Name: osmotic minipump implantation for increasing glucose concentration in mouse cerebrospinal fluid
Uploaded: 2023-04-07T14:20:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid at JoVE.com

National Institutes of Health grant DK124619 to KHC.
Start-up funds and pilot research award, Department of Medicine, University of Rochester, NY, to KHC.
The Del Monte Institute for Neuroscience Pilot Research Award, University of Rochester, to KHC.
University Research Award, Office of the Vice President for Research, University of Rochester, NY, to KHC.
MUR designed and performed the method, analyzed results, prepared graphs and figures, and wrote and edited the manuscript. KHC conceived and supervised the study, analyzed results, and wrote and edited the manuscript. KHC is the guarantor of this work. All authors approved the final version of the manuscript.

All mouse procedures were approved by the Institutional Animal Care and Use Committee at the University of Rochester and were performed according to the US Public Health Service guidelines for the humane care and use of experimental animals. Six weeks old C57BL/6J male mice used for this study were commercially obtained. All the animals were group housed (5 mice per cage) in a room with a 12 h day/night cycle and were given access to food and water ad libitum. After the mice were implanted with a cannula for infusing glucose into the lateral ventricle, they were single housed to prevent any damage to the implants from other mice.
1. Assembly of osmotic minipumps
<ol>
	<li>Prepare artificial cerebrospinal fluid (aCSF) according to the following protocol. Dissolve NaCl (8.66 g), KCl (0.224 g), CaCl2.2H2O (0.206 g), and MgCl2.6H2O (0.163 g) in 500 mL of sterile water to prepare solution A. Dissolve Na2HPO4.7H2O (0.214 g) and NaH2PO4.H2O (0.027 g) in 500 mL of sterile water to prepare solution B. Then, mix solution A and B in a 1:1 ratio to prepare the aCSF. 
	NOTE: This formulation of aCSF contains no glucose.</li>
	<li>To prepare the 50% glucose solution, add 50 g of glucose to 50 mL of aCSF in a beaker. Place the beaker on a hot plate and bring the temperature of the suspension to 60&#160;&#176;C. Mix the suspension with a magnetic stirrer until the glucose is completely dissolved.</li>
	<li>Add aCSF to the beaker to make the final volume 100 mL. Pass the solution through 0.22 &#181;m pore filter to sterilize it.</li>
	<li>Prepare a 0.9% saline (NaCl) solution and pass the solution through the 0.22 &#181;m filter.</li>
	<li>Place all the components of the minipump and infusion kit in a sterile tray and cut the tubing from the brain infusion kit to 1 inch in length with a sterile scissor. Hold the tubing with a sterile hemostat and push it on the top of one end of the cannula. Connect the other end of the tubing to the top side of the flow modulator. Cover both ends of the tubing with a small amount of glue for a stronger connection.</li>
	<li>Attach a 27 G needle to a 1 mL syringe and fill it with either aCSF or the glucose solution. Insert the 27 G needle into a small piece of tubing. Then, connect the tubing to the open end of the flow modulator and fill it with the corresponding solution until drops of the solution start coming out from the cannula. Remove any air bubbles trapped in the tubing.</li>
	<li>To fill the minipump, hold the pump upright and insert the syringe needle into the pump. Fill it with the corresponding solution until a drop of the solution starts forming at the opening of the minipump.</li>
	<li>Slowly insert the flow modulator into the pump while making sure not to introduce any air bubbles in the assembly.</li>
	<li>After the osmotic minipumps are assembled, transfer them to a 50 mL conical tube. Fill the tube with the sterile saline so that the pumps are completely submerged, while making sure that the cannula stays out of the saline solution. Four such assembled pumps can be placed in one 50 mL tube.</li>
	<li>Loosely place the cap on each tube and incubate the pumps at 37&#160;&#176;C to prime for at least 48 h.</li>
</ol>
2. Surgery to implant osmotic pumps
<ol>
	<li>Pre-surgery procedure
	<ol>
		<li>Sterilize the surgical instruments in an autoclave and let them cool down for at least 30 min before use. The autoclave conditions involve steam sterilization at 121 &#176;C for 30 min.</li>
		<li>Disinfect the surgery area with 70% ethanol. Wipe the knobs, ear bars, and stereotaxic frame surface. 
		NOTE: Surgery should be performed in an aseptic environment, keeping in mind to avoid touching non-sterile surfaces while performing surgery. Replace gloves if there is a suspicion of contamination.</li>
		<li>Place the mouse in an anesthesia chamber to anesthetize it with 3% isoflurane mixed in air for 3-5 min. Then, remove the mouse from the chamber and place it on a sterile drape. Maintain anesthesia via a nose cone at 1.5%-2%.</li>
		<li>Pinch the mouse hind paw to observe any reflex and verify the depth of anesthesia.</li>
		<li>Inject 5 mg/kg carprofen subcutaneously for analgesia.</li>
		<li>Use clean clippers to clip the surgery area on the mouse head. Rub the scalp first with povidone-iodine solution and then with alcohol swabs thoroughly for at least 2 min. Repeat this process at least three times.</li>
		<li>Apply an eye lubricant to prevent the eyes from drying during surgery.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Place the mouse on the stereotaxic frame and fit the head on the incisor bar. Cover the nose with the nose cone. Apply sterile drapes to secure the surgical area.</li>
		<li>Shift the flow of isoflurane to the nose cone. Continue to maintain the anesthesia at&#160;1.5%-2% isoflurane.</li>
		<li>Place a heating pad under the mouse to maintain the body temperature.</li>
		<li>Put on new sterile gloves and, with a scalpel, make a midline incision (~10-15 mm long) on the scalp. Expose the skull surface with a spatula while using cotton tips to wipe any bleeding. 
		NOTE: Use a cauterizer if bleeding profusely, which is rare.</li>
		<li>Rub with a cotton tip dipped in 3% hydrogen peroxide to expose the cranial sutures.</li>
		<li>Take note of bregma and lambda to navigate the skull coordinates. Set all the coordinates to zero at bregma. 
		NOTE: These coordinates were obtained from a mouse brain atlas14. The reference bregma-lambda distance in the atlas is 4.21 mm. To correct for age- and sex-based differences in skull size, the bregma-lambda distance for each mouse was measured and then divided by 4.21 mm to obtain a multiplication factor. For example, if the distance between bregma and lambda was 4.65 mm, division by 4.21 mm gave a multiplication factor of 1.10. All the coordinates obtained from the atlas were then multiplied with this factor to measure the actual coordinates that were used to drill the hole. In the above example, the resulting anterior-posterior (AP), and medial-lateral (ML) coordinates would be 0.55 and 1.1 mm, respectively (i.e., 1.1 x 0.5 or 1 mm).</li>
		<li>Use an electric drill to make a hole at these coordinates: 0.5 mm posterior to bregma and 1 mm lateral to midline toward the right.&#160; 
		NOTE: While drilling the hole in the skull, be careful not to rupture the dura mater. Make sure the drill bit is sterile. It can be placed with other instruments in the same autoclave pouch for sterilization.</li>
		<li>Insert a hemostat under the incised skin and push it to the place where the osmotic minipump will be implanted. Gently open and close the hemostat to make a pocket for the minipump.</li>
		<li>Use a sterile hemostat to pick&#160;a primed osmotic minipump from the tip of the flow modulator. Push the pump through the incised skin into the pocket. 
		NOTE: Be careful to not let the pump and the cannula touch any non-sterile surface.</li>
		<li>Apply some glue on the underside of the cannula and fix it in the cannula holder. Lower the cannula slowly through the drilled hole 2 mm ventral to the skull surface. Leave it there for at least 5 min to allow the glue to solidify.</li>
		<li>Before inserting the cannula down into the brain, make sure that the cannula is not clogged and that the solution is flowing properly. 
		NOTE: Sometimes, glucose may deposit at the tip of the cannula due to a very low flow rate, blocking the flow.</li>
		<li>With the help of a scissor, clip the top of the cannula making sure that it doesn&#39;t dislodge from the skull surface.</li>
		<li>Cover the cannula with a layer of dental cement for extra support.</li>
		<li>Use nylon sutures (5-0 size and 30 inches in length) to close the wound and apply topical antibiotic on the wound to prevent infection.</li>
		<li>Turn off the isoflurane and let the animal recover from the anesthesia. Then, transfer the animal to a new clean cage.</li>
	</ol>
	</li>
	<li>Post-surgery care
	<ol>
		<li>After surgery, monitor the mouse for at least 1 week.</li>
		<li>Administer 5 mg/kg carprofen subcutaneously, once every 24 hours for three days post-surgery.</li>
		<li>Let the wound heal completely before removing the sutures 7-10 days after surgery. 
		&#8203;NOTE: It was observed that mice with high CSF glucose took longer to heal. Trimming nails on mouse&#39;s hind paws minimizes the chance of injury to the site of the incision due to the scratching behavior of mice.</li>
	</ol>
	</li>
</ol>
3. Replacement of the minipumps
NOTE: Since, the minipumps used in this study last only for 4 weeks, the replacement of minipumps was also tested to extend the duration of glucose infusion, as it may be required in the case of long-term studies. This involved the following steps.
<ol>
	<li>Prepare the mice for surgery as detailed above.</li>
	<li>Make a small 1 cm vertical incision in the skin slightly above the pump.</li>
	<li>Insert a hemostat under the incised skin and pull the pump out.</li>
	<li>Remove the pump from the tubing and connect a new primed pump to the tubing.</li>
	<li>Insert the pump back inside and stitch the skin.</li>
	<li>Follow the same post-surgery care instructions as described above.</li>
</ol>
4. CSF collection procedure
<ol>
	<li>Preparation
	<ol>
		<li>Pull 1 mm diameter glass capillaries with a micropipette puller machine to obtain 0.5 mm diameter tips. The settings are: heat = 800, pull = 15, velocity = 5, and time = 200 units. Place the pulled capillaries in a sterile box till use.</li>
		<li>Attach a 27 G needle to a 1 mL syringe. Insert the pulled capillary on the needle and use a small adhesive tape to secure the connection.</li>
		<li>Firmly fix the syringe in a capillary holder of a micromanipulator.</li>
		<li>Next, prepare the mouse for surgery, as described in the pre-surgery procedure above.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Place the mouse on the stereotaxic frame. After fixing the head on the frame, as described previously, rotate the knobs to tilt the head so that the nose is facing downward. Apply sterile&#160;drapes to secure the surgical area.</li>
		<li>Make a small incision on the dorsal surface, starting from the intra-aural plane to the caudal side. 
		NOTE: Be careful not to damage the tubing while making the incision.</li>
		<li>Remove the support from the mouse body and let the mouse rest vertically so that the neck is fully extended dorsally.</li>
		<li>With the help of curved blunt forceps, gently bisect the posterior neck muscles from the midline to create a small window.&#160;Then, use a wet cotton tip applicator to gently displace the neck muscles from&#160;mid-line to&#160;the periphery.</li>
		<li>Observe whether the cisterna magna is exposed, which appears as a triangular window with a transparent dura membrane.</li>
		<li>Place the micromanipulator assembly on the stereotaxic frame next to the mouse while making sure that the capillary tip does not touch any surface.</li>
		<li>Gently break the capillary tip without disturbing the setup.</li>
		<li>While looking into the magnifying glass (magnification of 2.25x), rotate the corresponding knobs on the micromanipulator to slowly align and move the capillary tip toward the cisterna magna.</li>
		<li>Some resistance is felt once the capillary tip touches the cisterna magna membrane. Very slowly push the tip against the membrane with the help of the knobs. 
		NOTE: Be careful not to damage any blood vessels in the membrane. This step is very important to avoid any blood contamination in the CSF.</li>
		<li>Very gently pierce the membrane. CSF will start flowing into the capillary at once due to negative pressure in the capillary.</li>
		<li>Leave the setup for few minutes until ~10 &#181;L of CSF is collected in the capillary.</li>
		<li>Then, slowly draw the capillary out of the cisterna magna and gently press a sterile cotton tip applicator against the opening of the cisterna magna to stop CSF leakage.</li>
		<li>Carefully remove the capillary from the syringe and attach it to a microcap bulb dispenser. Press the bulb to transfer the CSF into a sterile microcentrifuge tube.</li>
		<li>Place a support under the mouse and rotate the stereotaxic knobs to level the head.</li>
		<li>Close the wound with nylon sutures.</li>
		<li>Inject 300 &#181;L of sterile saline subcutaneously before removing the mouse from the apparatus.</li>
		<li>Give post-operative care to the animals&#160;as described above, until it is time to remove the sutures-~7-10 days after surgery.</li>
	</ol>
	</li>
</ol>
5. Glucose assay
<ol>
	<li>Follow the protocol as described in the glucose colorimetric assay kit.</li>
	<li>Dilute the sodium phosphate buffer stock solution provided in the kit to 50 mM concentration in ultra-pure water.</li>
	<li>Prepare glucose standards in 0, 2.5, 5, 7.5, 10, 15, 20, 25, and 50 mg/dL ranges from the 1,000 mg/dL stock solution provided in the kit in diluted buffer solution.</li>
	<li>Prepare a 7 fold&#160;dilution of CSF samples in the buffer.&#160;For example, if the total CSF volume collected is 10 &#181;L, then the seven fold diluted volume will be 70 &#181;L.</li>
	<li>Pipette 15 &#181;L of glucose standards and diluted CSF in duplicates in a 96-well plate.</li>
	<li>Pipette 85 &#181;L of diluted buffer in each of the wells with standards and CSF.</li>
	<li>Add 6 mL of diluted buffer to the glucose calorimetric enzyme mixture vial and vortex it for a few seconds to mix thoroughly.</li>
	<li>Add 100 &#181;L of the enzyme mixture preparation to each well to start the reaction.</li>
	<li>Seal the plate with a cover sheet and tap on the plate gently to mix the reagents.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 10 min. Vials with high glucose start to turn violet immediately.</li>
	<li>After 10 min, remove the cover and measure the absorbance at 500-520 nm using a plate reader.</li>
	<li>Calculate the glucose concentration in the CSF samples by interpolating their values from the standard curve.</li>
</ol>
6. Blood glucose assay
<ol>
	<li>Make a small lateral nick to the mouse tail using a razor blade and use a drop of blood to measure the blood glucose with a glucometer, according to the manual instructions.</li>
</ol>

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The authors declare that they have no conflict of interests.

This article reports a detailed protocol to increase CSF glucose in mice by using osmotic minipumps connected to a cannula implanted in the lateral ventricle. The chronic infusion of glucose in the mouse brain through this procedure&#160;will be useful in delineating the effects of long-term hyperglycorrhachia on cognition, systemic glucose metabolism, and energy balance&#160;and for better understanding the pathogenesis of diabetes complications.
Chronic diabetes causes brain damage that inte...

Both type 1 and type 2 diabetes impair brain function1,2,3. For example, diabetes increases the risk of cognitive decline and neurodegenerative disorders, including Alzheimer&#39;s disease3,4. Moreover, people with diabetes have defective glucose sensing in the brain5,6. This defect contributes to the pathogenesis of hypoglycemia associated unawareness and an insufficient counter-regulatory response to hypoglycemia7,8, which can be fatal if not treated immediately.
Considering that diabetes increases glucose levels in the blood as well as in cerebrospinal fluid (CSF)9, it is important to determine whether one or both of these factors contribute to impaired brain function. Whether diabetes causes brain damage by high CSF glucose alone or in combination with other factors like insulin deficiency or insulin resistance is also an open question. Animal models of type 1 and type 2 diabetes show cognitive decline and neurodegeneration in addition to an affected energy balance and peripheral glucose metabolism10,11,12,13. However, from these models, it is not feasible to uncouple the selective effects of high CSF glucose versus blood glucose levels in mediating the complications of diabetes on brain function.
This protocol describes methods to develop a mouse model of hyperglycorrhachia to test the effects of chronically high CSF glucose levels on brain function, energy balance, and glucose homeostasis. The mouse model developed through this technique presents a tool for studies investigating the etiological role of dysregulated glucose homeostasis on neural and behavioral function.
Therefore, the proposed approach will be useful in understanding the direct effects of elevated CSF glucose levels in various pathophysiological conditions.

<table><tbody><tr><td>0.22 &amp;micro;m syringe filter</td><td>Membrane solutions</td><td>SFPES030022S</td><td></td></tr><tr><td>1 mL sterile Syringe (Luer-lok tip)</td><td>BD</td><td>309628</td><td></td></tr><tr><td>1 mL TB syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>100 mL Glass beaker</td><td>Fisher&amp;nbsp;</td><td>N/a</td><td></td></tr><tr><td>100% Ethanol (Koptec)</td><td>DLI</td><td>UN170</td><td>Use 70% dilution to clean the surgery area</td></tr><tr><td>50 mL conical tube</td><td>Fisher&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Allignment indicator</td><td>KOPF</td><td>1905</td><td></td></tr><tr><td>Alzet brain infusion kit</td><td>DURECT</td><td>Kit # 3; 0008851</td><td>Cut tubing in the kit to 1 inch length</td></tr><tr><td>Alzet osmotic pump</td><td>DURECT</td><td>2004</td><td>Flow rate 0.25 &amp;micro;L/h</td></tr><tr><td>Anesthesia system</td><td>Kent Scientific</td><td>SomnoSuite</td><td></td></tr><tr><td>Betadine solution</td><td>Avrio Health</td><td>N/A</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; . 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher&amp;nbsp;</td><td>C79-500</td><td></td></tr><tr><td>Cannula holder</td><td>KOPF</td><td>1966</td><td></td></tr><tr><td>Centering scope</td><td>KOPF</td><td>1915</td><td></td></tr><tr><td>Dental Cement Liquid</td><td>Lang Dental</td><td>REF1404</td><td></td></tr><tr><td>Dental cement Powder</td><td>Lang Dental</td><td>REF1220-C</td><td></td></tr><tr><td>D-glucose&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Electric drill</td><td>KOPF</td><td>1911</td><td>While drilling a hole avoid rupturing dura mater</td></tr><tr><td>Eye lubricant (Optixcare)</td><td>CLC Medica</td><td>N/A</td><td></td></tr><tr><td>Glass Bead sterilizer (Germinator 500)</td><td>VWR</td><td>101326-488</td><td>Place instruments in sterile water to let them cool before surgery</td></tr><tr><td>Glucose Assay Kit</td><td>Cayman chemical</td><td>10009582</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>H1009-500ml</td><td>Apply 3% H2O2 on skull surface to make the cranial sutures visible.</td></tr><tr><td>Hair Clipper</td><td>WAHL</td><td>N/A</td><td></td></tr><tr><td>heating pad</td><td>Heatpax</td><td>19520483</td><td></td></tr><tr><td>Hemostat</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Isoflurane (Fluriso)</td><td>Zoetis</td><td>NDC1385-046-60</td><td></td></tr><tr><td>KCl</td><td>VWR</td><td>0395-500g</td><td></td></tr><tr><td>Magnetic stand</td><td>WPI</td><td>M1</td><td></td></tr><tr><td>Magnifying desk lamp</td><td>Brightech</td><td>LightView Pro Flex 2</td><td></td></tr><tr><td>Metal Spatula</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; . 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher&amp;nbsp;</td><td>BP214-500</td><td></td></tr><tr><td>Micromanipulator (Right handed)</td><td>WPI</td><td>M3301R</td><td></td></tr><tr><td>Micromanipulator with digital display</td><td>KOPF</td><td>1940</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; . 7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher&amp;nbsp;</td><td>S373-500</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653-5Kg</td><td></td></tr><tr><td>NaH2PO&lt;sub&gt;4&lt;/sub&gt; . H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher&amp;nbsp;</td><td>S369-500</td><td></td></tr><tr><td>Neosporin</td><td>Johnson &amp;amp; Johnson</td><td>N/A</td><td>Apply topical oinment to prevent infection</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>DM-999</td><td></td></tr><tr><td>Rimadyl (Carprofen) 50mg/ml</td><td>Zoetis</td><td>N/A</td><td>5 mg/kg, subcutaneous, for analgesia</td></tr><tr><td>Scalpel</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Stereotaxic allignment system</td><td>KOPF</td><td>1900</td><td></td></tr><tr><td>Sterile 27 gauge needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Sterile cotton tip applicators (Solon)</td><td>AMD Medicom</td><td>56200</td><td></td></tr><tr><td>Sterile nylon sutures (5.0)</td><td>Oasis</td><td>MV-661</td><td>Use non-absorable suture for closing the wound</td></tr><tr><td>Sterile sharp scissors&amp;nbsp;</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Sterile surgical blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Surgical gloves (Nitrile)</td><td>Ammex</td><td>N/A</td><td>Change gloves if there is suspision of contamination</td></tr><tr><td>Tray</td><td>N/A</td><td>N/A</td><td></td></tr></tbody></table>

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.

Male mice were implanted with a cannula assembled to an osmotic minipump (Figure&#160;1) to chronically infuse aCSF or a 50% glucose solution into their lateral ventricles (Figure 2). CSF was collected 10 days after the surgery (Figure 3) to validate the efficacy of this procedure. The results showed an increase in the CSF glucose levels (mean: 327.7 mg/dL) in mice infused with 50% glucose compared to that (mean: 56.5 mg/dL) in mice...

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Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

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 ...

osmotic-minipump-implantation-for-increasing-glucose-concentration

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

1.3K Views.  University of Rochester School of Medicine and Dentistry. 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.

Video: Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

In order to study brain reorganization under pathological conditions we used miniosmotic pumps for direct protein delivery into the brain circumventing the blood brain barrier. Tract tracers are then injected to study alterations in brain connectivity under the influence of the protein.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and ...

Neuroscience

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 ...

Medicine

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...

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 ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

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, ...

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Source: Ajoy, R., Chou, S. Y. <a href="https://jove.unicartagenaproxy.elogim.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)
In this video, we demonstrate the assembly of a micro-osmotic pump brain infusion system followed by its surgical implantation in the mouse skull. This system enables continuous delivery of potential drugs and facilitates the study of their effects on the brain and brain tissue.

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

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Rodent Models

Adult

Microdialysis of Interstitial Fluid from a Mouse Brain to Isolate Extracellular Proteins

Source: Yamada, K. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57869">In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes.</a> J. Vis. Exp. (2018)
The video demonstrates the process of using a microdialysis probe to collect extracellular proteins from the interstitial fluid (ISF) of a mouse brain. A microdialysis probe, equipped with a high-molecular-weight cut-off semi-permeable membrane, is inserted into the brain of an anesthetized mouse via an implanted guide cannula. The probe, connected to pumps, allows a physiological buffer to perfuse the brain tissue and mix with the ISF. Proteins in the ISF diffuse across the semi-permeable membrane and are collected for analysis.

Source: Yamada, K. In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off ...

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Summary

Explore More Videos

Chapters in this video

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

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

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Continuous Drug Infusion System in Mouse Model: A Surgical Procedure to Implant Micro-osmotic Pump Infusion System in the Mouse Brain for Continuous Drug Delivery

Microdialysis of Interstitial Fluid from a Mouse Brain to Isolate Extracellular Proteins