setup and operation of the hyperglycemic and hypoglycemic clamps in mouse for monitoring blood glucose and insulin release

Installation chirurgicale de pinces hyperglycémiques et hypoglycémiques chez la souris

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.

Pleins feux sur l’auteur : Étude de l’homéostasie de la glycémie dans le cerveau murin à l’aide d’une technique de clamp hyperglycémique et hypoglycémique rentable

Pince hyperglycémique et pince hypoglycémique chez la souris consciente

Our research investigates how our brain regulates the homeo status of blood glucose levels, and what kind of neurons, neuropeptides, and neuron circuits do that. Additionally, how does our memory of food, psychological stress, pain, and other somatic sensations affect it? It is difficult to assess glucose metabolism precisely.We must check in insulin sensitivity or resistance, insulin secretion and counter regulatory responses. We should also check which tissue changes glucose uptake and glucose production regarding insulin sensitivity or resistance. We believe that not only insulin sensitivity, but also insulin secretion and counter regulatory responses are all affecting normal blood glucose levels, even in healthy conditions.We are trying to understand the whole picture of glucose regulation by the brain. Our protocol is low cost and does not need a special and expensive device to create a clamp. It'll enable researchers to implement the use of the clamp easily and quickly.Because this protocol helps researchers to use the clamp very easily, we expect our protocol will encourage more research in the entire field of life sciences.

Installation et fonctionnement des pinces hyperglycémiques et hypoglycémiques chez la souris pour la surveillance de la glycémie et de la libération d’insuline

Commencez par configurer le système de pompe pour les pinces hypoglycémiques ou hyperglycémiques. Pour ce faire, remplissez deux seringues Hamilton avec la perfusion d’insuline et la solution de glucose à 30 %. Connectez les seringues remplies aux deux longs tubes de la configuration du tube d’extension destinés à la perfusion de glucose et d’insuline.Après avoir fixé l’ensemble seringue aux pousse-seringues respectifs, versez chaque solution dans les tubes d’extension respectifs. Ensuite, remplissez de solution saline dans la seringue d’un millilitre et le tube d’extension pour prélever le sang de l’artère. Ensuite, connectez le tube d’extension pour perfusion au cathéter de la veine jugulaire préinstallé de la souris correctement anesthésiée.Le tube d’extension pour aspirer le sang au cathéter de l’artère carotide de l’animal. Utilisez du ruban de cellophane pour fixer les tubes ensemble. Placez la souris avec les tubes dans un bécher vide de 500 millilitres avec litière.Clamp l’extrémité supérieure du tube d’extension pour prélever du sang tout en le connectant à une nouvelle seringue. Extrayez 50 microlitres de sang, transférez-le dans un tube de 1,5 millilitre et mesurez la glycémie. Infusez ensuite 50 microlitres de solution saline.Pour prélever du sang pour les mesures hormonales, extrayez 50 microlitres de sang supplémentaires et transférez-les dans un tube de 1,5 millilitre sur de la glace. Après la mesure de la glycémie de zéro minute, démarrez la pompe à seringue à insuline pour perfuser la quantité appropriée d’insuline. Continuez à mesurer la glycémie et modifiez le débit de perfusion de glucose toutes les 5 à 10 minutes pendant la période de surveillance de 120 minutes jusqu’à ce qu’un état d’équilibre soit atteint.Pour le clamp hyperglycémique, suivez les étapes décrites pour le clamp hypoglycémique, à l’exception de la perfusion d’insuline et d’une glycémie ajustée à l’état d’équilibre.

Research

JoVE Journal

Medicine

Setup and Operation of the Hyperglycemic and Hypoglycemic Clamps in Mouse for Monitoring Blood Glucose and Insulin Release

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, ...

Recherche

Médecine

Surgery to Implant Hypoglycemic and Hyperglycemic Clamp Tubings in Mouse

Chirurgie pour implanter des tubes de clamp hypoglycémiques et hyperglycémiques chez la souris