Bone functional changes in intact, ovariectomized, and ovariectomized, hormone-supplemented adult cynomolgus monkeys (Macaca fascicularis) evaluated by serum markers and dynamic histomorphometry.

Several parameters of bone mass and function were investigated in three experiments involving intact, ovariectomized, or hormone-supplemented ovariectomized female cynomolgus monkeys. Ovariectomized animals had increased serum levels of alkaline phosphatase and acid phosphatase compared with intact and hormone-supplemented animals. Vertebral bone mass measured ex vivo by dual-photon absorptiometry was reduced by 11-19% in ovariectomized animals compared with intact and hormone-supplemented animals. The most dramatic effects observed with ovariectomy were markedly increased (30-60%) bone formation rates in vertebral cancellous bone, primarily caused by higher activation frequency of basic multicellular units of bone. In addition, combined resorption and reversal periods were decreased and formation period increased in untreated ovariectomized animals. Changes in static histomorphometry parameters were less dramatic, cancellous bone volume being 1-14% lower in ovariectomized animals compared with intact or ovariectomized hormone-supplemented animals. The data indicate that changes in bone resorption are primarily responsible for the lower bone mass of estrogen deficiency and increased bone mass in hormone-supplemented animals. Bone changes in ovariectomized cynomolgus monkeys resemble those in women after menopause and similarly respond positively to hormone supplementation. As such, cynomolgus monkeys are an excellent model for studying the basic mechanisms of osteoporosis and for the development of suitable therapeutic regimens.

Immunolocalization of noncollagenous bone matrix proteins in lumbar vertebrae from intact and surgically menopausal cynomolgus monkeys.

The noncollagenous matrix proteins, composing about 10% of the organic matrix of bone, are considered important for cell matrix organization and regulation of mineralization in bone. In the present study, seven of the major noncollagenous bone matrix proteins were localized immunohistochemically in serial sections of lumbar vertebrae from 24 (12 intact and 12 ovariectomized) adult female cynomolgus monkeys (Macaca fascicularis). Osteocalcin was the only protein restricted to bone cells and mineralized bone matrix. Bone sialoprotein was present in both bone and calcified cartilage, and all the other proteins were distributed in soft tissues as well as bone. Staining for both osteocalcin and bone sialoprotein was present diffusely throughout the bone matrix, but osteonectin, osteopontin, matrix gla protein, decorin, and biglycan staining was concentrated along bone surfaces. Osteoid was negative for osteocalcin and bone sialoprotein, but all other proteins had areas of positive immunostaining within osteoid. All proteins except biglycan exhibited strong immunostaining of a subset of active osteoblasts, suggesting that they may be markers of osteoblast maturity or state of activation. The pattern of immunostaining in intact and surgically menopausal monkeys was similar, except that staining for matrix proteins concentrated along bone surfaces appeared to be more widely distributed in the surgically menopausal monkeys, probably due to the higher rate of bone formation in these animals.

Effects on bone of oral hormone replacement therapy initiated 2 years after ovariectomy in young adult monkeys.

The purpose of this study was to determine the effects of oral estrogen replacement therapy with conjugated equine estrogens (CEE), alone or in combination with continuous medroxyprogesterone acetate (MPA), on lumbar spine bone mineral content (BMC) and density (BMD) and on serum chemistries in ovariectomized cynomolgus monkeys when therapy is initiated following a 2 year hypoestrogenic period. Study design was done in the form of a randomized, placebo-controlled, nonhuman primate paraclinical trial. Monkeys were subjects in an experiment designed to study the effects of a lipid-lowering diet combined with hormone replacement therapy on atherosclerosis. Initially, they were ovariectomized and fed a high-fat diet for 24 months. They were then were allocated to three treatment groups by stratified randomization and were fed a diet containing reduced dietary fat for an additional 28 months. Treatment groups consisted of: (1) an untreated group (ovx, n = 24); (2) a CEE-treated group (CEE, n = 19); and (3) a CEE plus continuous MPA group (CEE + MPA, n = 20). Lumbar spine BMC and BMD values were measured by dual-energy x-ray absorptiometry at baseline and 4, 10, 16, 22, and 28 months of treatment. Serum chemistries were relevant to bone metabolism at 22 and 28 months. Rates of gain in BMC and BMD were greater (p < 0.05) in hormone-supplemented animals (groups 2 and 3) than in untreated ovx animals during the first 16 months of treatment, resulting in increased BMC and BMD measurements in these groups. Serum markers of bone metabolism were significantly lower (p < 0.05) in the hormone-treated groups (groups 2 and 3) compared with ovx animals after 22 and 28 months of treatment, indicating reductions in bone turnover rate. Oral estrogen replacement with CEE at doses similar to those taken by women leads to significantly increased BMC and BMD in monkeys, even when therapy is begun 2 years after ovariectomy. Most of the increase occurred during the first 16 months of treatment. The addition of MPA to the CEE regimen provided no additional benefit.

Bovine achondrogenesis: evidence for defective chondrocyte differentiation.

A survey study of growth cartilage abnormalities in bovine bone dysplasias revealed that a disorder in Holstein cattle called bulldog calf closely resembles human achondrogenesis Type II. Substantial amounts of Type I collagen and other non Type II collagens were detected in the bulldog cartilage which was comprised primarily of extensive vascular canals and cells having the characteristics of hypertrophic and degenerative chondrocytes normally found in the growth plate. It is proposed that chondrocytes throughout the bulldog growth cartilage prematurely differentiate into hypertrophic cells that degenerate and predispose the cartilage to vascular invasion and the formation of cartilage canals. The presence of these canals probably accounts for most of the observed collagen abnormalities.

The effects of hormone replacement therapy on carbohydrate metabolism and cardiovascular risk factors in surgically postmenopausal cynomolgus monkeys.

Controversy exists regarding the effects of estrogen and estrogen/progestin replacement therapies on glucose tolerance and insulin resistance. Also unknown are whether changes in glucose tolerance and insulin resistance with hormone therapy affect arterial glycation and atherosclerosis. We studied ovariectomized female monkeys fed a lipid-lowering diet and given either no hormone replacement therapy (n = 25) or conjugated equine estrogens (CEE) alone (n = 22) or combined with medroxyprogesterone acetate ([MPA] n = 21) for 30 months. Monkeys receiving combined hormone replacement had significantly higher fasting glucose and insulin levels and higher insulin responses to a glucose challenge compared with controls or those given estrogen alone. Monkeys given estrogen-only therapy had lower body weights, lower measures of abdominal adiposity, and decreased serum androgen concentrations. However, due to the effective dietary lipid decrease, there was no additional effect of hormone treatment on atherosclerosis. Also, there was no correlation between either arterial glycation or insulin levels and atherosclerosis extent. Thus, although there were adverse effects of combined hormone replacement therapy on carbohydrate metabolism, we were unable to determine whether these effects altered the extent of atherosclerosis.

Osteoarthritis in cynomolgus macaques: a primate model of naturally occurring disease.

The objective of the present study was to determine if naturally occurring osteoarthritis of the knee joints that is similar to the condition in humans develops in cynomolgus macaques. Knee joints from 58 young adult (mean age, 7.4 years) female cynomolgus macaques were studied with x-ray densitometry, high-detail radiography, and histology. The animals studied were subjects in a triad designed to examine the effects of the administration of sex steroids on atherosclerosis; except for a control group, the monkeys had been either ovariectomized or treated with sex steroids for 2 years. Therefore, the data were analyzed to determine if these treatments, both of which can influence bone density, affected the severity of osteoarthritis. There was a high prevalence of osteoarthritic lesions, morphologically similar to those seen in humans. Bone changes were more common and severe than cartilage changes and morphologically appeared to precede the cartilage changes. Treatment with testosterone resulted in increased body weight, body mass index, and bone mineral content in the femur and tibia but did not affect the severity of osteoarthritis. These data indicate that naturally occurring osteoarthritis developed in the knee joints of cynomolgus macaques; these animals may be a useful model for the study of osteoarthritis in humans.

Effects of an oral contraceptive combination with or without androgen on mammary tissues: a study in rats.

OBJECTIVES: Oral contraceptive (OC) therapy has long been known to produce hypoandrogenemia. However, androgens are not part of any OC therapy available to women. This project was designed to evaluate the effects of low-estradiol containing OC, with or without methyltestosterone (MT), on cell proliferation and progesterone receptor (PgR) expression in mammary gland epithelia of virgin female rats. METHODS: Sixty rats were divided into four groups. One group received OCs, whereas a second group received OC plus MT. A third group of rats was treated with an antiandrogen to mimic the hypoandrogenemic effects caused by OC therapy. All treated groups were compared with age-matched untreated controls. RESULTS: After 15 weeks of treatment, no inflammatory, precancerous, or cancerous lesions were observed in any treatment group. OC plus MT therapy caused significant suppression of epithelial proliferation, a reduction in the number of proliferating cell nuclear antigen-labeled cells, and an increase in the number of PgR-labeled cells. CONCLUSIONS: Our results suggest that a medication containing an estrogen-progestin-androgen combination has antiproliferative effects in mammary glands of experimental animals that could prove to have breast-protective potential in women.

Uterine infarctions in cynomolgus monkeys (Macaca fascicularis).

Uterine infarctions have not been reported in domestic animals, and there are few reports in the human medical literature. In a retrospective study, uterine infarctions were identified in 9 of 323 (2.8%) female cynomolgus monkeys (Macaca fascicularis) necropsied over a 13-year period. The infarctions were grossly visible, after fixation, on the serosal surface of the uterus in 2 monkeys; the remainder were first recognized in histologic sections. Histologically, the lesions consisted of well-demarcated regions of endometrial and myometrial necrosis and of hemorrhage. All affected monkeys had histologic evidence of a previous pregnancy, which included enlarged myometrial vessels with an expanded perivascular matrix. In all monkeys with uterine infarctions, there was clinical evidence of severe systemic illness, which included trauma, diarrhea, hypovolemia, or septicemia. The major pathologic findings in affected monkeys included cutaneous or skeletal muscle necrosis (n = 5), enterocolitis (n = 4), pulmonary edema or diffuse alveolar damage (n = 3), and intestinal amyloidosis (n = 1). Histopathologic evidence of intravascular fibrin thrombi in multiple organs of 5 monkeys was consistent with a diagnosis of disseminated intravascular coagulopathy (DIC). Based on these findings, it appears that uterine infarction is an uncommon finding in cynomolgus monkeys and may occur secondary to a severe systemic illness, predisposing to DIC.

Psoriatic plaques in Macaca fascicularis.

An adult male cynomolgus monkey (Macaca fascicularis) developed erythematous plaques with adherent white scales on the skin. Microscopically, the epidermis had regular acanthosis with marked parakeratosis, focal absence of stratum granulosum, and spongiform pustules and Munro-like microabscesses. These histologic findings were suggestive of psoriasis vulgaris.

Oral contraceptive treatment inhibits the normal acquisition of bone mineral in skeletally immature young adult female monkeys.

The purpose of the present study was to determine the effects of oral contraceptive therapy on bone density and serum markers of bone metabolism in a prospective, longitudinal study of young adult female cynomolgus monkeys. Two hundred and seven intact cynomolgus monkeys were randomized to two groups, and fed an atherogenic diet containing either no drug (Control) or a triphasic oral contraceptive regimen (Contraceptive). Measurements of bone density were carried out by dual-energy X-ray absorptiometry at 10-month intervals (0, 10, and 20 months) and serum bone biomarkers were determined at 5-month intervals over the 20-month time course. No significant differences in these variables were observed prior to treatment. Both groups of animals gained bone mineral during the study, indicating that peak bone mass had not been reached at baseline. Contraceptive-treated animals gained less spinal (lumbar vertebrae 2-4) bone mineral content and density and less whole-body bone mineral content than Controls over the course of the study. Significant depressive effects of contraceptive treatment on gains in BMC and BMD were observed during each 10-month interval of the study. Bone metabolism was inhibited in the Contraceptive group, as reflected by marked reductions (approximately 40%) in serum osteocalcin and alkaline phosphatase levels along with moderate reductions in serum acid phosphatase and calcium. The results suggest that triphasic oral contraceptive treatment of young adult female monkeys that have not reached peak bone mass inhibits net bone accretion and/or growth by reducing bone metabolism. Thus, prolonged continuous oral contraceptive use in skeletally immature females may lead to a lower peak bone mass--an effect which could increase the risk of fractures in later life.

Maternal factors affecting reproduction in a breeding colony of cynomolgus macaques (Macaca fascicularis).

A breeding colony of cynomolgus macaques (Macaca fascicularis), composed of imported and colony-born animals and established for 9 years, was evaluated for maternal factors associated with reproductive failure. The factors evaluated included age, gravidity, parity, origin, previous stillbirths, clinical incidents and type of housing. The effects of each factor on pregnancy rate (PR), stillbirth rate (SR), infant mortality rate (IMR) and pregnancy success (PS) were evaluated. The overall colony rates were: PR = 53%, SR = 22%, IMR = 22%, and PS = 60%. Neonatal death rate for the group was 12%. Pregnancy rate was most affected by maternal factors. Clinical incidents occurring during pregnancy were associated with a significant increase in the stillbirth rate, but did not affect infant mortality rate. Maternal age did not affect any of the measures of reproductive output. Pregnancy rate peaked at 6-8 years of age and decreased thereafter, while pregnancy success peaked at 9-11 years of age. Gravidity and parity had a positive linear relationship with pregnancy rate.

The effect of androstenedione/estrone supplementation on cortical and cancellous bone in the young intact female monkey: a model for the effects of polycystic ovarian disease on the skeleton?

The goal of this study was to determine the effects of chronically elevated blood androstenedione and estrone levels on the quality and quantity of both cancellous (trabecular) and cortical bone in a young (mean age 9.4 years) female primate model (M. fascicularis). Thirteen intact female monkeys received continuous androstenedione/estrone supplementation via subcutaneous implants over a 24-month period to simulate the human condition known as polycystic ovarian disease (PCOD). A group of 16 untreated intact age-matched female monkeys served as controls. Lumbar spine and whole body bone mineral density (BMD) status was determined mid-study by dual photon absorptiometry (DPA); subsequent analysis of the bone related to data obtained following the 2-year treatment period without further BMD measurement. Bone markers, including serum acid phosphatase, total bone alkaline phosphatase, bone gla protein and tartrate-resistant acid phosphatase were measured at the end of the study. At necropsy, the lumbar vertebrae and femora were recovered in order to analyze the bone mineral quality and quantity of cancellous and cortical bone respectively and to compare these with the control group. Mineralization profiles of the vertebrae and femora were obtained using the density fractionation technique. Chemical analysis of the three largest fractions retrieved by density fractionation was performed to evaluate differences in %Ca, %P, Ca/P ratio and mineral content (%Ca + %PO4) between control and experimental groups. In addition, unfractionated bone powder was examined by X-ray diffraction to identify any changes in crystal size. Coronal sections of vertebrae were analyzed for structural parameters using histomorphometry and image analysis. Cross-sections taken at the midshaft diaphyseal femora were analyzed for structural macroscopic and intracortical parameters. There was a significant increase in BMD at the L2-L4 region in the treatment group compared with the control groups (p < 0.005) as measured at 1 year into the trial. Serum acid phosphatase was significantly lower (p < 0.05) in the treatment group compared with the controls near study termination. A nonsignificant shift in the mineralization profile of the vertebrae towards less dense bone was observed in the treatment group, while there was a significant shift in the mineralization profile towards more dense bone in the treated femora compared with controls (p < 0.05) after a 2-year period. There was no difference between treatment and control groups in terms of size/strain of the cortical or cancellous bone crystal as detected by X-ray diffraction. There was a significant increase in cancellous bone area (B.Ar.) (p < 0.02) and a significant increase (p < 0.05) in mean trabecular width with a corresponding decrease in trabecular separation (p < 0.03) in the experimental group compared with the controls. There were no significant changes in osteoid parameters (perimeter, area or width) or eroded perimeter measurements in the experimental group compared with the controls. In the experimental group, trabecular strut analysis showed a significant increase in the number of nodes (p < 0.02) and in the total strut length (p < 0.003) compared with the controls. There was also a significant increase in the node to node (p < 0.04) and node to terminus (p < 0.004) strut length in the treatment group compared with the controls. A significant increase in B.Ar. without concurrent indices of ongoing remodelling differing from controls suggests that cancellous bone of the vertebral body in the treated young female primate had been receptive to the anabolic stimulus of androstenedione/estrone supplementation over the 2-year period. In contrast, macroscopic parameters of cortical bone such as perimeter, area and width were preserved over the 2-year course, while intracortical remodeling was evident with increased percent porosity (p < 0.001), osteonal bone (p < 0.01) and osteonal density (p < 0.01) observed in the treatment group compared with the controls. The endocrine profile of both elevated androstenedione and estrone levels in an intact female primate of reproductive age may identify differential effects of the condition known as polycystic ovarian disease on the skeletal compartments.

Effects of oestrogens and progestogens on coronary atherosclerosis and osteoporosis of monkeys.

We have used the cynomolgus macaque as a model for the study of the effects of endogenous and exogenous sex steroid hormones on atherosclerosis and osteoporosis. As in human beings, premenopausal female cynomolgus macaques develop much less extensive coronary artery atherosclerosis than their male counterparts. Furthermore, surgical menopause results in a more atherogenic plasma lipoprotein pattern and an approximate doubling of atherosclerosis extent. Frequent pregnancy, a hyperoestrogenic state, results in an approximate 50% reduction in atherosclerosis extent. Physiological replacement with 17 beta-oestradiol alone or in combination with progesterone prevents the increase in coronary artery atherosclerosis extent associated with ovariectomy. This effect is independent of plasma lipoprotein concentrations and appears to be accounted for, at least in part, by an inhibitory effect of oestrogen replacement therapy on the uptake and degradation of LDL by the artery wall. Also, as in human beings, treatment with certain types of combination oral contraceptives results in marked decreases in plasma HDL-C concentration. Nonetheless, coronary artery atherosclerosis extent is reduced in monkeys by oral contraceptive treatment, and this effect is most pronounced among animals at highest risk due to theoretically adverse plasma lipoprotein profiles. It appears that, as with oestrogen replacement therapy, this effect can be accounted for, at least in part, by an inhibition of the uptake and degradation of low density lipoprotein by the artery wall. The monkey also appears to be a good model for studies of postmenopausal bone loss. As in women, surgical menopause results in significant diminution of bone mineral density and bone mineral content. Also, serum biomarkers of bone turnover (total alkaline phosphatase, acid phosphatase, tartrate-resistant acid phosphatase and osteocalcin) are increased in surgically postmenopausal monkeys, indicating increased bone turnover resulting from the surgical menopause. These increases in bone loss and indices of bone turnover were prevented by physiological oestrogen replacement therapy. Cynomolgus monkeys seem to be exceptionally useful models for studies of the effects of sex steroid hormones on atherosclerosis and osteoporosis, two major public health problems in postmenopausal women.

Gestational diabetes mellitus in a cynomolgus monkey with group A streptococcal metritis and hemolytic uremic syndrome.

Gestational diabetes mellitus was diagnosed retrospectively in a ten-year-old female cynomolgus monkey. The mother developed severe purulent group A streptococcal metritis resulting in fetal sepsis. After parturition, the mother died from signs consistent with hemolytic uremic syndrome.

Accuracy and reproducibility of lumbar bone mineral status determined by dual photon absorptiometry in live male cynomolgus macaques (Macaca fascicularis).

Dual photon absorptiometry (DPA) was used to determine the in vivo bone mineral content (BMC) and bone mineral density of lumbar vertebrae (L2-4) in feral adult male cynomolgus macaques (Macaca fascicularis, n = 20). Following in vivo DPA scans, all animals were euthanized and the lumbar spine segment excised. The excised lumbar vertebrae, plus spinal cord, were formalin-fixed and measured three times by ex vivo DPA. The first ex vivo scan matched the monkey's own in vivo soft tissue beam attenuation ratio (Rs, ranging from 1.38 to 1.45). The second ex vivo scan was made creating a constant obese-like condition (Rs = 1.38), and the third creating a constant lean-like condition (Rs = 1.45). All scans were taken at a 1.0 mm point resolution, 1.0 mm line spacing, a 6 cm width, and a scan speed of 5 mm/sec. The second lumbar vertebra (L2) was ashed and the ash weight was compared to the measured L2BMC of the in vivo and ex vivo DPA analyses. Noninvasive in vivo DPA proved to be adequate in measuring the lumbar bone mineral content in male cynomolgus macaques. Ex vivo DPA at a high and constant Rs value of 1.45 provided for the best accuracy of formalin-fixed wet bone tissue when compared to the bone ash weight.

Effects on bone of surgical menopause and estrogen therapy with or without progesterone replacement in cynomolgus monkeys.

The influence of estrogen replacement therapy on bone loss of surgically postmenopausal cynomolgus macaques was evaluated histomorphometrically using the first (L-1) lumbar vertebra and ex vivo dual photon absorptiometry of the third (L-3) lumbar vertebra. The animals were a subgroup of a larger study on the effects of estrogen replacement therapy on diet-induced coronary artery atherosclerosis. The three experimental conditions were as follows: untreated females with oophorectomy, females with oophorectomy treated with continuous estrogen replacement therapy plus cyclic progesterone, and females with oophorectomy treated with estrogen replacement therapy. Bone mineral density (grams per square centimeter) of L-3, when covaried for body mass index (body mass index, body weight/(trunk length/100)2), was significantly lower for the oophorectomy group compared with the group treated by estrogen replacement therapy plus progesterone and estrogen replacement therapy groups (p = 0.018). When covaried for body mass index, trabecular bone volume percentage of a midsagittal section of L-1 was not significantly different between groups, but the adjusted mean was greatest in the estrogen replacement therapy plus progesterone group, followed closely by the estrogen replacement therapy group, and was least in the oophorectomy group. When covaried for body mass index, trabecular plate number was significantly lower (p = 0.022) and mean trabecular plate separation was significantly higher (p = 0.033) in the oophorectomy group. Thus both estrogen replacement therapy and estrogen replacement therapy plus progesterone provided overall protection against surgical menopause--associated bone mass loss. Cynomolgus macaques are an extremely useful animal model for estrogen replacement therapy use in prevention of postmenopausal bone loss.

Cerebral infarction in two cynomolgus macaques (Macaca fascicularis) with hypernatremia.

Hypernatremia resulting in neurologic symptoms ranging from lethargy to coma, and with underlying lesions of cerebral hemorrhage and thrombosis, has been reported in human beings. Herein we report two cases of cerebral infarction with venous thrombosis in cynomolgus monkeys. Both animals were severely hypernatremic because of water deprivation, with serum sodium levels of 185 and 193 meq/liter, respectively. At necropsy, there were bilateral multiple hemorrhagic and malacic areas visible on the surface of the cerebrum and extending into the parenchyma, primarily involving the occipital lobes. These lesions were interpreted microscopically as infarcts because, in addition to hemorrhage and necrosis, multiple thrombi were present in small and medium-sized veins of gray matter and meninges. The pathogenesis of hypernatremia-induced cerebral lesions is believed to involve cellular dehydration that caused shrinkage of the brain. Because the vasculature of the brain is tightly adherent to the skull, this shrinkage results in tearing of blood vessels, with consequent hemorrhage and thrombosis.