Resources for genetic management and genomics research on non-human primates at the National Primate Research Centers (NPRCs).

The National Primate Research Centers (NPRCs) established Working Groups (WGs) for developing resources and mechanisms to facilitate collaborations among non-human primate (NHP) researchers. Here we report the progress of the Genome Banking and the Genetics and Genomics WGs in developing resources to advance the exchange, analysis and comparison of NHP genetic and genomic data across the NPRCs. The Genome Banking WG has established a National NHP DNA bank comprising 1250 DNA samples from unrelated animals and family trios from the 10 NHP species housed within the NPRC system. The Genetics and Genomics WG is developing SNP arrays that will provide a uniform, highly informative, efficient and low-cost method for rhesus and long-tailed macaque genotyping across the eight NPRCs. This WG is also establishing a Biomedical Informatics Research Network-based portal for shared bioinformatics resources including vital statistics, genotype and population data and information on the National NHP DNA bank.

The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex.

The anesthetic gas nitrous oxide (N2O) and the volatile anesthetic isoflurane (ISO) are commonly used in surgical procedures for human infants and in veterinary and laboratory animal practice to produce loss of consciousness and analgesia. Recent reports indicate that exposure of the developing brain to general anesthetics that block N-methyl-D-aspartate (NMDA) glutamate receptors or potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. In the present study, the question arises whether a relatively low dose of ISO alone or its combination with N2O entails significant risk of inducing enhanced apoptosis. In addition, the role of L-carnitine to attenuate these effects was also examined. Postnatal day 7 (PND-7) rat pups were exposed to N2O (75%) or a low dose of ISO (0.55%) alone, or N2O plus ISO for 2, 4, 6 or 8 h with or without L-carnitine. The neurotoxic effects were evaluated 6 h after completion of anesthetic administration. No significant neurotoxic effects were observed for the animals exposed to N2O or ISO alone. However, enhanced apoptotic cell death was apparent when N2O was combined with ISO at exposure durations of 6 h or more. Co-administration of L-carnitine (300 or 500 mg/kg, i.p.) effectively protected neurons from the anesthetic-induced damage. These data indicate that 6 h or more of inhaled anesthetic exposure consisting of a combination of N2O and ISO results in enhanced neuronal apoptosis, and L-carnitine effectively blocks the neuronal apoptosis caused by inhalation anesthetics in the developing rat brain.

Technique for culturing Macaca mulatta peripheral blood lymphocytes for fluorescence in situ hybridization of whole chromosome paints.

The rhesus monkey (Macaca mulatta) has long been an important model in biomedical and behavioral research. The biomedical importance of M. mulatta is due to its 93% genetic similarity with humans and its complex social behavior. The recent sequencing of the M. mulatta genome has enhanced its role in biological research. However, the use of the macaque as an experimental model in cytogenetic assays has been problematic due to difficulties in obtaining large numbers of well-spread cells in metaphase without the use of extremely toxic mitogens such as staphylococcal enterotoxin A (SEA). Here we describe a technique for culturing and producing sufficient numbers of cells in metaphase using the common mitogens phytohemagglutinin (PHA), concanavalin A (ConA), and T-cell growth factor (TCGF) which act synergistically to induce M. mulatta T-lymphocyte division. Using this method we have obtained a mitotic index in 48 h cultures of 12.0+/-2.2 metaphase cells/100 cells (n=5 animals). Fluorescence in situ hybridization with whole chromosome painting of M. mulatta cells was performed with human whole-chromosome probes that labeled the following chromosomes for human (M. mulatta): 1(1), 2q(12), 2p(13), 4(5) pairs in red, and 3(2), 5(6) and 6(4) pairs in green. In humans this probe combination simultaneously paints 3 chromosome pairs in red and 3 in green, whereas in M. mulatta 4 chromosome pairs are labeled in red and 3 pairs are labeled in green. Using this method we show a baseline frequency of 0.026 translocations per 100 whole-genome cell equivalents in peripheral blood lymphocytes obtained from unexposed adolescent non-human primates. This method will add to the usefulness of M. mulatta as an animal model in biomedical research.

Daily treatment with human recombinant parathyroid hormone-(1-34), LY333334, for 1 year increases bone mass in ovariectomized monkeys.

PTH stimulates bone formation to increase bone mass and strength in rats and humans. The aim of this study was to determine the skeletal effects of recombinant human PTH-(1-34) [rhPTH-(1-34)] in monkeys, as monkey bone remodeling and structure are similar to those in human bone. Adult female cynomolgus monkeys were divided into sham-vehicle (n = 21), ovariectomized (OVX)-vehicle (n = 20), and OVX groups given daily s.c. injections of rhPTH-(1-34) at 1 (n = 39) or 5 (n = 41) microg/kg for 12 months. Whole body bone mineral content was measured, as was bone mineral density (BMD) in the spine, proximal tibia, midshaft radius, and distal radius. Serum and urine samples were also analyzed. rhPTH-(1-34) treatment did not influence serum ionized Ca levels or urinary Ca excretion, but depressed endogenous PTH while increasing serum calcitriol levels. Compared to that in the OVX group, the higher dose of rhPTH-(1-34) increased spine BMD by 14.3%, whole body bone mineral content by 8.6%, and proximal tibia BMD by 10.8%. Subregion analyses suggested that the anabolic effect of rhPTH-(1-34) on the proximal tibia was primarily in cancellous bone. Similar, but less dramatic, effects on BMD were observed with the lower dose of rhPTH-(1-34). Daily s.c. rhPTH-(1-34) treatment for 1 yr increases BMD in ovariectomized monkeys without inducing sustained hypercalcemia or hypercalciuria.

Use of peripheral quantitative computed tomography for densitometry of the femoral neck and spine in cynomolgus monkeys (Macaca fascicularis).

Quantitative computed tomography (QCT) allows for the separate densitometric examination of cortical and cancellous bone in vivo. With the new peripheral QCT (pQCT) instrument (the Norland/Stratec XCT-3000A), we evaluated the clinically relevant axial sites of spine and femoral neck in nonhuman primates in vivo. The reproducibility was good (coefficient of variation [CV] <3% at both sites for cortical, trabecular, and total bone mineral density [BMD]; CV 3%-7% for bone mineral content [BMC] and cross-sectional bone area). One hundred sixty intact female cynomolgus monkeys (M. fascicularis) were scanned at the femoral neck. There was less variability among monkeys in cortical BMD (mean 802 mg/mL, CV 6%) as opposed to trabecular BMD (mean 334 mg/mL, CV 28%) or transition zone BMD (mean 457 mg/mL, CV 12%). Scans were performed on lumbar vertebrae (L-4, L-5, and L-6) from five monkeys in vivo and ex vivo. Removal of soft tissue increased measured BMD. Decreasing voxel size from 0.4 mm to 0.2 mm increased measured BMD by diminishing the partial volume effect. Factor analysis demonstrated the expected relationships between pQCT parameters and physical measurement of bone mass and volume ex vivo. Preliminary results in eight ovariectomized and eight reproductively intact monkeys revealed a lower transition zone BMD at the femoral neck, and lower total BMD of the vertebral body in estrogen-deficient animals.

The anesthetic isoflurane decreases ionized calcium and increases parathyroid hormone and osteocalcin in cynomolgus monkeys.

The effects of anesthetics on calcium metabolism in cynomolgus monkeys were studied. Eight adult female cynomolgus monkeys were used in a crossover design. Blood was collected from each of the monkeys at four timepoints: (1) while conscious; (2) following induction of anesthesia with ketamine, ketamine and atropine, isoflurane, or no anesthetic; (3) at 30 min; and (4) 120 min thereafter. Four experiments were performed with a 1 week washout period between sessions, such that each monkey received each treatment. Potassium was lower in anesthetized monkeys than in those that remained conscious. Cortisol, although high, did not differ among anesthetic treatments. Ketamine and ketamine/atropine did not consistently affect ionized calcium or parathyroid hormone (PTH) concentrations. Isoflurane decreased ionized calcium (0.05 mmol/L), and increased PTH and osteocalcin twofold. The serum inorganic fluoride concentration was higher in monkeys anesthetized with isoflurane than with ketamine/atropine, which may partially account for the decrease in ionized calcium with isoflurane. The increases in PTH and osteocalcin are presumably secondary to the decrease in ionized calcium.

Levormeloxifene prevents increased bone turnover and vertebral bone loss following ovariectomy in cynomolgus monkeys.

Levormeloxifene, a nonsteroidal selective estrogen receptor modulator (SERM), has been evaluated for its effects on bone in cynomolgus monkeys (Macaca fascicularis). Adult female monkeys were imported from Indonesia and randomized into six groups of 25-28 animals each (n = 158). Animals in one group were sham ovariectomized (sham) and received vehicle. Animals in the remaining five groups were ovariectomized and received either vehicle (ovx); 17beta-estradiol at 0.016 mg/kg (est); or levormeloxifene at 0.5 (L1), 1 (L2), or 5 (L3) mg/kg. Lumbar spine and whole body bone mass were measured by dual-energy X-ray absorptiometry (DXA) pretreatment and at 6 and 12 months following the initiation of treatment. Bone mass at the femoral neck was measured by peripheral quantitative computed tomography (pQCT) at 0 and 12 months. Serum markers of bone turnover, including bone-specific alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP), and urinary collagen C-terminal extension peptides (CrossLaps), were measured at 0, 6, and 12 months. Ovariectomy resulted in an increase in these markers; the increase was prevented by estradiol or levormeloxifene. Estradiol or levormeloxifene inhibited loss of lumbar spine bone mineral density (BMD) following ovariectomy compared with untreated monkeys (ovx -5.0%; sham -0.4%; est +0.2%; L1 -3.6%, L2 -2.0%, L3 -2.5%). Estradiol, but not levormeloxifene, prevented loss of BMD at the femoral neck (ovx -7.4%; sham -3.1%; est -3.6%; L1 -8.0%, L2 -6.5%, L3 -7.8%), and whole body bone mineral content (BMC) (ovx -7.6%; sham -1.9%, est -2.9%; L1 -6.2%, L2 -6.1%, L3 -6.7%). Bone loss at each site was correlated with bone turnover as measured by serum and urine biomarkers. There was no dose effect of levormeloxifene.

Effect of surgical removal of subcutaneous tumors on survival of rats.

Mammary and other subcutaneous tumors were surgically removed from aged Sprague-Dawley rats in an attempt to extend survival. The surgical technique was straightforward, and survival following mastectomy was good (17/21). The number of mammary and pituitary tumors in sexually intact rats and those that had previously undergone ovariectomy were compared. The frequency of mammary tumors was significantly lower in ovariectomized vs sexually intact rats (2/47 vs 24/49), as was the frequency of pituitary adenomas (2/46 vs 27/41). Survival to 630 days of age was higher in ovariectomized than in sexually intact rats (42/47 vs 29/49), although tumors did not contribute significantly to mortality.

The effects of chronic methylphenidate administration on operant test battery performance in juvenile rhesus monkeys.

Methylphenidate (MPH) is an amphetamine derivative widely prescribed for the treatment of attention deficit-hyperactivity disorder. Recent concern over its genotoxic potential in children [11] spurred a study on the effects of chronic MPH treatment in a nonhuman primate population and the studies reported here were conducted in conjunction with that study in the same animals. Here, the focus was on the ability of juvenile rhesus monkeys to learn how to perform a battery of operant behavioral tasks while being treated chronically with MPH. Performance of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB) was used to quantify the learning of tasks thought to model specific aspects of cognitive function including learning, motivation, color and position discrimination, and short-term memory. The OTB tasks designed to assess these specific behaviors included Incremental Repeated Acquisition (IRA), Progressive Ratio (PR), Conditioned Position Responding (CPR), and Delayed Matching-to-Sample (DMTS), respectively. Juvenile males (n=10/group) pressed levers and press-plates for banana-flavored food pellets. Subjects were treated orally, twice a day, five days per week (M-F) for 66 weeks with escalating doses (0.15 mg/kg initially, increased to 2.5 mg/kg for the low dose group and to 12.5 mg/kg for the high dose group) and tested in OTB tasks 30 to 60 min after the morning dose. The findings indicate that MPH at doses up to 2.5 mg/kg twice per day were well tolerated (performance was no different than controls) but at doses of 12.5 mg/kg twice per day there was a significant decrement in OTB performance, characterized by decreases in both percent task completed and response rates for all tasks. These effects of MPH seem primarily due to decreases in motivation to perform for food, which is not surprising given the well known appetite suppressing effects of amphetamine-like stimulants. Thus, the current data do not strongly suggest cognitive impairments following chronic MPH administration.