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

Installazione chirurgica di pinze iperglicemiche e ipoglicemizzanti nei topi

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.

Autore in primo piano: Indagine sull'omeostasi della glicemia nel cervello murino utilizzando una tecnica di clamp iperglicemica e ipoglicemica conveniente

Pinza iperglicemica e pinza ipoglicemica nei topi coscienti

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.

Installazione e funzionamento dei morsetti iperglicemici e ipoglicemici nel topo per il monitoraggio della glicemia e del rilascio di insulina

Iniziare impostando il sistema di pompaggio per le pinze ipoglicemiche o iperglicemiche. A tale scopo, riempire due siringhe Hamilton con l'insulina infusa e la soluzione di glucosio al 30%. Collegare le siringhe riempite ai due tubi lunghi della configurazione del tubo di prolunga destinato all'infusione di glucosio e insulina.Dopo aver collegato il gruppo siringa alle rispettive pompe a siringa, riempire ciascuna soluzione nei rispettivi tubi di prolunga. Quindi, riempi la soluzione fisiologica nella siringa da un millilitro e nel tubo di prolunga per prelevare il sangue dall'arteria. Successivamente, collegare il tubo di prolunga per l'infusione al catetere della vena giugulare preinstallato del topo adeguatamente anestetizzato.Il tubo di prolunga per prelevare il sangue al catetere dell'arteria carotide dell'animale. Usa del nastro di cellophane per fissare insieme i tubi. Posiziona il mouse con i tubi all'interno di un becher vuoto da 500 millilitri con lettiera.Clamp l'estremità superiore del tubo di prolunga per prelevare il sangue mentre lo si collega a una nuova siringa. Estrarre 50 microlitri di sangue, trasferirlo in una provetta da 1,5 millilitri e misurare il livello di glucosio nel sangue. Quindi infondere 50 microlitri di soluzione fisiologica.Per raccogliere il sangue per le misurazioni ormonali, estrarre altri 50 microlitri di sangue e trasferirlo in una provetta da 1,5 millilitri su ghiaccio. Dopo la misurazione della glicemia a zero minuti, avviare la pompa a siringa per insulina per infondere la quantità appropriata di insulina. Continuare a misurare il livello di glucosio nel sangue e modificare la velocità di infusione del glucosio ogni 5-10 minuti durante il tempo di monitoraggio di 120 minuti fino a raggiungere uno stato stazionario.Per il morsetto iperglicemico, seguire i passaggi illustrati per il morsetto ipoglicemico, escludendo l'infusione di insulina e un livello di glucosio nel sangue allo stato stazionario aggiustato.

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, ...

Ricerca

Medicina

Surgery to Implant Hypoglycemic and Hyperglycemic Clamp Tubings in Mouse

Intervento chirurgico per impiantare tubi di bloccaggio ipoglicemici e iperglicemici nel topo