动物造模 z-bo 2022-02-04 409

　　Introduction: Due to the relative lack of insulin, chronic functional carbohydrate metabolism disorders lead to diabetes. Non-human primates (NHPS) can naturally develop type 2 diabetes (T2DM) in a manner similar to the progression and onset of T2DM in humans. Metabolic disorders NHPs have been used in diabetes and obesity research in many studies. The NHP model also plays an important role in screening new compounds for regulating food intake, blood sugar and/or body weight. The data of the NHP model can discover and verify new mechanisms or treatment strategies and targets for metabolic diseases. In biomedical research, especially in large animals, the use of implantable telemetry devices for continuous glucose monitoring is very limited. Traditional methods, such as handheld blood glucose meters, clinical chemistry analyzers, or ANOLX analyzers, are usually used for blood glucose measurement. These conventional methods require regular blood sampling. Bleeding may cause stress and a significant decrease in blood volume, and periodic sampling may lose some key data points during the sampling interval. Therefore, "no day and night", no stress, no exercise animal blood glucose real-time measurement has some unique advantages in preclinical research. In addition, detecting glucose directly from the blood may reduce or eliminate many of the problems often encountered in interstitial glucose sensing. The HD-XG implantable glucose device can continuously measure the blood sugar, temperature and exercise activity of rodents for more than 2 months. In this study, a modified HD-XG transmitter device was used to observe the circadian rhythm, diet, stress process, and blood glucose changes of drug exposure in conscious and exercise cynomolgus monkeys (or macaques). An electrochemical glucose oxidase sensor placed in one of the monkey's femoral arteries provides continuous real-time blood glucose measurement. The temperature sensor embedded in the transmitter body provides continuous real-time temperature information. The change in signal intensity caused by the subject's physical activity provides a continuous real-time activity count calculated in DEM/MX2.

　　Method: Implantation of HD-XG telemetry device: An implantable telemetry device is composed of a glucose sensor lead, a reference lead and the device body, and is packaged and sterilized. Sterilize the operating room and surgical instruments one day before the operation. Before implantation of the device, animals were fasted, ketamine (15 mg/kg) and buprenorphine 0.01 mg/kg were injected intramuscularly, and additional ketamine (5 mg/kg) was given during the operation. During the operation, the animal's body temperature was maintained through a thermostatically controlled warm water circulation pad, and the temperature was monitored and maintained at 37°C. Shave the skin of the surgical site and disinfect the surgical area. Monitor vital signs during surgery, such as heart rate, blood pressure, oxygen saturation, and breathing rate. Make a small incision in the femoral area and carefully dissect a branch of the femoral artery. Insert the glucose sensor electrode of a HD-XG device into the artery branch, and its tip reaches the femoral artery, and fix the artery branch and sensor electrode together. The reference electrode and the device body are subcutaneously fixed near the femoral artery. Then suture the incision and cover it with gauze. The monkey puts on the monkey vest, and the transmitter is placed in the vest pocket. The monkey regained consciousness and was sent back to the cage. 0.01 mg/kg buprenorphine is injected intramuscularly within 6-12 hours after implantation for 2 days, and if necessary, amoxicillin 7 mg/kg is injected intramuscularly. Closely monitor the health of the animals during the week. After the operated animal was returned to the cage and completely recovered from anesthesia, food and water were provided.

　　Device calibration: Fibrin (soluble) or tissue in the glucose sensor area of the electrode placed in the artery can affect the glucose reading. In order to obtain the best performance and accuracy of glucose readings, during the study, the implanted HD-XG device must be used for reference measurements with the NoVA STATROBD blood glucose meter from time to time during the study. The calibration reference value is recorded as mg/dL. The calibration algorithm converts the telemetry (nA) data into a value equivalent to the appropriate mg/dL result.

　　Multi-point calibration: Multi-point calibration is required to establish a linear relationship between sensor output and blood glucose level at the beginning of initial data collection and at the end of the study. During the calibration period, the blood glucose level should differ by at least 100 mg/dL to reduce calibration errors caused by inaccurate glucose reference. Our study uses intravenous glucose to obtain a multipoint calibration.

　　Single-point calibration: During the study, a single-point calibration is performed to prevent non-physiological changes in baseline glucose values over time. Non-physiological changes include enzyme instability due to sensor drift or fibrin and tissue growth on the sensor electrode. At the same time of the day and during the period when the animal's blood glucose is relatively stable, calibration is performed at least twice a week.

　　Data acquisition: The implanted HD-XG glucose electrode continuously detects blood sugar and records electrical signals. After calibration with the glucose level measured by the NOVA STATROBT blood glucose meter, the recorded electrical signal is converted to glucose concentration. At the same time, the NOVA STATROPT blood glucose meter measures the blood glucose concentration during various glucose tests or drug challenges to further verify the telemetry data. In order to detect insulin resistance and β-cell insulin response to acute hyperglycemia, an intravenous glucose tolerance test (IVGTT) was performed. The experimental animals were fasted overnight (approximately 16 hours) and were initially anesthetized with ketamine at 10 mg/kg (I.M), and an additional dose of anesthesia (5 mg/kg, I.M) was added if needed during the operation. When the blood glucose is stable for about 30 minutes by observing the HD-XG glucose signal, the glucose solution (0.25 g/kg = 0.5 mL/kg 50% glucose) is infused through the cranial vein within 30 seconds, and then the system is flushed with 5 ml heparin saline to drain the glucose .

　　Result: Verification of the blood glucose level measured by the implanted HD-XG device: In order to verify the reliability of the telemetry method, the blood glucose level was measured at different time points and with or without various challenges one week after the device was implanted. In normal (n=3) and diabetic (n=2) monkeys, 187 blood glucose parameters were collected by telemetry and blood glucose meter methods. The blood glucose level measured by the telemetry method is highly correlated with the blood glucose value measured by the blood glucose meter, which shows that the telemetry method using the implantable HD-XG device is reliable for continuous blood glucose monitoring.

　　Blood glucose fluctuations in daily activities: With the method of clinical hand-held blood glucose meters, it is impossible to continuously monitor blood glucose day and night in awake, stress-free and freely active monkeys. A sensor was successfully placed in the femoral artery and the HD-XG device body was implanted in a subcutaneous space. This monitoring can continue until 10 weeks later. Interestingly, the blood glucose circadian rhythm pattern of normal blood glucose monkeys (n=3) is different from the blood glucose circadian rhythm observed from diabetic monkeys (n=2). The average blood sugar level of normal blood sugar animals dropped to a low level within 2 hours from 3 am, and then remained at a low level until the afternoon feeding at 3 pm. However, the average blood sugar level of diabetic animals gradually increased to a new high level around 7 am, and then maintained at a relatively high level until after 9 pm. Both normal blood sugar (n=3) and diabetic (n=2) animals did not have obvious postprandial hyperglycemia, but the blood sugar level increased by 20-30% after feeding in the afternoon.

　　Experimental process or blood glucose response to drugs: To check whether the experimental process will cause stress and change blood sugar levels, a fasted monkey moved out of his cage, grabbed its collar and let it sit on a monkey chair. This method increases the blood glucose level of normal blood glucose monkeys by about 30 mg/dL for about 10 to 20 minutes. However, the same method increased the blood glucose level of diabetic animals by 50-70 mg/dL (n=2) for more than 2 hours. Oral or nasal lavage is the commonly used method of administration of NHPs. To test whether such a procedure would also cause stress and change blood sugar levels, a fasted monkey was placed on the monkey’s chair and performed a false oral gavage (a feeding tube was placed in the stomach without giving food, medicine or Solution). A separate procedure resulted in an increase of 30-50% in both normal blood sugar and diabetic monkey blood sugar. However, in diabetic monkeys (60 minutes, n=2), program-induced hyperglycemia lasted longer than normal blood sugar monkeys (approximately 20 minutes, n=3). Stress-induced hyperglycemia is most likely caused by neuronal alertness and hormone secretion in the central or lactation process. To test whether stress hormones can cause hyperglycemia, monkeys implanted with HD-XG devices were given intravenous injections of norepinephrine and angiotensin II. After blood sugar stabilized for 30 minutes, 0.4μg/kg/min norepinephrine solution was injected intravenously for 40 minutes. Blood sugar gradually increased in normal blood sugar (L02) and diabetic (J04) monkeys, reaching the peak value near the end of the infusion. After norepinephrine was stopped, the hyperglycemia response to norepinephrine slowly subsided, and the blood glucose level of diabetic animals was lower than the norepinephrine level at the end of the infusion.

　　Conclusion: The HD-XG telemetry method has been successfully used to continuously monitor and record real-time blood glucose in a conscious, free-moving NHPS. This new technology allows researchers to detect circadian rhythms and changes in blood sugar due to physical activity or natural behaviors such as eating and sleeping, as well as stress or medication. Through this telemetry method, we can continuously collect blood glucose data for up to 10 weeks. Therefore, the use of this telemetry technology can help us further understand glucose metabolism under more natural physiological conditions.