食蟹猴血液学及血清生化指标日间节律性变化

目的研究食蟹猴血液学及血清生化指标日间节律性变化,为正确全面评估血液学及血清生化测定结果提供一定的实验事实依据。方法在9：30（am）～10：30（am）、12：30（pm）～13：30（pm）、15：30（pm）～16：30（pm）及19：30（pm）～20：30（pm）4个时间段对12只食蟹猴（雌雄各半）采血。采用全自动血细胞分析仪及血液生化分析仪分别测定血液学及血清生化指标。利用高效液相色谱（HPLC）方法分析血浆中皮质醇含量。结果雌雄食蟹猴下午白细胞数（WBC）及尿素氮（BUN）显著升高;雌性食蟹猴下午血红蛋白（HGB）及血细胞压积（HCT）显著降低,天门冬氨酸氨基转换酶（AST）及丙氨酸氨基转换酶（ALT）活性显著升高;雄性食蟹猴下午血糖（GLU）及血浆皮质醇显著降低。结论雌雄食蟹猴血液学及血清生化部分指标存在明显的日间节律性。     

H5N1接种恒河猴和食蟹猴后外周血细胞变化的分析

目的测定H5N1型禽流感病毒感染恒河猴、食蟹猴后,其外周血细胞的变化,为H5N1模型猴提供基础数据及研究参考。方法健康合格食蟹猴、恒河猴各4只,经滴鼻方式接种H5N1病毒107TCID50,确认发病后,在不同时间点进行血细胞及T淋巴细亚群的分析。结果与接种H5N1病毒前比较,接种后白细胞总数（WBC）在第6天时有所降低,至第9天时回升;红细胞总数（RBC）在第3天有所降低,之后回升;淋巴细胞比例及数量分别在第6天、第9天升高并达到最高值。至第9天时,CD4^＋T细胞数明显高于接种前,CD8^＋T细胞数上升显著,导致CD4^＋/CD8^＋T细胞比例下降,甚至在2只食蟹猴出现了比例倒置。结论实验用猴感染H5N1后,可导致WBC,CD4^＋,CD8^＋T等血液细胞的变化,应作为H5N1模型动物的检测指标。     

The effects of ketamine anesthesia on hematological and serum biochemical values in female cynomolgus monkeys (Macaca fascicularis)

The effects of ketamine anesthesia (15 mg/kg body weight) on hematological and serum biochemical values were examined in six female cynomolgus monkeys (Macaca fascicularis) who were born in the wild. As control, another six female cynomolgus monkeys of the same origin were injected with physiological saline. The white blood cell count, total protein concentration, albumin concentration and calcium concentration decreased after the injection of ketamine, whereas the red blood cell count, hematocrit value, hemoglobin concentration, total cholesterol concentration, free cholesterol concentration, triglyceride concentration, transaminase activities (GOT, GPT) and alkaline phosphatase activity were not affected. A transient increase of the serum glucose level was observed within 10 minutes after ketamine injection. The relationship between these effects of ketamine anesthesia and serum cortisol levels measured by radioimmunoassay was discussed.     

Changes in hematologic values in 11 months after birth in the cynomolgus monkeys

Changes in hematological values were studied with 131 healthy cynomolgus monkeys aged less than 11 months. The parameters measured were erythrocyte count (RBC), hematocrit value (Ht), mean corpuscular volume (MCV), hemoglobin concentration (Hb), total leucocyte count (WBC), and differential leucocyte count. No remarkable change was found with RBC during the whole period of this study. Relatively high values were obtained with Ht, MCV, and Hb in the newborns aged 0 to 7 days. These values continued to decrease until 3 months of age, after which the values increased again attaining approximately adult levels at 11 months of age. WBC was very high at birth and then decreased to the minimum level at 3 days of age. It was followed by gradual increase until about 4 months of age at which a nearly adult level was attained. Lymphocyte counts were smaller than neutrophil counts on the day of birth. However, this numerical relation was inverted 2 days after birth and the lymphocyte counts became markedly larger than the neutrophil counts about 1 week after birth. Additionally, the values obtained from the cord blood of 6 Cesarean-delivered newborns were compared with those from the blood taken 5 hours after cesarean delivery.     

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis)

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.     

The repeated procedure of weaning and peer group formation causes accumulation of stress and changes of plasma cortisol level and natural killer activity in squirrel monkeys (Saimiri sciureus)

The psychological stress was evaluated in repeated and unrepeated procedures of weaning as well as forming peer group in squirrel monkeys. The repeated procedure included the process of increasing the period of separation or formation stepwise during four weeks. The plasma cortisol levels and natural killer (NK) activities were monitored during experiment to evaluate the stress in infant monkeys. The plasma cortisol level rapidly increased two to three times as much as basal level and kept high levels throughout experiment in repeated group. In the infants of unrepeated group, significant increase of cortisol level and decrease of NK activity were observed in day-1, but both of them returned to the basal level at day-7. Both cortisol level and NK activity did not show any change with exception of decrease in NK activity at day-7 in infants who were introduced into peer group without repeated procedure. On the other hand, both cortisol level and NK activity increased during the repeated procedure. These results indicate that both weaning and forming peer group induce the psychological stress in infant squirrel monkeys, resulting in changes of plasma cortisol level and NK activity. Repeating the procedure of separation or introduction applied in this study caused the accumulation of stress. Although plasma cortisol level increased in infants exposed to both weaning and forming peer group, the changing pattern of NK activity differed between them. This finding suggests that social or psychological stress show two different effects on immune function, suppression, and enhancement depending on the level of stress.     

中老年食蟹猴群体自发型糖尿病的筛选

筛选440只中老年偏胖食蟹猴群体中自发糖尿病个体,并探讨食蟹猴群体中糖尿病粗筛的方法。以调查基础血糖值为基础,推断疑似糖尿病血糖值,后经OGTT(口服糖耐量)和尿检结果验证该血糖值是否准确。结果显示中老年偏胖食蟹猴群体血糖值为(3.88±0.98)mmol/L,其中56只食蟹猴血糖值大于5.0mmol/L,被初步定为糖尿病个体。这些个体全部糖耐量异常,且36只(69.23%)出现尿糖阳性,证明血糖值大于5.0mmol/L可作为本群体食蟹猴糖尿病的粗筛标准。由于针对中老年偏胖食蟹猴群体,患病率为12.72%(56/440),高于我国糖尿病患病率(9.7%)。虽然该实验的糖尿病血糖指标并不适用于所有食蟹猴群体,但是该筛选的流程简单快捷,对动物损伤小,可适用于大群体糖尿病的筛选。     

Ratio of leptin to adiponectin as an obesity index of cynomolgus monkeys (Macaca fascicularis).

Obesity is responsible for inducing various metabolic diseases. Laboratory-bred cynomolgus monkeys exhibit spontaneous onset of obesity. However, to date, no blood chemistry index to identify the state of obesity in cynomolgus monkeys has been determined. In the present study, to determine such an index, we measured the serum levels of two adipocyte-derived hormones, leptin and adiponectin, and evaluated the relationship between these hormones and other serum energy metabolic factors (i.e. insulin, total protein, glucose, total cholesterol and triglyceride) as well as the percentage of body fat (%Fat) in mature cynomolgus monkeys. Both in females and males, leptin was positively correlated with insulin and %Fat, and adiponectin was negatively correlated with insulin and %Fat. In female cynomolgus monkeys, leptin, adiponectin, and glucose were selected as the most important determinants for %Fat in multiple regression analysis, and in male cynomolgus monkeys, leptin was selected. The ratio of leptin to adiponectin (L/A ratio) was significantly elevated in the animals with %Fat over 40 (P < 0.01). The results indicate that L/A ratio is a potential index for comprehensively identifying obesity in cynomolgus monkeys.     

Effects of fasting on hematologic and clinical chemical values in cynomolgus monkeys (Macaca fascicularis)

Background Fasting is an important pre‐analytical factor that may affect clinical pathology parameters in toxicological and pharmacological studies. Little information is available on how fasting affects clinical pathology parameters in cynomolgus monkeys (Macaca fascicularis).The aim of this study was to evaluate the influence of fasting on clinical pathology parameters in healthy adult cynomolgus monkeys. Methods Five female and six male cynomolgus monkeys were fasted for 0, 8, 16, and 24 hours. Changes in body weight (BW), core hematologic, and serum clinical chemical parameters were evaluated. Results The BW significantly decreased after 24 hours of fasting. Significant decreases in red blood cell count, hemoglobin, hematocrit, and mean corpuscular volume and increases in mean cell hemoglobin and mean cell hemoglobin concentration were observed at 16 hours in males. In females, increasing the duration of fasting caused a significant time‐dependent increase in platelets. Blood urea nitrogen showed significant decreases in female and male monkeys after fasting. Alkaline phosphatase increased in females after fasting. Aspartate transaminase significantly increased both in females and males at 8 hours. In females, alanine transaminase and lactate dehydrogenase significantly increased at 8 hours. Albumin significantly decreased in males 24 hours, but increased in females 16 hours after fasting. Serum glucose and triglyceride were not affected by fasting. Serum calcium decreased and inorganic phosphorus increased in males after fasting. Conclusion These results suggested that clinical pathology data would vary after fasting. The decision to feed or fast before blood collection for clinical pathology tests should be made based on careful consideration.     

Relationship between body weight and hematological and serum biochemical parameters in female cynomolgus monkeys (Macaca fascicularis).

Obesity is a risk factor triggering a variety of metabolic diseases. Cynomolgus monkeys (Macaca fascicularis) exhibit spontaneous onset of obesity in adulthood, similar to such onset exhibited by humans. To clarify the characteristics accompanying obesity in female cynomolgus monkeys, we used simple and multiple regression analyses to determine the relationship between body weight and hematological and serum biochemical parameters as well as obesity-related hormones, namely, leptin and insulin. Simple regression analysis showed that body weight was significantly (P < 0.05) correlated with leptin level, insulin level, hemoglobin concentration, hematocrit values, mean corpuscular volume, glucose concentration, and triglyceride concentration. In addition, a multiple regression model containing leptin level, insulin level, mean corpuscular volume, and red blood cell count explained 66.9% of the variance in body weight. Therefore, female cynomolgus monkeys show similar obesity characteristics to humans, i.e., obesity is associated with enhanced synthesis and excretion of leptin in adipocytes, high risk of diabetes mellitus, and high levels of hematocytes. Our results indicate that female cynomolgus monkeys are good models for studying obesity in humans.     

The effect of transportation stress on circulating corticosteroids, enzyme activities and hematological values in laboratory dogs.

Stress during transportation was monitored in truck-transported Beagle dogs "IVTK HD". Cortisol (F), corticosterone (B), red and white blood cell parameters, and enzyme activities in plasma were estimated. Plasma levels of cortisol and corticosterone were increased significantly during transportation and returned to basal values overnight post arrival. Hematocrit, hemoglobin, erythrocytes and leukocytes increased moderately. No obvious differences in enzyme activities were observed. Only alpha-HBDH values dropped significantly at the end of the 9.5 h transport period. This study confirms, although individual reaction is remarkably different, transportation as a potent stressor for Beagle dogs.     

Sensitivity to stress-induced reproductive dysfunction is associated with a selective but not a generalized increase in activity of the adrenal axis

Stress-induced reproductive dysfunction is a relatively common cause of infertility in women. In response to everyday life stress, some individuals readily develop reproductive dysfunction (i.e., they are stress sensitive), whereas others are more stress resilient. Female cynomolgus monkeys, when exposed to mild combined psychosocial and metabolic stress (change in social environment + 20% reduced calorie diet), can be categorized as stress sensitive (SS; they rapidly become anovulatory in response to stress), medium stress resilient (MSR; they slowly become anovulatory in response to prolonged stress), or highly stress resilient (HSR; they maintain normal menstrual cycles in response to stress). In this study, we examined whether increased sensitivity to stress-induced reproductive dysfunction is associated with elevated adrenal axis activity by measuring 1) the diurnal release of ACTH and cortisol, 2) ACTH and cortisol in response to an acute psychological stress, 3) the percent suppression of cortisol in response to dexamethasone negative feedback, 4) the diurnal release of ACTH and cortisol following exposure to mild psychosocial and metabolic stress, 5) the concentration of cortisol in hair, and 6) adrenal weight. SS monkeys (n = 5) did not differ from MSR (n = 5) or HSR (n = 7) monkeys in any measurement of baseline HPA axis activity or the integrated measurements of chronic HPA axis activity. However, MSR + SS monkeys (n = 10) did secrete more cortisol than HSR monkeys during the daytime hours (1000-1800) following exposure to a novel social environment and reduced diet. We conclude that increased activity of the HPA axis is unlikely to be the primary mechanism causing increased sensitivity to stress-induced reproductive dysfunction.     

Age-related changes in hematological and serum biochemical values in cynomolgus monkeys (Macaca fascicularis) bred and reared using the indoor individually-caged system

Blood samples were collected from 1000 or more apparently healthy cynomolgus monkeys of different ages. All the monkeys had been bred and reared under uniform environmental conditions at the Tsukuba Primate Center for Medical Science, NIH, Japan. Biochemical and hematological data were determined with these samples. The calculated arithmetic means and standard deviations were listed for each item of measurement performed (Tables 3, 4 and 5). The correlation between the obtained value and the age was analyzed using a statistical significance level of 0.1% (Table 6). It was revealed that hemoglobin concentration, mean corpuscular volume, alubuminglobulin ratio and triglyceride concentration increased with age, whereas red blood cell count and serum alkaline phosphatase activity decreased with age in both sexes.     

Glucoregulatory hormones in man at high altitude

Concentrations of glucose, lactic acid, free fatty acid (FFA), insulin, cortisol and growth hormone (GH) in the blood were monitored in 15 euglycaemic men (sojourners, SJ) at sea level (SL) and while at altitudes of 3500 m and 5080 m, in acclimatised low landers (ALL) and in high altitude natives (HAN). In SJ, blood glucose and insulin concentrations showed a significant increase on the 3rd and 7th day after arrival at high altitude (HA), thereafter returning to sea level values and remaining the same during the entire period of their stay at 3500 m. Subsequently, on arrival at higher altitude (5080 m) the glucose concentrations again showed an increase over the preceding values and returned to SL values on day 41 while at 5080 m. A significant increase in cortisol concentrations was seen on day 3 after arrival at HA and the increased levels were maintained until day 21 at 3500 m. The cortisol concentrations on day 30 after arrival at 5080 m came down to SL values and remained unchanged thereafter. No appreciable change in GH and FFA was seen during the sojourn at HA. On the other hand, blood lactic acid concentration decreased significantly. There was no difference between the fasting glucose concentrations in ALL at 3500 m and in HAN at 3500 m and 4200 m compared to values of SJ at SL, whereas ALL at 4200 m had higher glucose values. Concentrations of plasma insulin and GH in ALL and HAN were higher than the values of SJ at SL, whereas cortisol values did not show any difference. These observations indicated that at HA the glucose values were high for the insulin concentration observed and might have been due to increased secretion of GH by the pituitary gland.     

Effects of single and multiple injections of ketamine hydrochloride on serum hormone concentrations in male cynomolgus monkeys

Using simulated short- and long-term effect studies, we evaluated the effect of ketamine anesthesia on serum cortisol, testosterone, and immunoreactive luteinizing hormone (ILH) and bioactive LH (BioLH) concentrations in adult male cynomolgus monkeys. Cortisol, testosterone, and ILH were measured by use of radioimmunoassay, and BioLH was measured by use of a radioreceptor assay method. For the acute effect, the first group (eight monkeys) was given four successive intramuscular injections of ketamine (10, 5, 5, and 5 mg/kg of body weight at 0, 30, 60, and 110 min respectively). Blood samples were taken at 0, 15, 30, 45, 60, and 120 min. For the long-term effect, the second group (10 monkeys) was given a single injection of ketamine (10 mg/kg) once a week for 4 consecutive weeks. Blood samples were taken 5 to 10 min after each injection, then were used to determine the variation in hormone concentrations among the monkeys (inter-individual variation) and within each monkey (intra-individual variation). There were no statistically significant differences in serum cortisol, testosterone, ILH, and BioLH values between the first blood sample (before the ketamine injection) and sequential blood samples in monkeys of the first group. Although intra-individual variation in the hormones (i.e., hormonal change within each monkey) was not statistically significant, inter-individual variation (among the monkeys) was significantly (0.00001 < P < 0.033) different in monkeys of the second group. These results indicate that an adequate number of animals must be used to minimize animal-to-animal variability. Our results confirm that ketamine is a suitable anesthetic agent to immobilize male cynomolgus monkeys in experimental studies (short- and long-term studies) aimed at elucidating hormonal changes.     

The relationship of retrobulbar hematomas to vision in cynomolgus monkeys

An experimental model has been developed to measure the effect of retrobulbar hematomas on functional vision in cynomolgus monkeys. In this model, functional vision was quantitated using flashed evoked visual potentials in five monkeys following creation of retrobulbar hematomas. In one monkey used as a control, functional vision remained impaired for 180 minutes following induction of retinal ischemia by increased intraorbital pressure. In two monkeys in which increased intraorbital pressure was relieved by anterior chamber paracentesis following 15 minutes of retinal ischemia, flashed evoked visual potential promptly returned to baseline level. In two additional monkeys in which increased intraorbital pressure was relieved following 30 minutes of retinal ischemia, flashed evoked visual potentials improved but never returned to baseline levels. This study demonstrates the usefulness of flashed evoked visual potentials in measuring functional vision in cynomolgus monkeys. This experimental model should prove useful in evaluating the effects of increased intraorbital pressure on functional vision and the effect of intervention on impaired vision due to retrobulbar hematomas. Further studies with larger numbers of animals are needed to clarify these preliminary studies and document longer-term effects of retinal ischemia secondary to retrobulbar hematomas.     

Evidence for morphine-induced galactorrhea in male cynomolgus monkeys

We evaluated the effect of a daily injection of morphine hydrochloride on galactorrhea in male cynomolgus monkeys. Three groups of three monkeys (nine total) were used. The treatment schedule was separated into three periods: pre-treatment, treatment, and post-treatment. Each group of monkeys was subcutaneously injected daily with 1.5, 3.0, or 6.0 mg/kg (monkey weight) of morphine for 74–130 days, respectively, during the treatment period, and with saline during the pre-treatment and post-treatment periods. We then measured the prolactin (PRL) and testosterone (T) levels from weekly blood samples that were taken 20 hours after injection. No statistically significant differences in either the PRL or T level were detected throughout the treatment period. However, monkeys treated with 3.0 and 6.0 mg/kg/day showed a decrease in T level and an increase in PRL level during the early post-treatment period. Seven of the nine monkeys produced a milk-like secretion from their mammary gland (a symptom of galactorrhea) during the treatment and post-treatment periods. For several months of post-treatment period (average 6.75 months), we monitored the time-course changes in PRL and T levels in all monkeys for 10 hours after a single injection of morphine at the same dose given during the treatment period. Morphine induced a sudden increase in the PRL level (peaked within 30 minutes) and a gradual decrease in the T level (leveled off within 6.5–10 hours), and then returned to basal levels. These results indicate that morphine does not cause a long-term effect on hormonal changes and that a morphine-induced transient rise in PRL levels accompanied by a decrease in T levels can induce spontaneous galactorrhea in male cynomolgus monkeys.     

Internal carotid and vertebral arterial flow velocity in men at high altitude

Cerebral blood flow increases at high altitude, but the mechanism of the increase and its role in adaptation to high altitude are unclear. We hypothesized that the hypoxemia at high altitude would increase cerebral blood flow, which would in turn defend O2 delivery to the brain. Noninvasive Doppler ultrasound was used to measure the flow velocities in the internal carotid and the vertebral arteries in six healthy male subjects. Within 2-4 h of arrival on Pikes Peak (4,300 m), velocities in both arteries were slightly and not significantly increased above sea-level values. By 18-44 h a peak increase of 20% was observed (combined P less than 0.025). Subsequently (days 4-12) velocities declined to values similar to those at sea level. At altitude the lowest arterial O2 saturation (SaO2) and the highest end-tidal PCO2 was observed on arrival. By day 4 and thereafter, when the flow velocities had returned toward sea-level values, hemoglobin concentration and SaO2 were increased over initial high-altitude values such that calculated O2 transport values were even higher than those at sea level. Although the cause of the failure for cerebral flow velocity to increase on arrival is not understood, the subsequent increase may act to defend brain O2 transport. With further increase in hemoglobin and SaO2 over time at high altitude, flow velocity returned to sea-level values.     

Discriminant analysis of hematological and serum biochemical values in cynomolgus monkey (Macaca fascicularis) bred and reared under the indoor individually-caged conditions

The data on hematological and serum biochemical properties of laboratory-bred cynomolgus monkeys (Macaca fascicularis) at different ages were analyzed by discriminant analysis. All the animals had been bred and reared under uniform environmental conditions at Tsukuba Primate Center for Medical Science, N.I.H., Japan. The items used were as follows: red blood cell count (RBC), hematocrit value (Ht), hemoglobin concentration (Hb), mean corpuscular volume (MCV), white blood cell count (WBC), glutamic oxaloacetic transaminase activity (GOT), glutamic pyruvic transaminase activity (GPT), total protein concentration (TP), albumin concentration (ALB), albumin-globulin ratio (A/G), blood urea nitrogen (BUN), glucose concentration (GLU), total cholesterol concentration (TCHO), free cholesterol concentration (FCHO), triglyceride concentration (TG) and alkaline phosphatase activity (ALP). In total, 1086 animals in 10 age groups were examined. Data analyses were done with respect to the difference of sex. Discrimination was possible by Mahalanobis' generalized distance between centroids of groups. In canonical discriminant analysis (discriminant analysis with reduction of dimensionality), age was highly correlated to the value of the first canonical variate. From the approximate relative value of the eigenvector of the first canonical variate, the most discriminant variables are WBC, TP, ALB, A/G, TCHO, FCHO, TG, and ALP. It can be concluded that periodic measurement of these 8 parameters is necessary and sufficient to monitor the physiological conditions of growing monkeys.