This work was supported by the Leading Initiative for Excellent Young Researchers (from MEXT); a Grant-in-Aid for Scientific Research (B) (Grant Number JP21H02352); Japan Agency for Medical Research and Development (AMED-RPIME, Grant Number JP21gm6510009h0001, JP22gm6510009h9901); the Uehara Memorial Foundation; Astellas Foundation for Research on Metabolic Disorders; Suzuken Memorial Foundation, Akiyama Life Science Foundation, and Narishige Neuroscience Research Foundation. We also thank Nur Farehan Asgar, Ph.D, for editing a draft of this manuscript.

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Kumamoto University.
NOTE: For pain relief, ibuprofen was given in drinking water (0.11 mg/mL) for 48 h, and buprenorphine (0.05-0.1 mg/kg i.p.) was given 30 min before surgery. Sterile conditions include gloves, masks, and autoclaved instruments sterilized with ethylene oxide between animals. The surgery was performed on a heating pad set at 37 &#176;C and covered by a new lab mat for each animal. Before the surgery, the surgical area was cleaned with a betadine&#160;solution and alcohol. All surgical instruments were sterilized with an autoclave (for no more than two surgeries). Before making the incision, mice were checked to ensure they were fully anesthetized. The depth of anesthesia for each mouse was assessed prior to and during surgery by a toe pinch.&#160;The acclimatization period was no more than 5 min each time. Follow the instructions of the IACUC at the respective institution.
1. Preparation of tubings for the jugular vein and carotid artery
<ol>
	<li>Sterilize all tubings, polypropylene supplies (e.g., pipet tips), and sutures with an autoclave or ethylene oxide. For the jugular vein, connect 8 cm of tubing1 (see Materials) and 3 cm of tubing2 using glue (Figure 2A). Put 2 mm of tubing1 on the top because tubing 2 (polyethylene) is too hard and may damage blood vessels (Tubing1.1).</li>
	<li>Extension tube for the jugular vein (Tubing1.2) to infuse glucose and insulin
	<ol>
		<li>Prepare two 30 cm tubes and one 10 cm tube with tubing2 (Figure 2A). Cut off the sharp end of a 20 &#956;L pipette tip (or anything that has a narrow tip end with OD &#60; 0.5 mm to connect with tubing 1.1) and place three tubing2 (30 cm, 30 cm, and 10 cm) together in it. Seal with adhesive glue.</li>
		<li>Place 5 cm of tubing1 at the other end of tubing2.</li>
	</ol>
	</li>
	<li>For carotid artery&#160;(Tubing1.3): Stretch tubing2 to make it thinner. Connect 8 cm of tubing1 and 3 cm of the stretched tubing2 using glue (Figure 2A). Make a mark at 9 mm from the tip.</li>
	<li>Needle with non-sharp end that connects tubing to the syringe: Make a scratch near the sharp end of the needle (23 G) with a metal file, gently bend it back and forth a few times with pliers, and break it. Do the same cut in the middle of the needle to make a piece of metal connector (Figure 2A).</li>
	<li>Extension tube for the artery to draw blood (Tubing set1.4): Connect 10 cm of tubing1 with the metal connector and the non-sharp needle that was made in step 1.4. 
	&#8203;NOTE: All tubings and sutures are sterilized with an autoclave or ethylene oxide</li>
</ol>
2. Surgery
<ol>
	<li>Anesthetize a mouse with isoflurane (1.5-2.0%) or ketamine/xylazine (ketamine 10 mg/mL, xylazine 1 mg/mL in 0.9% sterile saline, 0.1 mL/10 g of body weight (BW) via intraperitoneal [ip] injection). Keep the mouse on the warm pad (37 &#176;C) to reduce physical stress. Wait for deep anesthesia for 5-10 min. Confirm the depth of anesthesia by pedal reflex, respiration, and heart rate, and response to stimuli by toe pinching.&#160;During surgery, surgical tape should be&#160;used to secure the nose cap to the operating table for continuous inhalation of anesthesia.&#160;Apply ophthalmic ointment to eyes to prevent dryness while under anesthesia.</li>
	<li>Fill heparinized saline (100 U/mL) in tubing1.1 and 1.3 and connect them with a 1 mL syringe with a non-sharp needle (Figure 2B). Close the end of tubing3 (see Table of Materials) by melting it with a soldering iron, which will be used as caps for tubing1.1 and tubing1.3.</li>
	<li>Shave and wipe the first (interscapular on the back) and second (neck in the front) incision area with three cycles of 10% betadine followed by a preoperative preparation with sterile 70% alcohol. Make a small vertical midline incision 5 mm cephalic to the sternum, blunt dissect the tissues, and expose the artery. Separate the vagus nerve from the artery. This reduces the negative effects of removing the vagus nerve on glucose metabolism. 
	NOTE: The step from the ventral incision to the step where the catheter is inserted into the vein and secured with silk sutures is performed under a microscope.</li>
	<li>Place two silk sutures under the artery. Stop blood flow by tying one suture tightly on the cranial side (Figure 2C-1) and the other loosely at the caudal side (Figure 2C-2), which is enough to stop blood flow but enough to open again later. Place one more suture under the artery (Figure 2C-3).</li>
	<li>Cut the artery near Figure 2C-1 with spring scissors and place tubing1.3 into the artery. Make a loose tie on both the artery and tubing (Figure 2C-3, do not tie firmly. The tubing will be inserted deeper into the artery). Open the knot on the caudal side (Figure 2C-2) to insert the tube until the 9 mm mark reaches the knot in the middle (Figure 2C-3). Securely tie all ligatures and flush with heparinized sterile saline.</li>
	<li>Expose the right jugular vein from the same incision as the right carotid artery for the jugular catheter. Isolate the cranial end and ligate it with silk suture (Figure 2D-1, Table of Materials). Place another piece of suture at the caudal end of the exposed vein (Figure 2D-2). Cut the vein near the mark Figure 2D-1&#160;with spring scissors.</li>
	<li>Insert the catheter (not too deep to prevent penetrating vessels), tie it, and confirm visually that it samples blood. Flush with heparinized sterile saline (0.2 mL) and visually confirm that no blood remains in the catheter.</li>
	<li>Place the mouse on the new sterile surgical drape to prevent infection from the first wound. Turn over the mouse, wipe with three cycles of betadine followed by a preoperative preparation with sterile alcohol, and make a small incision between the shoulder blades.</li>
	<li>Pass a needle holder under the skin from the incision on the back to the ventral side. Clamp the catheters with the needle holder, pass them under the skin, and bring them back. Clean the incision site and close the ventral incisions with a synthetic&#160;suture (diameter 0.15-0.2 mm). Clamp the venous catheter with micro serrefine at the incision site between the shoulder blades.</li>
	<li>Cut the catheter 1 cm above the clamp, flush it with heparinized saline, and close it with a cap (step 2.4). Follow the same procedure for the arterial catheter. Close the dorsal incision with a synthetic&#160;suture (diameter 0.15-0.2 mm).</li>
	<li>Place the mouse in a warm, clean cage (Figure 2E). Perform postoperative care daily.</li>
</ol>
3. Recovery
<ol>
	<li>Single-house the mouse because another mouse may bite the catheter in the group housing.
	<ol>
		<li>To reduce stress by social isolation, keep mice with environmental enrichments (e.g., shelters).&#160;Perform postoperative care daily. To relieve pain, distress, and discomfort, provide postoperative care, including analgesia (ibuprofen in drinking water (0.11 mg/mL).</li>
		<li>Observe mice for signs of infection, such as festering, lethargy, or pain at the incision site. Most healthy mice begin walking and feeding about 2 h after surgery. A hunched posture, ruffled fur, and decreased food intake may indicate pain. If these signs are observed, immediately euthanize the mice by decapitating them under deep anesthesia or carbon dioxide asphyxiation.</li>
	</ol>
	</li>
	<li>Blood will enter the catheter in the artery and form a clot. To maintain catheter lines, remove the clot daily, following steps 3-6.</li>
	<li>Fill a 1 mL syringe and 23 G needle (non-sharp end) with heparinized saline (100 U/mL). Using an induction chamber,&#160;anesthetize the mouse lightly with isoflurane (1.0%-1.5%). Then, remove the mouse from the chamber and perform clot removal under isoflurane anesthesia with a nose cap.</li>
	<li>Clamp the tubing for the artery on the back of the mouse with micro serrefine, remove the cap, and extract the blood and clots. Flush the catheter with heparinized saline using another 1 mL syringe with a 23 G needle (non-sharp end) and re-cap it. Clean the venous line like the arterial line in the case of severe clotting.</li>
	<li>Do the same procedure to clean up the catheter once a day for 3-5 days.</li>
	<li>Check the body weight of the mouse. If the body weight reduces by more than 10% from the day of the surgery, apply supportive care and try to improve the normal body condition score, and use the mouse for another experiment.&#160; 
	&#8203;NOTE: The weight loss of animals was less than 10% in the experiments in this study. Weight loss will strongly affect systemic glucose metabolism. Thus, it is recommended to wait for the body weight recovery or remove from the clamp experiment.</li>
</ol>
4. Set up the pump system (for hypoglycemic clamp)
<ol>
	<li>Prepare 1 U/mL insulin in 0.1% BSA saline, 30% glucose in saline, and heparinized saline.</li>
	<li>Measure the body weight of the mouse. Calculate the volume for 1 U/mL insulin to make the insulin infusate (10 mU/kg/min). Infuse 1.7 &#956;L/min of insulin solution into the mice in the clamp experiment. To make 300 &#956;L of insulin infusate, the required volume for 1 U/mL insulin (&#956;L) is 2.647 (&#956;L/g) x Body weight (g). A volume of 300 &#956;L of insulin infusate is enough to finish the clamp experiment on 1 mouse. Table 1 shows an example of making insulin infusate.</li>
	<li>Fill insulin infusate and 30% glucose in each Hamilton syringe, connect them with tubing 1.2, and set the syringe on the syringe pump (Figure 3A). Fill each solution in tubing 1.2. Fill saline in a 1 mL syringe and tubing set 1.4 (Figure 2A).</li>
	<li>Anesthetize the mouse with isoflurane (1.0%-1.5%) and connect tubing1.2 to the venous catheter and tubing set1.4 to the arterial catheter (Figure 3A). Use cellophane tape to hold the tubes together.</li>
	<li>Place the mouse in an empty 500 mL beaker. 
	&#8203;NOTE: For hyperglycemic clamp, use saline instead of 1 U/mL insulin.</li>
</ol>
5. Hypoglycemic clamp
<ol>
	<li>Measure blood glucose levels every 5-10 min, as shown in Figure 3B, and collect blood samples for hormone measurements at -15 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min. Follow step 5.2 to measure blood glucose. 
	NOTE: It is not necessary to measure blood glucose at all time points, but it is recommended to measure it at least every 10 min. Measure blood glucose every 5 min when its level and glucose infusion rate are not steady.</li>
	<li>Clamp the top end of the tubing set1.4 and connect a new syringe, extract 50 &#956;L of blood, and place it in a 1.5 mL tube for washing.&#160;Measure blood glucose level. 
	NOTE: This is the blood diluted by the saline solution in the tube because of catheter washing after the previous sampling. A 50 &#956;L volume is enough to replace diluted blood with pure blood.</li>
	<li>Store the diluted blood (red blood cells). Wash the pooled blood (~500 &#956;L) with saline (see steps 5.11-5.12) and return to the body to prevent hypoxia.</li>
	<li>The catheter is connected to the artery with high blood pressure, so when the clamp is loosened, blood will flow out and be used to measure glucose with a handy glucose meter. Infuse 50 &#956;L of saline to keep enough amount of blood fluid.</li>
	<li>For blood sampling, extract an additional 50 &#956;L of blood and place it in a 1.5 mL tube on ice. Infuse 100 &#956;L (50 &#956;L of replacement + 50 &#956;L of sampling) of saline.</li>
	<li>After 0 min of blood glucose measurement, start the insulin syringe pump. First, infuse 30 mU/kg/min in bolus for 2 min (5.1 &#956;L /min), then 10 mU/kg/min (1.7 &#956;L /min) infusion for the rest of the duration.</li>
	<li>Measure blood glucose level and change the glucose infusion rate every 5-10 min for 120 min.</li>
	<li>Create a steady state where the glucose infusion rate does not change, and the blood glucose level is 50 mg/dL.</li>
	<li>After collecting the blood sample at t = 120 min, infuse a euthanasia agent into the jugular vein and collect tissues for RNA or protein analysis.</li>
	<li>Centrifuge blood at 1000 x g for 5 min and measure hormone concentration such as insulin or c-peptide using an ELISA kit according to the manufacturer&#39;s protocol.</li>
	<li>For washing blood, centrifuge the blood at 1000 x g for 3 min. Remove the supernatant, add 500 &#956;L of heparinized saline, and perform pipetting.</li>
	<li>Repeat the wash again and place the washed red blood cells in 1 mL syringe for the artery. The blood cells will be reinfused automatically when 50 or 100 &#956;L of saline is infused in steps 5.2 and 5.3. Monitor hematocrit carefully to prevent hypoxia. 
	&#8203;NOTE: For blood glucose measurement, a commercial handy glucose meter was used.</li>
</ol>
6. Hyperglycemic clamp
<ol>
	<li>Follow the steps as the hypoglycemic clamp technique, but without insulin infusion and with blood glucose adjusted to 250-300 mg/dL. Measure blood glucose levels every 5-10 min, as shown in Figure 3B, and collect blood samples for hormone measurements at -15 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min.</li>
	<li>Connect a new syringe to the tubing set1.4, extract 50 &#956;L of blood, and place it in a 1.5 mL tube for washing.</li>
	<li>For blood sampling, extract an additional 50 &#956;L of blood and place it in a 1.5 mL tube on ice. Infuse 100 &#956;L (50 &#956;L of replacement + 50 &#956;L of sampling) of saline.</li>
	<li>After 0 min of blood glucose measurement, start the glucose syringe pump. First, infuse 30 g/kg/min in bolus for 2 min (5.1 &#956;L /min), then 10 g/kg/min (1.7 &#956;L /min) for the rest of the duration.</li>
	<li>Measure the blood glucose level and change the glucose infusion rate every 5 -10 min for 120 min. Create a steady state where the glucose infusion rate does not change, and the blood glucose level is 250-300 mg/dL, a clamped hyperglycemic state.</li>
</ol>

Surgical Installation of Hyperglycemic and Hypoglycemic Clamps in Mice

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The authors declare no competing interests.

The method described here is a simple one that can be done with pipette tips, syringes, and other items found in ordinary laboratories. Although researchers may need to purchase additional tubes and pumps, expensive equipment is not needed. Thus, this protocol of catheterization and clamp is easier to start compared to previous reports12,13,14.
The clamp technique was developed around 1970 and has been used in mice and humans15. It is a useful method for accurately measuring glucose metabolism and is said to be the gold standard. However, it is not a common technique used by many researchers. The hyperinsulinemic-euglycemic clamp technique has been reported in mice13 and rats14, but the method of catheterization here is different, and readers can choose the easier one for their experiments. One of the purposes of this paper is to reduce the hurdle for starting an experiment. Therefore, we provided detailed information on the materials of handmade catheters, surgical procedures, and an example of experimental time course. These are informative for the researcher who attempts to perform clamp in the first time.
Insulin secretion in obesity and diabetes is stage-dependent. Many reports suggest insulin secretion is increased in obesity to reduce blood glucose in the insulin-resistant state16, but &#946;-cell function will be damaged in Type 2 DM17,18. In fact, the number and area of pancreatic islets and insulin secretion have been reported to be increased in obesity mouse models, such as mice fed with a high-fat diet19 or leptin-deficient mice20. In these mouse models, which have an obvious phenotype, differences can be determined by examining blood insulin levels 15-30 min after glucose administration in the GTT. However, in some cases, it is not easy to determine the differences in insulin secretion. For example, if transgenic (Tg) mice have a blood glucose level of 500 mg/dL while a WT mouse has 300 mg/dL and both mice have the same blood insulin level, can we say insulin secretion decreases in Tg? In this case, we cannot compare insulin secretion ability unless blood glucose levels are the same using the method introduced here. This is one of the reasons why there is no established theory as to when &#946;-cell function begins to deteriorate in the transition from obesity to diabetes. We can also measure insulin secretion by primary culture of the pancreas21 or ex vivo22. However, it will ruin the effect of the central nervous system on insulin release because the innervation of the vagus nerve will be removed. Post-absorptive insulin secretion is well-known, but the brain and autonomic nervous system also regulate insulin release23. The experiment to analyze the latter should be performed in an unanesthetized, unrestrained, painless blood collection. This is also why the vagus nerve has to be separated from the carotid artery in step 2.3.
Diabetes mellitus has been reported to cause hyperglycemia due to insulin resistance and increased secretions of glucagon24 and other counter-regulatory hormones25. In addition, repeated episodes of hypoglycemia in humans with diabetes due to failures of insulin dosage or other causes can lead to a condition called recurrent hypoglycemia, in which patients are prone to hypoglycemia26. It has been suggested that the rate of fall in glycemia in hypoglycemic clamp affects peripheral or central detection of the hypoglycemia27. Slow onset hypoglycemia may be appropriate to study the role of glucose sensors in portal-mesenteric veins, while a very rapid decrease in blood glucose may be for the study of brain glucose sensors27. 1 U/mL Insulin is used in lean C57BL mice. But, a higher insulin concentration will be needed in obese mice because they have insulin resistance, and 1 U/mL is not enough to decrease blood glucose levels.
In the one-compartment model of the blood glucose pool (Figure 1A), the amount of absorbed glucose may affect the rate of glucose input14. Thus, the rate of glucose production, one of the main objectives of measuring the hyperinsulinemic-euglycemic clamp, can be affected by the duration of fasting. However, long fasting time may increase the release of counter-regulatory hormones. Hence, researchers set fasting time according to their analysis purpose. Another clamp method includes blood sampling from the tail, which is simple because only an intravenous cannula needs to be inserted14. However, blood is collected in a restraint, which causes a moderate amount of restraint stress and an increase in plasma catecholamines and other stress hormones14. In addition, it is preferable to measure hormone concentrations in the blood flow in the center of the body rather than in the blood at the extremities. Therefore, blood sampling from arteries in free-moving mice is the best for measuring glucose metabolism physiologically. The mice do not move around, and swivel is not needed when they are acclimated to the experimental environment. However, it is recommended to use a swivel to prevent entangling infusion and sampling lines. Tubing1.2 and tubing set1.4 (Figure 3A) are not good for using a swivel. The system should be improved if the researcher is required to use a swivel. Reinfusion of blood cells does not influence the established steady state of blood glucose and insulin. The present method can also be applied to metabolomics studies using isotopes. For example, if 13C-glucose is continuously infused into the vein, the systemic metabolic turnover rate and intracellular intermediate metabolites can be measured28. Thus, this is a useful method to analyze glucose metabolism.

Glucose is an important source of energy for cells, and a lack of glucose can lead to a variety of symptoms and complications. In the event of low glucose (hypoglycemia, generally less than 70 mg/dL in fasting blood glucose level, but should not be determined by a single value1), the most common symptoms include weakness, confusion, sweating, and headache. It can also disrupt cerebral function and increase the risk of cardiovascular events and mortality2. Conversely, hyperglycemia is a medical condition in which the plasma glucose concentration exceeds normal levels (generally &#62; 126 mg/dL in fasting blood glucose level3). This can occur in individuals with diabetes who have either a deficit in insulin production or utilization. Hyperglycemia can lead to diabetic ketoacidosis, which occurs when the body cannot use glucose for energy but instead breaks down fatty acids for fuel. The hyperglycemic hyperosmolar state also increases mortality4. Long-term hyperglycemia can cause damage to blood vessels, nerves, and organs, leading to the development of several chronic complications such as cardiovascular disease, retinopathies, and kidney diseases. Thus, the blood glucose concentration must be maintained in a tight range between 100 mg/dL and 120 mg/dL.
Blood glucose is regulated by the balance between glucose input and output in a one-compartment model (Figure 1A). Glucose input includes absorbed glucose from food and glucose production from the liver, kidneys, and small intestine. Glucose output comprises glucose uptake in tissues and glucose disposal from the kidneys. Both the amount of glucose input and output are regulated by endocrine hormones. For example, glucagon, corticosterone, and catecholamines, known as counter-regulatory hormones, are released when blood glucose levels decrease5. They stimulate the breakdown of glycogen and the synthesis of glucose, mainly from the liver; these processes are known as glycogenolysis and gluconeogenesis, respectively. Hyperglycemia increases insulin release from pancreatic &#946;-cells and stimulates glucose uptake in the muscles, adipose tissues, and heart6,7,8,9. Exercise increases insulin-independent glucose uptake10. The sympathetic nervous system increases glucose uptake in muscles and brown adipose tissue6,11. To measure the ability to regulate glucose metabolism in peripheral tissues, researchers typically use the glucose tolerance test (GTT) and the insulin tolerance test (ITT) (Figure 1B,C). In GTT, two factors must be considered: insulin release and insulin sensitivity (Figure 1B). However, the glucose concentration curve during the 120 min test is different in each mouse, which may affect different amounts of hormone release. In ITT, blood glucose is regulated by both insulin sensitivity and the release of counter-regulatory hormones. Therefore, it is difficult to determine the precise meaning of glucose metabolism, insulin release, and insulin sensitivity in GTT and ITT, in situations where blood glucose levels are not constant.
To overcome these problems, it is desirable to keep blood glucose at a constant level (or &#34;clamp&#34;). In hyperglycemic clamp, glucose is infused into the bloodstream to raise blood glucose levels to a specific level and then maintained at that level for a period of time. The amount of infused glucose is adjusted based on measurements of blood glucose levels every 5-10 min to maintain a steady state. This technique is particularly useful for understanding the parameters of insulin secretion at a clamped glucose level. Hypoglycemic clamp is a method to maintain low blood glucose levels by infusing insulin. Glucose is infused at a variable rate to maintain a specific blood glucose level. If the mouse cannot recover from hypoglycemia, more glucose should be infused.
Although there are many advantages to performing hyperglycemic and hypoglycemic clamps, the surgical and experimental procedures are considered technically difficult. Thus, few research groups have been able to do them. We aimed to describe these methods for researchers with financial and workforce constraints to start these experiments at a lower budget.

<table><tbody><tr><td>Adhesive glue</td><td>Henkel AG &amp;amp; Co. KGaA</td><td>LOCTITE 454</td><td></td></tr><tr><td>ELISA kit (C-peptide)</td><td>Morinaga Institute of Bilogical Science Inc</td><td>M1304</td><td>Mouse C-peptide ELISA Kit</td></tr><tr><td>ELISA kit (insulin)</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>633-03411</td><td>LBIS Mouse Insulin ELISA Kit (U-type)</td></tr><tr><td>Handy glucose meter</td><td>Nipro Co.</td><td>11-777</td><td>Free Style Freedom Lite</td></tr><tr><td>Insulin (100U/ml)</td><td>Eli Lilly &amp;amp; Co.</td><td>428021014</td><td>Humulin R (100U/ml)</td></tr><tr><td>Mouse</td><td>Japan SLC Inc.</td><td>C57BL/6NCrSlc</td><td>C57BL</td></tr><tr><td>Suture</td><td>Natsume seisakusho</td><td>C-23S-560 No.2</td><td>Sterilized</td></tr><tr><td>Syringe Pump</td><td>Pump Systems Inc.</td><td>NE-1000</td><td></td></tr><tr><td>Synthetic suture</td><td>V&amp;Ouml;MEL</td><td>HR-17</td><td></td></tr><tr><td>Tubing1</td><td>AS ONE Corporation</td><td>9-869-01</td><td>LABORAN(R) Silicone Tube</td></tr><tr><td>Tubing2</td><td>Fisher Scientific</td><td>427400</td><td>BD Intramedic PE Tubing</td></tr><tr><td>Tubing3</td><td>IGARASHI IKA KOGYO CO., LTD.</td><td>size5</td><td>Polyethylene tubing size5</td></tr></tbody></table>

hyperglycemic clamp and hypoglycemic clamp in conscious mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively.
Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

The hypoglycemic clamp study was performed in male C57BL/6N mice (8 weeks old, more than 25 g BW) 3 h fasted at the start of the experiment (Figure 4A,B). The initial blood glucose level was 136 mg/dL (t = -15 min). If it is less than 90 mg/dL, it may be either because the surgery did not go well, or the arterial catheter was inserted too deep, or blood clots have entered the blood flow. The mouse condition after surgery affects the energy metabolism in the mouse. Physiological glucose metabolism cannot be measured under poor health conditions. C57BL is more prone to clotting after surgery; mice that have a higher body weight, such as FVB or ICR mice, are less likely to lose weight due to clot washing after surgery. Thus, it&#39;s better for beginners to use FVB mice for practicing clamp procedures. C57BL mice require more intensive care. The 9 mm catheter insertion into the artery has not given us any trouble in collecting blood, even in FVB and ICR mice. Insulin was started at t = 0 min, and blood glucose levels decreased (Figure 4A). The GIR had not been stable between t = 0 min and t = 70 min. Subsequently, it became a steady state after 80 min.
The hyperglycemic clamp study was also performed in male C57BL/6N mice (8 weeks old, more than 25 g BW) 3 h fasted at the start of the experiment (Figure 4C,D). After measuring blood glucose and collecting blood samples at t = -15 and t = -5 min, glucose was infused from t = 0 min. The blood glucose level became 250 mg/dL at t = 40 min. Steady state continued from t = 30 min to the end.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65581/65581fig01.jpg" /> 
Figure 1: Regulation of blood glucose levels. (A) Conceptual diagram illustrating the regulation of blood glucose in the body. (B,C) Regulation of glucose metabolism in (B) glucose tolerance test (GTT) and (C) insulin tolerance test (ITT). Several factors regulate blood glucose levels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65581/65581fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65581/65581fig02v2.jpg" /> 
Figure 2: Preparation of tubings and steps of the surgery. (A) Images of tubings for carotid artery and jugular vein. Tubing numbers match the protocol step numbers. (B) Method of preparing the tubings for surgery. (C, D) The numbered positions where the silk threads are ligated to insert the catheter into&#160;(C) artery and (D) vein. Positions of the silk threads in the cranial (C-(1)) and caudal side of&#160;the artery (C-(2)), and one more in the middle of them (C-(3)) (procedure 2-4). Positions of&#160;the silk threads in the cranial (D-(1)) and caudal side of the vein (D-(2)), and one more in&#160;the middle of them (D-(3)) (procedure 2-6). (E) Diagram of arterial and venous cannulas exiting the back <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65581/65581fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65581/65581fig03.jpg" /> 
Figure 3: Setting of the clamp. (A) Diagram showing the setup of tubings, pumps, syringes, and the mouse. (B) Sheet to record blood glucose levels and glucose infusion rate. 50 &#956;L of blood is collected at t = -15 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min. The insulin infusion rate was 5.1 &#956;L/min from t = 0 to 2 min and changed to 1.7 at t = 2 min. Blood glucose level was measured every 10 min. The blood glucose was measured every 5 min when its level was not stable. The glucose infusion rate was changed each time blood glucose was measured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65581/65581fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65581/65581fig04.jpg" /> 
Figure 4: Representative results of hypoglycemic clamp and hyperglycemic clamp. (A) Blood glucose levels and (B) glucose infusion rate of hypoglycemic clamp. (C) Blood glucose levels and (D) glucose infusion rate of the hyperglycemic clamp. Data represent mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65581/65581fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Calculation</td>
			<td>For 20 g mouse</td>
		</tr>
		<tr>
			<td>1 U/mL insulin</td>
			<td>(2.647 x body weight) &#956;L</td>
			<td>52.9 &#956;L</td>
		</tr>
		<tr>
			<td>0.1% BSA saline</td>
			<td>300 &#956;L - volume of insulin (&#956;L)</td>
			<td>247.1 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: An example calculation for preparing insulin infusate.

Watch this Scientific Journal Video about Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice at JoVE.com

Author Spotlight: Investigating the Blood Glucose Homeostasis in Murine Brain Using a Cost-Effective Hyperglycemic And Hypoglycemic Clamp Technique

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, ...

hyperglycemic-clamp-and-hypoglycemic-clamp-in-conscious-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  Kumamoto University. Ons onderzoek onderzoekt hoe onze hersenen de homeostatus van bloedglucosespiegels reguleren en wat voor soort neuronen, neuropeptiden en neuronencircuits dat doen. Bovendien, hoe beïnvloedt ons geheugen van voedsel, psychologische stress, pijn en andere somatische gewaarwordingen het? Het is moeilijk om het glucosemetabolisme precies te beoordelen.We moeten de insulinegevoeligheid of -resistentie, de insulinesecretie en de tegenregulerende reacties controleren. We moeten ook nagaan w...

Video: Auteur in de schijnwerpers: onderzoek naar de bloedglucosehomeostase in de hersenen van muizen met behulp van een kosteneffectieve hyperglykemische en hypoglykemische klemtechniek

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

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