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

चूहों में Hyperglycemic और Hypoglycemic क्लैंप की सर्जिकल स्थापना

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.

लेखक स्पॉटलाइट: एक लागत प्रभावी हाइपरग्लाइसेमिक और हाइपोग्लाइसेमिक क्लैंप तकनीक का उपयोग करके murine मस्तिष्क में रक्त ग्लूकोज होमियोस्टेसिस की जांच करना

Hyperglycemic क्लैंप और Hypoglycemic दबाना सचेत चूहों में

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.

सेटअप और रक्त ग्लूकोज और इंसुलिन रिलीज की निगरानी के लिए माउस में hyperglycemic और hypoglycemic clamps के संचालन

हाइपोग्लाइसेमिक या हाइपरग्लाइसेमिक क्लैंप के लिए पंप सिस्टम स्थापित करके शुरू करें। ऐसा करने के लिए, इंसुलिन इन्फ्यूसेट और 30% ग्लूकोज समाधान के साथ दो हैमिल्टन सीरिंज भरें। ग्लूकोज और इंसुलिन जलसेक के लिए मतलब विस्तार ट्यूब सेटअप के दो लंबे ट्यूबिंग के लिए भरा सीरिंज कनेक्ट करें.संबंधित सिरिंज पंप के लिए सिरिंज विधानसभा संलग्न करने के बाद, संबंधित विस्तार ट्यूबिंग में प्रत्येक समाधान भरें. अगला, धमनी से रक्त खींचने के लिए एक मिलीलीटर सिरिंज और विस्तार ट्यूब में खारा भरें। अगला, ठीक से संवेदनाहारी माउस के पूर्व स्थापित जुगुलर नस कैथेटर के लिए जलसेक के लिए विस्तार ट्यूब कनेक्ट.जानवर के कैरोटिड धमनी कैथेटर में रक्त खींचने के लिए विस्तार ट्यूब। ट्यूबों को एक साथ सुरक्षित करने के लिए सिलोफ़न टेप का उपयोग करें। बिस्तर के साथ एक खाली 500 मिलीलीटर बीकर के अंदर ट्यूबिंग के साथ माउस रखें.रक्त खींचने के लिए एक्सटेंशन ट्यूब के ऊपरी छोर को जकड़ें, जबकि इसे एक नई सिरिंज से जोड़ते हैं। 50 माइक्रोलीटर रक्त निकालें, इसे 1.5 मिलीलीटर ट्यूब में स्थानांतरित करें और रक्त शर्करा के स्तर को मापें। फिर 50 माइक्रोलीटर खारा डालें।हार्मोन माप के लिए रक्त एकत्र करने के लिए, अतिरिक्त 50 माइक्रोलीटर रक्त निकालें और इसे बर्फ पर 1.5 मिलीलीटर ट्यूब में स्थानांतरित करें। शून्य मिनट रक्त शर्करा माप के बाद, इंसुलिन की उचित मात्रा को संक्रमित करने के लिए इंसुलिन सिरिंज पंप शुरू करें। रक्त शर्करा के स्तर को मापते रहें और 120 मिनट की निगरानी के समय के दौरान हर 5 से 10 मिनट में ग्लूकोज जलसेक दर को तब तक बदलें जब तक कि एक स्थिर स्थिति न हो जाए।हाइपरग्लाइसेमिक क्लैंप के लिए, हाइपोग्लाइसेमिक क्लैंप के लिए प्रदर्शित चरणों का पालन करें, इंसुलिन जलसेक को छोड़कर, और एक समायोजित स्थिर राज्य रक्त शर्करा का स्तर।

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

माउस में हाइपोग्लाइसेमिक और हाइपरग्लाइसेमिक क्लैंप ट्यूबिंग प्रत्यारोपित करने के लिए सर्जरी