of E and P on TPH protein in the dorsal raphe of macaques correlates with the previously observed effect at the level of mRNA expression. P had no effect on the stimulatory action of E, whereas MPA blocked the stimulatory effect of EE. Tamoxifen acted as a potent antiestrogen on TPH protein expression. If TPH protein mass influences serotonin synthesis, then these steroids will impact many autonomic systems that are regulated by serotonin.

Can puzzle feeders be used as cognitive screening instruments? Differential performance of young and aged female monkeys on a puzzle feeder task.

Conventional cognitive testing of monkeys is time-consuming and involves single-caging and food or water deprivation. Here we report a novel test of global cognitive performance that can be completed in a short time period without food/water or social restrictions. Nine mazes of increasing difficulty were developed using a standard puzzle feeder, and the maze-solving performance of ten young and five aged female cynomolgus monkeys (Macaca fascicularis) was tested. The young monkeys solved maze configurations at higher levels of difficulty and solved the first level of difficulty more quickly than aged monkeys. This task discriminated performance by age in nonhuman primates as do more conventional forms of cognitive testing and indicates that this task may be a quick and easy assessment of global cognitive function.

Effects of hormone replacement therapy and social stress on body fat distribution in surgically postmenopausal monkeys.

OBJECTIVE: To investigate the effects of hormone replacement therapy (HRT) and social stress on body fat distribution in an animal model of women's health, the female cynomolgus macaque (Macaca fascicularis). DESIGN/SUBJECTS: Adult female cynomolgus monkeys were ovariectomized and fed an atherogenic diet for two years while housed in social groups of 3-8 monkeys each. Animals were then fed a lipid-lowering diet and randomized into four experimental groups: a baseline group which was necropsied immediately and not included in the study reported here, 26 females fed diet only (CONTROL), 22 females fed diet plus conjugated equine estrogens (CEE), and 21 females fed the diet plus CEE and medroxyprogesterone acetate (CEE + MPA). Treatment lasted 30 months. MEASUREMENTS: During the last nine months of treatment, social status was determined three times at three month intervals. At the end of the study, whole body obesity and fat distribution patterns were determined using anthropometry and computerized tomography (CT). RESULTS: The addition of a progestin to the estrogen replacement regimen administered to surgically postmenopausal monkeys, increased all anthropometric and CT measures of obesity except intra-abdominal fat. HRT had no effect on patterns of fat distribution. Socially-dominant, ovariectomized females were more obese than subordinates using both anthropometric and CT measurements of whole body obesity. Dominant females were more likely to have their fat deposited centrally as measured anthropometrically. However, CT measures revealed a trend for dominants to preferentially deposit fat in the subcutaneous abdominal depot in contrast to subordinates who deposited fat in the intra-abdominal depot. CONCLUSIONS: The results of this study suggest that progestins, when administered in combination with estrogens, may increase fat deposition, particularly in subcutaneous depots. In addition, the social stress experienced by subordinate monkeys, may have mild effects on fat deposition patterns, even after removal of ovarian function as a factor. These observations may have implications for treatment recommendations in postmenopausal women. Lastly, CT may measure different characteristics of fat distribution than skinfolds and circumferences.

Behavioral and neurobiological effects of estrogen replacement therapy and a history of triphasic oral contraceptive exposure.

The effects of contraceptive steroids and estrogen replacement therapy on behavior and neuroendocrine function were evaluated in adult female cynomolgus monkeys. During the 'premenopausal' phase of the experiment, the animals were assigned to either treatment with a triphasic oral contraceptive (OC) for 24 months or the untreated control group. The monkeys were then ovariectomized and half of each of the premenopausal groups were randomly assigned to either treatment with conjugated equine estrogens (ERT) or the untreated control group for 12 months (the 'postmenopausal' phase). All evaluations were completed during the postmenopausal phase of the experiment. Both types of exogenous steroid treatments appeared to increase cardiovascular and hypothalamic-pituitary-adrenal responses to stress in socially dominant but not socially subordinate females. A history of triphasic OC administration increased contact aggression received, and reduced the prolactin response to fenfluramine, suggesting reduced serotonergic activity, for at least a year following the cessation of triphasic OC treatment.

Social subordination stress, behavior, and central monoaminergic function in female cynomolgus monkeys.

BACKGROUND: Social subordination in female cynomolgus monkeys is stressful and activates the hypothalamic-pituitary-adrenal axis. In a previous experiment behavioral depression was observed in a subset of subordinates. METHODS: In the experiment reported here behavioral and physiological indicators of stress were evaluated in dominant and subordinate female cynomolgus monkeys, and brain dopaminergic activity was assessed, as reflected in the prolactin response to haloperidol, a dopamine2 (D2) receptor antagonist. RESULTS: Subordinates were aggressed more, spent more time in fearful scanning of the social environment, spent less time as the recipients of the active affiliative behavior of being groomed, had more variable heart rates in response to a novel environment, and were hypercortisolemic compared to dominants. Prolactin responses to haloperidol challenge were lower in subordinates than dominants, an observation consistent with the hypothesis that subordinate females have decreased D2 receptor function. CONCLUSIONS: These observations suggest that social subordination is stressful and may alter brain dopaminergic function in primates. The neurophysiological characteristics of social subordinates may contribute to their susceptibility to depression.

Effects of chronic social separation on cardiovascular disease risk factors in female cynomolgus monkeys.

A lack of social support is associated with increased risk of coronary heart disease morbidity and mortality in human beings. Similarly, chronic social separation (single cage housing) potentiates atherosclerosis in female monkeys. Under the hypothesis that autonomic arousal and/or ovarian impairment may mediate this effect (as both are associated with increased atherosclerosis), heart rate and luteal phase plasma progesterone concentrations were measured in 12 female cynomolgus monkeys that were first socially housed, then individually housed, and finally returned to their original social groups. Afternoon heart rates increased during social separation compared to the social groupings (P < 0.001). Increased heart rates could not be explained by activity levels, which were lower during social separation than in social groupings (P < 0.001). Ovarian function (i.e. luteal-phase progesterone concentrations) was not influenced by housing condition. Single caging reduced the extent of social signaling, even though animals were in visual and auditory contact. Rates of affiliative behaviors increased and time spent alone decreased in post-reunion social groups compared to pre-separation social groups (P's < 0.01). The results indicate that chronic social separation in this group-living species may exacerbate atherosclerosis via altered autonomic activity, as evidenced by higher heart rates during social separation.

Expression of estrogen receptor alpha and beta transcripts in female monkey hippocampus and hypothalamus.

Understanding mechanisms of estrogen effects on cognition is critical for designing therapies for post-menopausal women and others with dementia. Hippocampus, an area important to cognitive function, responds robustly on estrogen. ERbeta and ERalpha transcripts were detected in the hippocampus and hypothalamus of an ovariectomized female monkey at a relatively high ERbeta/ERalpha ratio. These results suggest that ERbeta may play a role in mediating estrogen effects in the primate hippocampus and hypothalamus.

17alpha-Dihydroequilenin increases hippocampal dendritic spine density of ovariectomized rats.

The effects of estradiol and 17alpha-dihydroequilenin on the apical dendrite spine density of pyramidal cells of the CA1 region of rat hippocampus were compared. 17alpha-Dihydroequilenin was as effective as estradiol in increasing spine densities relative to controls. 17alpha-Dihydroequilenin is not uterotrophic like estradiol but does have beneficial effects on the cardiovascular system, suggesting that it may be an effective single-agent hormone replacement therapy to treat menopausal symptoms and reduce chronic disease risk in menopausal women.

Behavior and physiology of social stress and depression in female cynomolgus monkeys.

The hypothesis that social subordination is stressful, and results in a depressive response in some individuals, was examined in socially housed female cynomolgus monkeys. Social status was manipulated such that half of the previously subordinate females became dominant and half of the previously dominant females became subordinate. Current subordinates hypersecreted cortisol, were insensitive to negative feedback, and had suppressed reproductive function. Current subordinates received more aggression, engaged in less affiliation, and spent more time alone than dominants. Furthermore, they spent more time fearfully scanning the social environment and displayed more behavioral depression than dominants. Current subordinates with a history of social subordination were preferentially susceptible to a behavioral depression response. The results of this experiment suggest that the stress of social subordination causes hypothalamic-pituitary-adrenal and ovarian dysfunction, and support the hypothesis that chronic, low-intensity social stress may result in depression in susceptible individuals.

Discriminative stimulus effects of ethanol and 3 alpha-hydroxy-5 alpha-pregnan-20-one in relation to menstrual cycle phase in cynomolgus monkeys (Macaca fascicularis).

The present study was designed to characterize the discriminative stimulus effects of ethanol and the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in nonhuman primates as a function of menstrual cycle phase. Female cynomolgus monkeys (Macaca fascicularis) were trained in a two-lever procedure to discriminate 1.0 g/kg ethanol (IG, 30 min pretreatment) from water using food reinforcement. A cumulative dosing procedure was used to assess changes in the potency of ethanol and an endogenous anxiolytic steroid in the follicular versus the luteal phase of the menstrual cycle. Plasma progesterone and allopregnanolone levels were determined within 24 h of testing to verify phase of menstrual cycle. The monkeys were more sensitive to the discriminative stimulus effects of ethanol and the ethanol-like effects of the endogenous neuroactive steroid allopregnanolone during the luteal phase of the menstrual cycle. These findings suggest that changes in the endogenous levels of ovarian-derived progesterone and allopregnanolone alter sensitivity to the discriminative stimulus effects of ethanol.

Social stress, depression, and brain dopamine in female cynomolgus monkeys.

Socially subordinate adult female cynomolgus monkeys are hypercortisolemic, the targets of aggression, fearful, vigilant, receive little positive affiliative contact, exhibit pathological behaviors indicating anxiety, and are disengaged in the social events around them. Subordinates also have altered dopaminergic activity that may be due to decreased D2 receptor binding. Dopaminergic activity indices were more closely associated with affiliative than agonistic behaviors.

Psychosocial factors, sex differences, and atherosclerosis: lessons from animal models.

OBJECTIVE: Premenopausal women, compared with men, are relatively spared from coronary heart disease and the underlying atherosclerosis. Our purpose has been to elucidate the reason for this difference and to explore the role of behavioral factors in this phenomenon. METHODS: Studies employed socially housed cynomolgus macaques (Macaca fascicularis) fed an atherogenic diet and subjected to behavioral observations. Ovariectomy, with or without hormone replacement, was used to test specific hypotheses about estrogen's role in the protection of females from atherosclerosis and coronary heart disease. RESULTS: Female macaques, like women, are resistant to atherosclerosis. However, this resistance is modified by social status-dominant monkeys develop little atherosclerosis, whereas subordinates resemble males in the amount of lesion that occurs. Subordinate females also are characterized by hypercortisolemia, behavioral dysfunction, and impaired ovarian function; the resulting low concentrations of circulating estrogen perhaps explain their accelerated atherosclerosis. Notably, atherosclerosis is exacerbated in ovariectomized monkeys but is suppressed in association with pregnancy, a hyperestrogenic state. Moreover, exogenous estrogen (an oral contraceptive) inhibits atherosclerosis in premenopausal social subordinates. CONCLUSIONS: To the extent that our results apply to women, they highlight the potential importance of behavioral stressors and their effects on estrogen activity in the premenopausal development of atherosclerosis. The triad of hypercortisolism, ovarian impairment, and psychiatric morbidity found in monkeys also occurs in women and may represent a high-risk state for disorders of the cardiovascular system and perhaps, other estrogen-sensitive tissues.