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

Surgical Installation of Hyperglycemic and Hypoglycemic Clamps in Mice

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.

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

Hyperglykemisk klemme og hypoglykemisk klemme i bevisste mus

Oppsett og drift av hyperglykemiske og hypoglykemiske klemmer i mus for overvåking av blodsukker og insulinfrigjøring

Begynn med å sette opp pumpesystemet for de hypoglykemiske eller hyperglykemiske klemmene. For å gjøre dette, fyll to Hamilton sprøyter med insulininfusat og 30% glukoseoppløsning. Koble de fylte sprøytene til de to lange slangene i forlengelsesrøroppsettet som er ment for glukose- og insulininfusjon.Etter at sprøyteenheten er festet til de respektive sprøytepumpene, fylles hver oppløsning i de respektive forlengelsesslangene. Deretter fyller du saltvann i en milliliter sprøyte og forlengelsesrøret for å trekke blod fra arterien. Deretter kobler du forlengelsesrøret for infusjon til det forhåndsinstallerte jugularvenkateteret til riktig bedøvet mus.Forlengelsesrøret for å trekke blod til dyrets halspulsåreketer. Bruk cellofantape for å feste rørene sammen. Plasser musen med slangene inne i et tomt 500 ml beger med sengetøy.Klem den øverste enden av forlengelsesrøret for å trekke blod mens du kobler det til en ny sprøyte. Trekk ut 50 mikroliter blod, overfør det til et 1,5 milliliter rør og mål blodsukkernivået. Tilsett deretter 50 mikroliter saltvann.For å samle blod for hormonmålinger, trekk ut ytterligere 50 mikroliter blod og overfør det til et 1,5 milliliter rør på is. Etter måling av null minutt blodglukose, start insulinsprøytepumpen for å tilføre riktig mengde insulin. Fortsett å måle blodsukkernivået og endre glukoseinfusjonshastigheten hvert 5. til 10. minutt i løpet av overvåkingstiden på 120 minutter til en jevn tilstand er nådd.For den hyperglykemiske klemmen, følg trinnene som er demonstrert for den hypoglykemiske klemmen, unntatt insulininfusjon, og et justert steady state blodsukkernivå.

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, ...

Surgery to Implant Hypoglycemic and Hyperglycemic Clamp Tubings in Mouse

Kirurgi for å implantere hypoglykemiske og hyperglykemiske klemmeslanger i mus