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.

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.

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.

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.

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.

Changes in bone turnover during the menstrual cycle in cynomolgus monkeys.

It is well established that estrogen deficiency at menopause results in increased bone turnover, which is reflected in increased concentrations of markers of bone formation and bone resorption in serum and urine. Since serum 17beta-estradiol concentrations vary markedly throughout the menstrual cycle, one would expect to see changes in bone turnover as well. Studies in humans have not yielded consistent results, perhaps because of differences in diet and activity throughout the test period. Therefore, we examined changes in bone biomarkers throughout the menstrual cycle in cynomolgus macaques. Seven intact female cynomolgus macaques (Macaca fascicularis) were evaluated. Vaginal swabs for menstrual blood were performed 3 times/week to determine the stage of the reproductive cycle. Blood and urine were collected at weekly or biweekly intervals for a total of eight samples per monkey for analysis of serum 17beta-estradiol, progesterone, parathyroid hormone (PTH), osteocalcin, bone-specific alkaline phosphatase (BSAP), and urinary CrossLapstrade mark. Cycle lengths were determined, and collection days were adjusted to a standardized length of 28 days for all monkeys. Values for bone biomakers were evaluated as % mean for each monkey cycle. By fitting the data to a sine wave (cosinor analysis), serum osteocalcin, BSAP, and urinary CrossLaps demonstrated significant cycles with peaks at days 2.6, 7.3, and 27.8, respectively. Serum osteocalcin and urinary CrossLaps were inversely correlated to serum 17beta-estradiol. Urinary CrossLaps were significantly lower in the week just prior to and during ovulation when estradiol was elevated. No rhythm was detected in serum PTH. A peak in bone resorption when serum 17beta-estradiol is at its nadir is consistent with the hypothesis that estrogen decreases bone turnover.

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.

Toxicology and pharmacokinetics of DTGM, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor, in cynomolgus monkeys.

We developed a fusion toxin consisting of the catalytic and translocation domains of diphtheria toxin linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (DTGM) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the toxicity and pharmacokinetics of DTGM in cynomolgus monkeys (Macacca fascicularis), which possess cross-reactive GM-CSF receptors. Four groups of young adult monkeys (6 males and 12 females) were treated with five daily bolus iv infusions of 1, 5, 7.5, and 10 microgram/kg DTGM. Monkeys (2 males and 2 females) treated at 1 microgram/kg/day showed no significant side effects. Monkeys (2 males and 2 females) treated at 5 microgram/kg/day showed Grade 1-2 thrombopenia (NCI common toxicity criteria) on day 9. In contrast, monkeys (6 females) treated at 7.5 microgram/kg/day developed Grade 3 neutropenia, Grade 1-2 thrombopenia, Grade 1-3 anemia, and Grade 1-3 hypoalbuminemia. The neutropenia developed by day 4 in the 7.5 microgram/kg/day monkeys and by day 3 or 5 in the 10 microgram/kg/day monkeys and resolved in both groups by day 9, but the thrombopenia, anemia, and hypoalbuminemia persisted until day 16. Monkeys (2 male and 2 female) treated with 10 microgram/kg/day showed Grade 4 neutropenia that resolved by day 8 and Grade 2-3 anemia, hypoalbuminemia, and thrombopenia. Three of the animals developed sepsis. DTGM plasma half-life was 30 min with a peak concentration of 0.1 microgram/mL or 2 nM (1000-fold higher than the IC50 in vitro for AML blasts). Immune responses were minimal in all animals tested at 14 and 28 days with anti-DTGM levels <1 microgram/mL. All four animals at 10 microgram/kg died or were euthanized, and necropsies were performed. Animals necropsied on days 4 and 6 showed marked apoptosis and hypoplasia in the marrow, which was completely resolved for animals necropsied on day 9. No injury to other organs, including kidney, heart, liver, central nervous system, or lung, was seen. The drug was selectively toxic to malignant or differentiated myeloid cells with little toxicity to myeloid progenitors or other organs. Minimal effects in nontarget tissues make DTGM a promising candidate chemotherapeutic agent.

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.

Evaluation of a nonhuman primate model to study circadian rhythms of calcium metabolism.

We evaluated primate models for the study of circadian rhythms in calcium and bone metabolism. Blood and urine were collected from two cynomolgus macaques every 4 h for 24 h. Studies were initiated at three different clock times to separate the effects of repeated experimental sampling from circadian effects. Also, samples were collected from seven monkeys at times of expected maxima and minima. Some parameters exhibited the expected circadian rhythm with increases at night (serum total calcium) or in the early morning (urinary collagen cross-links). Others displayed the effects of the experimental procedure, either increasing (urinary creatinine and phosphorus) or decreasing (osteocalcin, urinary calcium) with repeated sampling. Serum phosphorus, cortisol, and type I procollagen were influenced by both clock time and experimental procedures. Alkaline phosphatase and parathyroid hormone did not show any differences with time or sampling. This data is consistent with findings in humans that bone resorption increases at night and that endogenous corticosteroids decrease bone formation. The usefulness of the monkey model is limited by the physiological stress of sample collection in these subjects.

Proliferative enteropathy of rabbits: the intracellular Campylobacter-like organism is closely related to Lawsonia intracellularis.

The intracellular Campylobacter-like organism associated with proliferative enteropathy of rabbits is closely related to Lawsonia intracellularis, the primary pathogen in porcine intestinal adenomatosis. Polymerase chain reaction primers based on the 16S rRNA gene of L. intracellularis were used to amplify DNA harvested from intestinal tissues of rabbits with severe proliferative intestinal lesions containing curved argentophilic intracellular bacteria. Sequencing of a 180-nucleotide DNA fragment of the 550-base pair-amplified polymerase chain reaction product revealed >98% similarity between the organism associated with the rabbit disease and the homologous sequence found in L.intracellularis.

Evaluation of cecal ligation as a model of mucoid enteropathy in specific-pathogen-free rabbits.

Mucoid enteropathy is a serious disease of rabbits, the cause of which is unknown. Ligation of the cecum has been reported to cause a mucoid enteropathy-like syndrome in 70% of conventional rabbits. The goal of this study was to evaluate this model of mucoid enteropathy in Pasteurella-free, coccidia-free rabbits for use in future studies. Five rabbits served as unoperated controls (group 1). Eight rabbits underwent ligation of the cecum, with large vessels and nerves spared (group 2). In six rabbits the distal branch of the ileocecocolic vessels and nerve were incorporated into the cecal ligature (group 3). At necropsy 3 to 5 days after surgery, all group-3 rabbits had copious amounts of clear, gelatinous mucus in the colon. Only one group-2 rabbit had grossly evident mucus hypersecretion, whereas none of the group-1 rabbits did. Group-3 rabbits had areas of necrosis in the cecum; this was not seen in group-1 or group-2 rabbits. Rabbits of groups 2 and 3 had inflammation of the distal portion of the colon. In specific-pathogen-free rabbits cecal ligation alone did not reliably stimulate mucus hypersecretion but induced a disease similar to natural mucoid enteropathy. Cecal ligation including vessels provided a reproducible syndrome of mucus hypersecretion; however, the severe cecal necrosis was not consistent with the naturally acquired disease.

Mucus secretagogue activity in cecal contents of rabbits with mucoid enteropathy.

Cecal filtrates from rabbits with cecal ligation-induced mucoid enteropathy have been reported to cause goblet cell hyperplasia in intestinal explants. This study was performed to see whether such filtrates would stimulate mucus secretion from intestinal explants. Filtrates were prepared from cecal contents of five control rabbits, five rabbits that had undergone cecal ligation, and five rabbits that had had the distal branch of the ileocecocolic vessels and nerve incorporated into the cecal ligature. After incubation of each with ileal and colonic explants from five healthy rabbits, the amount of mucus in the media/filtrate solution was measured with an enzyme-linked lectin assay. Significantly (P < 0.05) more mucus was secreted by colonic explants in the presence of cecal filtrates from either of the cecal ligation groups compared with controls. With pooled filtrates from each group, these results were confirmed by measuring secretion of radiolabeled mucus from explants that had been preincubated with [3H]glucosamine. The secretagogue activity was found to be precipitable by 85% ammonium sulfate and destroyed by heat treatment (100 degrees C for 30 min) or acid treatment (pH 1.0 for 30 min). Very little, if any, secretagogue activity was present in cecal vein serum. The results support the hypothesis that the cecal contents of rabbits with cecal ligation-induced mucoid enteropathy stimulate mucus secretion from colonic explants taken from healthy rabbits.

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.