Name: evaluating the effect of continuous drug infusion into the mouse brain on peripheral glucose levels
Uploaded: 2025-03-31T15:00:43.000+00:00
Duration: 58 s
Description: Source: Ajoy, R., et. al. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;
1.&#160;Preparation of Micro-Osmotic Pump Infusion Systems
NOTE: Prepare the pump, artificial cerebrospinal fluid (aCSF) buffer, and drug (Met-CCL5/RANTES protein solution (10 ng/mL in aCSF)) under sterile conditions using buffers filtered with 0.2 &#181;m filters and conduct all the procedures under the culture hood with gloves. The surgery procedures are conducted as follows:
<ol>
	<li>Prepare the micro-osmotic pumps one day before surgery: Fill the brain micro-osmotic pump with artificial cerebrospinal fluid (aCSF) with a 1 mL syringe and blunt-head needle provided along with the kit. Immerse the micro-osmotic pump in aCSF and place it on a shaker with gentle shaking overnight. 
	&#160;CAUTION: The pump should be filled with aCSF, and air bubbles should be avoided inside the pump (Figure 1A).</li>
	<li>Before starting surgery, prepare the recombinant Met-CCL5/RANTES protein solution (10 ng/mL, diluted in aCSF) for the experiment. Remove aCSF from the pump and fill the pump with the drug solution slowly until excess leaks out. 
	&#160;NOTE: 15 mL aCSF or Met-CCL5/RANTES solution is sufficient for 5-8 pumps. 
	&#160;CAUTION: Repeat the procedure to ensure that the pump is completely filled with the drug without bubbles inside.</li>
	<li>Cut the catheter tubes into the desired length and attach them to the brain infusion kit&#39;s blunt-end brain infusion needle. Fill the infusion kit and tubes with the drug.</li>
	<li>Finally, assemble and attach the brain infusion kit to the micro-osmotic pump. 
	&#160;CAUTION: No bubbles should be formed in the tube or the pump (Figure 1A).</li>
	<li>Immerse the entire osmotic pump-brain fusion set in aCSF in a sterilized 50 mL tube to prevent the pump from drying out. The osmotic pump-brain fusion set is now ready to be used for surgery. 
	&#160;NOTE: The micro-osmotic pump systems can be used for long-term drug infusion. This ensures a safe and convenient mode of drug delivery into the mouse brain.</li>
</ol>
2.&#160;Intracerebral Ventricular Surgery &#8211; implantation of the micro-osmotic pump
CAUTION: Sterilize the surgical environment with 75% ethanol and ensure that the people involved in the study are wearing sterile gloves and a clean lab coat. Surgical tools/ instruments must be autoclaved and dried before use and subsequently sterilized with 75% ethanol in-between mice surgeries.
<ol>
	<li>Weigh the mouse and anesthetize it using intra-peritoneal injection (IP) with Ketamine/Xylazine (Ketamine 50 mg/kg, Xylazine 10 mg/kg). 
	&#160;CAUTION: Mice body weights lower than 24 g are not recommended for osmotic pump implantation surgery.</li>
	<li>Mount and fix the mouse head onto the stereotactic apparatus (Figure 1B).</li>
	<li>Use a pair of surgical scissors and pincers to cut open the outer skin covering the skull. Use iodine to clean the peripheral skull.</li>
	<li>Separate the outermost layer of skin from the subcutaneous skin with the help of a pair of blunt-head pincers near the neck region for the osmotic pump-brain fusion set implantation (Figure 1C).</li>
	<li>Mark the infusion point with reference to the brain map (Figure 1D) using the stereotactic apparatus. In this experiment, the needle needs to be implanted in the 3rd ventricle region (Bregma: 0.0 mm lateral, 1.3 mm posterior, 5.7 mm ventral).</li>
	<li>Drill a hole using a nail drill around the area marked on the skull (Figure 1E). 
	&#160;CAUTION: Be careful not to break the mouse meninges and blood vessels, thus avoiding the disruption of micro-blood vessels in the brain.</li>
	<li>Place the micro-osmotic pump-brain fusion set containing aCSF (as control) or drug (Met-CCL5/RANTES protein solution) under the skin behind the neck region and insert the brain infusion needle into the drilled hole to infuse the drug into the mouse brain (Figure 1E). The needle will penetrate the meninges and get into the ventricle. Fix the needle in place on the skull using surface desensitizing gel (Figure 1F) and wait 1-2 min until the glue dries. Next, cut off the projecting part on top of the needle (Figure 1G-H).</li>
	<li>Use a tissue adhesive glue to heal the operation wound on the head. Apply 50 &#181;L of the glue on top of the wound, pull both sides of the skin together, and hold on for 30 s to allow the skin to seal (Figure 1I). 
	&#160;&#160;CAUTION: Use a 100% alcohol pad to clean the wound after surgery and 100 ul penicillin with streptomycin to prevent infection. NOTE: Mouse skin will form scar tissue and heal in a few days following the administration of the surgical glue. The main advantage of the glue is the avoidance of surgical stitches, which may cause skin irritation or inflammation.</li>
	<li>Place the mouse in a clean cage kept on a warm plate (heated up to 37 &#176;C) and wait until the mouse recovers from the anesthetic effect. 
	&#160;CAUTION: It is critical to maintain the mouse&#39;s body temperature to enhance the chance of survival after surgery.</li>
	<li>After a one-week recovery period, the mice will be ready for further experiments, such as the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT).</li>
</ol>
3. Oral Glucose Tolerance Test (OGTT)
NOTE: Perform the oral glucose tolerance test 7 days after the infusion of aCSF and MetCCL5/RANTES (10 ng/mL, 100 &#181;L). Maintain a 6 h fast for the mice before OGTT with sufficient water supply. Keep the animals on the same work bench where the experiments will be performed so that they can acclimatize to the environment to reduce stress during the procedure.
<ol>
	<li>Glucose solution preparation: Before conducting the experiment, prepare the glucose solution by dissolving 3.75 g glucose in 15 mL of distilled H2O.</li>
	<li>Set up a timetable to record the readings during the experimental procedure (Table 1). 
	&#160;NOTE: It is important to set up a timetable with proper intervals between each blood examination to allow accurate recording during the experiment.</li>
	<li>Weigh each mouse after fasting and calculate the appropriate amount of glucose to be injected. For example, if the mouse weighs 30 g, the amount of glucose solution to be administered should be 300 &#181;L.</li>
	<li>Prepare the following instruments at the workbench:
	<ol>
		<li>Glucometer (Press the start button to check the battery status; make sure it is functioning before the test.)</li>
		<li>Glucose chip</li>
		<li>Insulin syringe (0.3 mL Insulin syringe)</li>
		<li>Razor blades</li>
		<li>Timer</li>
	</ol>
	</li>
	<li>Once the bench is set up, measure and record the blood glucose level as follows: Put a clean and new glucose chip into the glucometer and press the start button to zero it.</li>
	<li>Pick up the mouse by the back of the neck and stroke the tail a few times to ensure sufficient blood flow to the tail region.</li>
	<li>Using a new razor blade to cut off a small piece of the tail and squeeze out one small drop of blood (about 10-20 &#181;L) into the glucose chip. The blood should fill the chip to allow accurate measurement. The glucometer will display the glucose level immediately. If the machine shows an &#34;error,&#34; repeat the procedure with a new glucose chip. 
	&#160;NOTE: The glucose chip requires only one drop of blood. When the blood sample needs to be collected more than once, simply apply pressure by running your fingers along the mouse&#39;s tail several times while holding the end of the tail directly on top of the chip to collect blood. It is not necessary to cut the tail end each time while collecting blood samples.</li>
	<li>Next, feed the mice glucose (0.25 g/mL) orally by using the intragastric gavage technique. The amount of glucose to be administered should be calculated by using the formula: 10X body weight (BW) &#181;L glucose solution (for example, if the mouse weighs 30 g, the amount of glucose solution to be administered will be 300 &#181;L). Start the timer immediately after administering oral glucose.</li>
	<li>Repeat the glucose measurement procedure at 15, 30, 60, 90, and 120 min.</li>
	<li>After all the glucose level readings have been recorded, discard the razor blades and glucose chips in a biohazard container. Put the food back into the mice cages and return them to the animal room.</li>
</ol>
Table 1. Timetable for Oral Glucose Tolerance Test (OGTT) recording
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#</td>
			<td>Mouse ID</td>
			<td>Body</td>
			<td>Glucose</td>
			<td>Start</td>
			<td>0</td>
			<td>15</td>
			<td>30</td>
			<td>60</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>weight</td>
			<td>&#956;L =10xBW</td>
			<td>time</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
			<td>mins</td>
		</tr>
		<tr>
			<td>1</td>
			<td>501</td>
			<td>25.8g</td>
			<td>258</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:00</td>
			<td>9:15</td>
			<td>9:30</td>
			<td>10:00</td>
		</tr>
		<tr>
			<td>2</td>
			<td>502</td>
			<td>25.3g</td>
			<td>253</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:07</td>
			<td>9:22</td>
			<td>9:37</td>
			<td>10:07</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>#1049SB</td>
			<td>The glue used to seal the lesion site on the mouse head</td>
		</tr>
		<tr>
			<td>LOCTITE 454 instant adhesive</td>
			<td>Durect Corporation</td>
			<td>#8670</td>
			<td>The glue used to fix the needle on the mouse skull</td>
		</tr>
		<tr>
			<td>Alzet Micro- Osmotic Pump</td>
			<td>Durect Corporation</td>
			<td>#9922</td>
			<td>0.11 &#956;l per hour, 28 days</td>
		</tr>
		<tr>
			<td>Brain infusion system</td>
			<td>Durect Corporation</td>
			<td>#8851</td>
			<td>1-3 mm, used to perfuse the drug in to the mice brain</td>
		</tr>
		<tr>
			<td>Glucometer</td>
			<td>Roche</td>
			<td>#06870244001</td>
			<td>Used to measure the blood glucose level</td>
		</tr>
		<tr>
			<td>Glucose chip</td>
			<td>Roche</td>
			<td>#06454011020</td>
			<td>Used to load the blood sample</td>
		</tr>
		<tr>
			<td>Evan&#39;s blue</td>
			<td>Sigma</td>
			<td>#MKBK0523V</td>
			<td>To demonstrate the drug infusion area</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>#3232145 C</td>
			<td>Used to administer insulin intraperitoneally</td>
		</tr>
		<tr>
			<td>MIO NE116 CONTROL UNIT 
			(nail drill)</td>
			<td>Mio System</td>
			<td>#E235-015</td>
			<td>To drill a hole in the skull of the mouse</td>
		</tr>
		<tr>
			<td>CCL5/Met-RANTES Protein</td>
			<td>R&#38;D</td>
			<td>#ADB0111081</td>
			<td>Recombinant Human CCL5, E-coli derived</td>
		</tr>
		<tr>
			<td>aCSF formula</td>
			<td>119 mM NaCl 
			26.2 mM NaHCO3 
			2.5 mM KCl 
			1 mM NaH2PO4 
			1.3 mM MgCl2 
			10 mM glucose</td>
			<td></td>
			<td>Filter sterilize with a 0.22 &#956;m filter apparatus, and store at 4&#176;C. 
			aCSF is stable for 3-4 weeks</td>
		</tr>
		<tr>
			<td>Phospho-IRS-1 Serine302 antibody</td>
			<td>Cell Signaling</td>
			<td>#12879</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>IRS-1 (D23G12) antibody</td>
			<td>Cell Signaling</td>
			<td>#12879</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Phospho-Akt Serine 473 antibody</td>
			<td>Cell Signaling</td>
			<td>#9916</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Akt (pan) (C67E7) antibody</td>
			<td>Cell Signaling</td>
			<td>#9916</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Animals: C57BL/6</td>
			<td>NAR Labs</td>
			<td></td>
			<td>Wild type mice strain used in the study</td>
		</tr>
	</tbody>
</table>

evaluating the effect of continuous drug infusion into the mouse brain on peripheral glucose levels

Source: Ajoy, R., et. al. <a href="https://app.jove.com/v/56410">Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain.</a> J. Vis. Exp. (2018)
This video demonstrates the effect of continuous drug infusion into the mouse brain on blood glucose levels. Glucose metabolism is assessed by collecting blood from a fasted mouse&#39;s tail and measuring glucose levels before and after oral glucose administration. Insulin tolerance is evaluated by injecting insulin in the mouse, with glucose levels recorded at specific intervals.

<img alt="Figure 1" src="/files/ftp_upload/23258/23258_Figure1.jpg" /> 
Figure 1. Osmotic pump preparation and implantation surgical procedure in mouse. (A) The brain infusion kit and pump preparation perfused with drug solution. Red arrows indicate the catheter tubes filled with liquid. (B) Fix and mount the mouse head onto the stereotactic apparatus. (C) Separate the outermost layer of the skin from the subcutaneou...

Evaluating the Effect of Continuous Drug Infusion into the Mouse Brain on Peripheral Glucose Levels

Source: Ajoy, R., et. al. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse ...

Begin with a mouse implanted with a micro-osmotic pump, which continuously delivers a drug directly into the brain over a set period.
This drug is an antagonist of a chemokine that binds to its receptor, blocking hypothalamic insulin signaling and impairing glucose metabolism.
Fast the mouse for the desired time interval.
Pick up the mouse and stroke the tail to ensure sufficient blood flow.
Cut off a small portion of the tail to obtain a drop of blood.
Place the blood drop onto the glucose chip inserted into a glucometer and record the glucose level.
Then, feed the mouse a glucose solution orally.
Measure the glucose levels at desired time intervals.
Persistently high glucose levels show impaired glucose metabolism by the drug.

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31 Views. Source: Ajoy, R., et. al. Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain. J. Vis. Exp. (2018) This video demonstrates the effect of continuous drug infusion into the mouse brain on blood glucose levels. Glucose metabolism is assessed by collecting blood from a fasted mouse's tail and measuring glucose levels before and after oral glucose administration. Insulin tolerance is evaluated by injecting insulin...

Video: Evaluating the Effect of Continuous Drug Infusion into the Mouse Brain on Peripheral Glucose Levels - Concept

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 ...

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Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and autism. As evidence suggests that remyelination can effectively ameliorate the disease symptoms, there is an increasing focus on drug development to promote the myelin regeneration process. Thus, a region-selectable and result-reliable drug delivery technique is required to test the efficiency and specificity of these drugs in vivo. This protocol introduces the osmotic pump implant as a new drug delivery approach in the lysolecithin-induced demyelination mouse model. The osmotic pump is a small implantable device that can bypass the blood-brain barrier (BBB) and deliver drugs steadily and directly to specific areas of the mouse brain. It can also effectively improve the bioavailability of drugs such as peptides and proteins with a short half-life. Therefore, this method is of great value to the field of central nervous system myelin regeneration research.

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Demyelination takes place in multiple central nervous system diseases. A reliable in vivo drug delivery technique is necessary for remyelinating drug testing. This protocol describes an osmotic pump-based method that allows long-term drug delivery directly into the brain parenchyma and improves the drug bioavailability, with broad application in remyelination research.

Demyelination has been identified in not only multiple sclerosis (MS), but also other central nervous system diseases such as Alzheimer's disease and ...

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.

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 ...

Evaluating the Effect of Continuous Drug Infusion into the Mouse Brain on Peripheral Glucose Levels

Transkrypcja

Evaluating the Effect of Continuous Drug Infusion into the Mouse Brain on Peripheral Glucose Levels

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Osmotic Pump-based Drug-delivery for In Vivo Remyelination Research on the Central Nervous System

Simple Continuous Glucose Monitoring in Freely Moving Mice