We thank Dvir Mintz DVM and the veterinary and husbandry staff in the animal facility, as well as members of our group, for fruitful discussions. This study was supported by an Israel Science Foundation grant 1541/21 awarded to D.B.Z. D.B.Z. is a Zuckerman STEM faculty.

The experiments were approved by the Hebrew University&#39;s Institutional Animal Care and Use Committee (IACUC).
NOTE: All tools must be sterilized, and handling of the cannula must be performed using a sterile technique. The protocol below is fine-tuned to a specific CGM. The protocol can be adapted to other CGMs.
1. Analgesic administration before the procedure
<ol>
	<li>Administer 5% dextrose and 0.45% saline with meloxicam at 5 mg/kg body weight subcutaneously.</li>
</ol>
2. Anesthesia administration
<ol>
	<li>Place the mouse in the induction chamber, closing the lid tightly. Set the induction of anesthesia in the induction chamber at 3% isoflurane at a flow rate of 500 mL/min.</li>
	<li>Once the mouse is unresponsive, remove the mouse from the chamber, and fit the nose cone to the mouse. Confirm the anesthesia level with an interdigital pinch. Set the concentration to 1%-1.5% isoflurane and the flow rate to 100 mL/min in a mouse weighing 30 g.</li>
	<li>Apply ophthalmic ointment to the eyes to prevent dryness during anesthesia.</li>
</ol>
3. Sensor preparation
<ol>
	<li>Mount the sensor onto the sensor mounting device to expose the sensor&#39;s tape and cannula side (Figure 1A). Be cautious since the needle is inserted into the cannula and exposed.</li>
	<li>Suture two 5-0 taper point sutures to the tape on both sides of the cannula (Figure 1A).</li>
</ol>
4. Hair removal and disinfection
<ol>
	<li>Shave an area of approximately 4 cm x 4 cm on the midline of the back of the mouse.</li>
	<li>Administer a depilatory cream to the shaved area to ensure complete hair removal.</li>
	<li>Wipe the skin, and disinfect it using an antiseptic solution containing 2% chlorhexidine gluconate and 70% isopropyl alcohol.</li>
</ol>
5. Dorsal skin preparation
<ol>
	<li>Make a 2 mm incision in the center of the shaved area above the spine using sharp scissors (Figure 1B).</li>
	<li>Briefly, insert small forceps with a blunt edge under the skin to form a small subcutaneous pocket so that the cannula can be inserted easily into the subcutaneous pocket (Figure 1B).</li>
	<li>Pass a suture from step 3.2 through the skin on each side of the incision (Figure 1C).</li>
</ol>
6. Sensor insertion
<ol>
	<li>Remove the sensor completely from the sensor mounting device (the cannula is void from the needle), and hold the sensor with forceps to keep the surrounding tape from sticking to itself.</li>
	<li>Carefully insert the cannula into the subcutaneous pocket.</li>
	<li>Pull the sutures on each side, and tighten and tie them to attach the sensor firmly in place, thus preventing the cannula from slipping out from the subcutaneous pocket once the adhesive tape loosens over time.</li>
</ol>
7. Sensor attachment and suturing
<ol>
	<li>Attach the sensor to the back firmly by tying the inner sutures and using the adhesive tape surrounding the sensor.</li>
	<li>Make eight discontinuous sutures around the sensor, attaching the border of the sensor&#39;s tape to the skin (Figure 1D).</li>
</ol>
8. Activation of the reader
<ol>
	<li>Once the sensor has been inserted, activate the reader by turning on the reader, pressing Start New Sensor, and swiping the sensor according to the manufacturer&#39;s instructions.</li>
	<li>The first reading can only be taken a few minutes after installing the CGM. In the case of this CGM, the first reading can be taken after 60 min.</li>
</ol>
9. Reading results
<ol>
	<li>Place the reader close to the mouse (there is no need to touch it). All data stored in the sensor are transmitted to the reader. 
	&#8203;NOTE: Different CGM devices may differ in the period of historic data saving capacity. In the case of this CGM, a maximum of 8 h can be stored between two readings.</li>
</ol>
10. Removing the sensor
<ol>
	<li>Anesthetize the mouse (see section 2).</li>
	<li>Cut the sutures connecting the sensor to the back of the mouse using sharp scissors.</li>
	<li>Remove and cut the sutures at the incision by gently removing the sensor.</li>
	<li>If necessary, use a single suture to close the incision in the back of the mouse.</li>
</ol>
11. Data analysis
<ol>
	<li>Data download: Download the data as per the instructions provided by the CGM manufacturer. 
	&#8203;NOTE: Each CGM has a different format, which may or may not be easily accessible to the user. This is an important consideration in choosing the CGM.</li>
	<li>For analysis with the software provided, format the data according to the instructions in the readme file on Github (https://github.com/mika-littor/CGM-in-Mice-Analysis.git).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64743/64743fig01.jpg" /> 
Figure 1: Attachment of the sensor to the mouse. (A) Two sutures marked by red arrows are passed through the sensor tape on both sides of the cannula on the bottom side of the CGM sensor, marked by a white arrow. (B) A small 2 mm incision is made in the center of the shaved area along the spine using sharp scissors. Small forceps with a blunt edge are briefly inserted under the skin to form a small subcutaneous pocket so that the cannula can be inserted subcutaneously. (C) The same sutures from A are passed subcutaneously on each side of the incision. The red arrows mark the sutures attached to the sensor as in A, the blue arrows mark the location the sutures passed through to the skin in the back of the mouse, and the black arrow shows the incision. (D) After the cannula is inserted, the inner sutures are tightened and tied close to the incision to secure the CGM. The sensor is then sutured to the skin. <a href="https://www.jove.com/files/ftp_upload/64743/64743fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Polonsky, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past+200+years+in+diabetes.">The past 200 years in diabetes.</a> New England Journal of Medicine. 367 (14), 1332-1340 (2012).</li><li>Rees, D. A., Alcolado, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+diabetes+mellitus.">Animal models of diabetes mellitus.</a> Diabetic Medicine. 22 (4), 359-370 (2005).</li><li>Pearson, J. A., Wong, F. S., Wen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+non-obese+diabetic+(NOD)+mouse+model+in+autoimmune+diabetes.">The importance of the non-obese diabetic (NOD) mouse model in autoimmune diabetes.</a> Journal of Autoimmunity. 66, 76-88 (2016).</li><li>Heydemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+murine+high+fat+diet+as+a+model+for+Type+2+diabetes+mellitus.">An overview of murine high fat diet as a model for Type 2 diabetes mellitus.</a> Journal of Diabetes Research. 2016, 2902351 (2016).</li><li>Kennard, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+mice+in+diabetes+research:+The+impact+of+experimental+protocols.">The use of mice in diabetes research: The impact of experimental protocols.</a> Diabetic Medicine. 38 (12), 14705 (2021).</li><li>Klueh, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+glucose+monitoring+in+normal+mice+and+mice+with+prediabetes+and+diabetes.">Continuous glucose monitoring in normal mice and mice with prediabetes and diabetes.</a> Diabetes Technology and Therapeutics. 8 (3), 402-412 (2006).</li><li>Wuyts, C., Simoens, C., Pinto, S., Philippaert, K., Vennekens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+glucose+monitoring+during+pregnancy+in+healthy+mice.">Continuous glucose monitoring during pregnancy in healthy mice.</a> Scientific Reports. 11, 4450 (2021).</li><li>Korstanje, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+glucose+monitoring+in+female+NOD+mice+reveals+daily+rhythms+and+a+negative+correlation+with+body+temperature.">Continuous glucose monitoring in female NOD mice reveals daily rhythms and a negative correlation with body temperature.</a> Endocrinology. 158 (9), 2707-2712 (2017).</li><li>Han, B. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+generation+of+human+stem+cell-derived+alpha+cells.">A method for the generation of human stem cell-derived alpha cells.</a> Nature Communications. 11, 2241 (2020).</li><li>Klueh, U., Liu, Z., Feldman, B., Kreutzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+fluid+physiology+as+it+relates+to+glucose+monitoring+technologies:+Importance+of+Interleukin-1+and+Interleukin-1+receptor+antagonist+in+short-term+glucose+sensor+function+in+vivo.">Interstitial fluid physiology as it relates to glucose monitoring technologies: Importance of Interleukin-1 and Interleukin-1 receptor antagonist in short-term glucose sensor function in vivo.</a> Journal of Diabetes Science and Technology. 4 (5), 1073 (2010).</li><li>Klueh, U., Antar, O., Qiao, Y., Kreutzer, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+interleukin-1/interleukin-1+receptor+antagonist+family+of+cytokines+in+long-term+continuous+glucose+monitoring+in+vivo.">Role of interleukin-1/interleukin-1 receptor antagonist family of cytokines in long-term continuous glucose monitoring in vivo.</a> Journal of Diabetes Science and Technology. 7 (6), 1538 (2013).</li><li>Klueh, U., Kaur, M., Qiao, Y., Kreutzer, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+tissue+mast+cells+in+controlling+long-term+glucose+sensor+function+in+vivo.">Critical role of tissue mast cells in controlling long-term glucose sensor function in vivo.</a> Biomaterials. 31 (16), 4540-4551 (2010).</li><li>Kogot-Levin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+metabolic+reprogramming+induced+by+sodium-glucose+cotransporter+2+inhibition.">Mapping the metabolic reprogramming induced by sodium-glucose cotransporter 2 inhibition.</a> JCI Insight. , 164296 (2023).</li></ol>

The authors have no conflicts of interest to disclose.

This protocol offers a simple, inexpensive method to monitor glucose levels in mice that does not require challenging microsurgery and does not involve bleeding or handling the mice. The method is easy to implement in every facility and does not cause mortality, pain, or excessive discomfort to the mice. The most critical step in the protocol is inserting the cannula of the glucose sensor under the skin of the mouse. The addition of a few sutures allows the cannula to stay in place for a longer time. The sensors are smal...

Diabetes mellitus (DM) is a devastating disease characterized by high blood glucose levels. Type 1 DM can be a result of an autoimmune attack on the insulin-producing beta cells in the pancreas. Type 2 DM and gestational DM, on the other hand, are characterized by a failure of the beta cells to secrete sufficient insulin in response to a rise in glucose levels1. The mouse is a common model organism used to study DM since it has similar physiology, and its normal glucose levels are close to those of humans. Furthermore, specific mouse strains may develop DM due to mutations in key signaling pathways or following exposure to specific diets, which enables disease modeling2,3,4.
Blood glucose is commonly measured in mice using glucometers designed for patients by extracting a small drop of blood (1-2 &#181;L) from the tip of the tail of the mouse. This method causes stress and requires handling of the mouse, which affects the glucose levels and prohibits the measurement of blood glucose levels in freely behaving mice or when the researcher is not close by5. Bleeding the mice may cause stress to nearby mice, particularly to mice of the same cage whose glycemia has not been measured yet, thus affecting the results. Mice respond differently depending on the handler, and the person measuring glucose may affect the glucose levels of the mice. These pitfalls call for careful experimental design and underlie some inconsistencies between experiments.
It is possible to measure glucose in freely moving mice with no bleeding by implanting glucose sensors into the aortic arch of the mice using state-of-the-art telemetry6. The resulting measurements are very good and can be sustained over a long period, but it is challenging to implant these sensors, and the telemetry system is expensive, leading to a moderate adoption of this methodology and no adoption in non-specialized labs. Subcutaneous or other glucose sensors that are tailored to the dimensions of the mice and their physiology have been developed in recent years, but these again require highly skilled experts and are in some cases costly6,7,8,9,10.
Commercial continuous glucose monitors (CGMs) that were originally developed to monitor the glucose levels of DM patients offer another option to measure glucose in freely moving mice, with lower cost and technical expertise requirements than implanted probes. Such probes have been used in basic research by a few labs5,11,12,13,14,15&#160;including our colleagues who used this protocol16. These devices typically include a sensor, a mounting device, a receiver, and a software application. The sensor has a cannula guiding the enzymatic glucosensor, adhesive tape, an energy source, short-term memory, and a wireless communication module that stores and sends the data to the receiver. The receiver can show the current glucose levels and sends the data to a server; this receiver can be a cellphone. The software application provides data for the patient and the medical care team on the glycaemia of the patient. In patients, the sensor is attached easily using the mounting device. The cannula is inserted subcutaneously by pressing the mounting device against the skin, and the sensor stays in place with the help of adhesive tape.
This is a detailed protocol for adapting a commercial CGM device to measure glucose levels in mice. This protocol describes how to surgically insert the glucose sensor and attach it to the mouse. Scripts for basic data analysis and data visualization are provided. The potential pitfalls, troubleshooting, and examples of standard results are provided. The protocol below is specific for a certain CGM but can be easily adapted to other types of commercial CGMs as they become available.

<table><tbody><tr><td>2%&amp;nbsp; Chlorhexidine Gluconate and 70%&amp;nbsp; Isopropyl Alcohol</td><td>3M</td><td>ID&amp;nbsp;7000136290</td><td></td></tr><tr><td>5% Dextrose and 0.45% Sodium Chloride Injection, USP</td><td>Braun</td><td>L6120</td><td></td></tr><tr><td>Castroviejo needle holder</td><td>FST</td><td>12061-02</td><td></td></tr><tr><td>Extra Fine Bonn scissors</td><td>FST</td><td>14084-08</td><td></td></tr><tr><td>FreeStyle Libre 1 reader</td><td>Abbott</td><td>ART27543&amp;nbsp;</td><td></td></tr><tr><td>FreeStyle Libre sensor</td><td>Abbott</td><td>ART36687</td><td></td></tr><tr><td>FreeStyle Libre sensor applicator</td><td>Abbott</td><td>ART36787</td><td></td></tr><tr><td>Gauze pads</td><td>Sion medical</td><td>PC912017</td><td></td></tr><tr><td>Graefe Forceps</td><td>FST</td><td>11052-10</td><td></td></tr><tr><td>Hair Removal Cream</td><td>Veet</td><td>3116523</td><td></td></tr><tr><td>High-fat high-sucrose diet</td><td>Envigo Teklad diets</td><td>TD.08811</td><td></td></tr><tr><td>Isoflurane, USP Terrell</td><td>Piramal</td><td>26675-46-7</td><td></td></tr><tr><td>Meloxicam 5 mg/mL</td><td>Chanelle Pharma</td><td>08749/5024</td><td></td></tr><tr><td>MiniARCO Clipper kit</td><td>Moser</td><td>CL8787-KIT</td><td></td></tr><tr><td>PROLENE Polypropylene Suture 5-0</td><td>Ethicon</td><td>8725H</td><td></td></tr><tr><td>Puralube Opthalmic Ointment</td><td>Perrigo</td><td>574402511</td><td></td></tr><tr><td>Q-tips&amp;nbsp;</td><td>B.H.W</td><td>271676</td><td></td></tr><tr><td>SomnoSuite Low-Flow Anesthesia System</td><td>Kent Scientific</td><td>SOMNO</td><td></td></tr></tbody></table>

simple continuous glucose monitoring in freely moving mice

Mice are a common model organism used to study metabolic diseases such as diabetes mellitus. Glucose levels are typically measured by tail-bleeding, which requires handling the mice, causes stress, and does not provide data on freely behaving mice during the dark cycle. State-of-the-art continuous glucose measurement in mice requires inserting a probe into the aortic arch of the mouse, as well as a specialized telemetry system. This challenging and expensive method has not been adopted by most labs. Here, we present a simple protocol involving the utilization of commercially available continuous glucose monitors used by millions of patients to measure glucose continuously in mice as a part of basic research. The glucose-sensing probe is inserted into the subcutaneous space in the back of the mouse through a small incision to the skin and is held in place tightly using a couple of sutures. The device is sutured to the mouse skin to ensure it remains in place. The device can measure glucose levels for up to 2 weeks and sends the data to a nearby receiver without any need to handle the mice. Scripts for the basic data analysis of glucose levels recorded are provided. This method, from surgery to computational analysis, is cost-effective and potentially very useful in metabolic research.

Surgical outcome 
Results from eight HSD:ICR mice (aged 8 weeks) fed a high-fat high-sucrose diet (HFHS) for 18 weeks and five lean HSD:ICR mice (aged 12 weeks) are shown. The device we used stores data for up to 8 h. Access to the local animal facility was restricted to 07:00-19:00, thus prohibiting data collection during the late P.M. hours, when the mice are active. The mice were, therefore, placed in a room with reverse lighting for 7 days before the surgical procedure, with dark hours between 8...

Watch this Scientific Journal Video about Simple Continuous Glucose Monitoring in Freely Moving Mice at JoVE.com

Simple Continuous Glucose Monitoring in Freely Moving Mice

Here, we describe a simple method to implant a commercial continuous glucose monitor designed for patients onto mice and provide the scripts to analyze the results.

Mice are a common model organism used to study metabolic diseases such as diabetes mellitus. Glucose levels are typically measured by tail-bleeding, which ...

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Research

JoVE Journal

Biology

5.0K Views.  The Hebrew University-Hadassah Medical School. Here, we describe a simple method to implant a commercial continuous glucose monitor designed for patients onto mice and provide the scripts to analyze the results.

Video: Simple Continuous Glucose Monitoring in Freely Moving Mice

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 ...

Medicine

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

Developmental Biology

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

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 ...

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 ...

Surgical Installation of Hyperglycemic and Hypoglycemic Clamps in Mice

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.

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.

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

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 &#181;L/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer&#39;s disease and/or other neurodegenerative disorders associated with diabetes.

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits ...

Oral Glucose Tolerance Test (OGTT): Measuring Glucose Metabolism in Mice

Source: Ajoy, R. and Chou, S. Y. <a href="https://www.jove.com/video/56410/studying-hypothalamic-insulin-signal-to-peripheral-glucose">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain</a>.&#160;J. Vis. Exp. (2018).
This video introduces how to perform an oral glucose tolerance test on a laboratory mouse.

Source: Ajoy, R. and Chou, S. Y. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

Assays for Qualitative and Quantitative Assessment of Mouse Urine Glucose Concentrations

This video demonstrates a procedure for collecting mouse urine and assessing its glucose levels. To evaluate the glucose level, immerse a test strip coated with enzymes and a chromogenic dye into the sample, initiating a color change that indicates the glucose levels. For quantifying urine glucose levels, introduce a urine sample to a multiwell plate with escalating glucose concentrations, add the reaction mixture, and incubate. Measure the absorbance to create a standard curve and compute the glucose levels based on the sample absorbance.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Spot ...