Name: osmotic minipump implantation for increasing glucose concentration in mouse cerebrospinal fluid
Uploaded: 2023-04-07T14:20:00
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 inf...

<ol>
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Molecular Basis of Disease. 1863 (1), 266-273 (2017).</li><li>Ernst, 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=Diabetic+db/db+mice+exhibit+central+nervous+system+and+peripheral+molecular+alterations+as+seen+in+neurological+disorders.">Diabetic db/db mice exhibit central nervous system and peripheral molecular alterations as seen in neurological disorders.</a> Translational Psychiatry. 3 (5), 263 (2013).</li><li>Wang, Y., Yang, Y., Liu, Y., Guo, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+impairments+in+type+1+diabetes+mellitus+model+mice+are+associated+with+synaptic+protein+disorders.">Cognitive impairments in type 1 diabetes mellitus model mice are associated with synaptic protein disorders.</a> Neuroscience Letters. 777, 136587 (2022).</li><li>Jolivalt, C. G., 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=Type+1+diabetes+exaggerates+features+of+Alzheimer's+disease+in+APP+transgenic+mice.">Type 1 diabetes exaggerates features of Alzheimer's disease in APP transgenic mice.</a> Experimental Neurology. 223 (2), 422-431 (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Paxinos and Franklin's The mouse brain in stereotaxic coordinates. , (2019).</li><li>Vinik, A. I., Maser, R. E., Mitchell, B. D., Freeman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+autonomic+neuropathy.">Diabetic autonomic neuropathy.</a> Diabetes Care. 26 (5), 1553-1579 (2003).</li><li>Furman, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptozotocin-induced+diabetic+models+in+mice+and+rats.">Streptozotocin-induced diabetic models in mice and rats.</a> Current Protocols. 1 (4), 78 (2021).</li><li>Grieb, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+streptozotocin+injections+as+a+model+of+Alzheimer's+disease:+in+search+of+a+relevant+mechanism.">Intracerebroventricular streptozotocin injections as a model of Alzheimer's disease: in search of a relevant mechanism.</a> Molecular Neurobiology. 53 (3), 1741-1752 (2016).</li><li>Kealy, J., 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=Acute+inflammation+alters+brain+energy+metabolism+in+mice+and+humans:+role+in+suppressed+spontaneous+activity,+impaired+cognition,+and+delirium.">Acute inflammation alters brain energy metabolism in mice and humans: role in suppressed spontaneous activity, impaired cognition, and delirium.</a> The Journal of Neuroscience. 40 (29), 5681-5696 (2020).</li><li>Dougherty, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;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&#160;</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&#160;</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 &#181;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>CaCl2 . 2H2O</td>
			<td>Fisher&#160;</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&#160;&#160;</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>H2O2</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>MgCl2 . 6H2O</td>
			<td>Fisher&#160;</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>Na2HPO4 . 7H2O</td>
			<td>Fisher&#160;</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653-5Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 . H2O</td>
			<td>Fisher&#160;</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Johnson &#38; 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&#160;</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...

Watch this Scientific Journal Video about Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid at JoVE.com

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 ...

This technique increases the Cerebrospinal Fluid or CSF glucose levels in mice without affecting their blood glucose concentration. This helps to investigate the direct effects of high CSF glucose levels on the animal's brain function. This technique can establish animal models to explore the theological role of higher CSF glucose on cognitive dysfunction.Leading to the discovery of new molecular targets to improve cognitive function. The method will also help determine whether high CSF glucose levels are sufficient to mediate phenotypes of neurodegenerative disorders in mice. Attention must be paid to the proper assembly and priming of the cannula setup and insertion of the cannula in the brain at the correct coordinates.To begin the surgery for Osmotic Minipump Implantation place the anesthetized mouse on the stereotaxic frame. Fit the animal's head on the incisor bar. Cover the nose with the nose cone to maintain the anesthesia and connect isofluorane flow to the nose cone at a rate of 1.5 to 2%Place a heating pad under the mouse to maintain the body temperature.After disinfecting the surgical area and making an incision expose the skull surface with a spatula and wipe the blood, if any, with cotton tips. Use a cauterizer if bleeding profusely. Rub with a cotton tip dipped in 3%hydrogen peroxide to expose the cranial sutures.Working under a microscope, take note of bregma and lambda to navigate the skull coordinates. Set all the coordinates to zero at bregma. Using an electric drill carefully and without rupturing the dura mater make a hole at the coordinates 0.5 millimeters posterior to bregma and 1 millimeter lateral to midline toward the right.Insert a hemostat and gently open and close it to make a pocket for the mini pump. Holding the pump from the tip of the flow modulator push it through the incised skin into the created pocket. Apply some glue on the underside of the cannula, and fix it in the cannula holder.Ensure that the cannula is not clogged and the solution is flowing properly. Then lower the cannula slowly through the drilled hole, two millimeters ventral to the skull surface. Leave it there for at least five minutes to allow the glue to solidify.With the help of scissors, clip the top of the cannula ensuring it doesn't dislodge from the skull surface. Cover the cannula with a layer of dental cement for extra support. For Cerebrospinal Fluid or CSF collection, place the properly anesthetized mouse on the stereotaxic frame.After fixing the head on the frame as demonstrated previously rotate the knobs to tilt the head so that the nose faces downward. After disinfecting the surgical area and making an incision remove the support from the mouse body and let the mouse rest vertically so that the neck is fully extended dorsally. Using curved blunt forceps, gently bisect the posterior neck muscles from the midline to create a small window.Then use a wet cotton tip applicator to gently displace the neck muscles from the midline to the periphery. Observe whether the cisterna magna is exposed as a triangular window with a transparent dura membrane. Place the micro manipulator assembly on the stereotaxic frame next to the mouse while ensuring the capillary tip does not touch any surface.Gently break the capillary tip without disturbing the setup. Rotate the corresponding knobs on the micro manipulator to align slowly and move the capillary tip toward the cisterna magna. 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 being careful not to damage any blood vessels in the membrane. As the membrane is pierced, CSF flows into the capillary at once due to negative capillary pressure. Leave the setup for a few minutes until approximately 10 microliters of CSF collects in the capillary.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 micro centrifuge tube. Place the support under the mouse and rotate the stereotaxic knobs to level the head.Close the wound with nylon sutures. Inject 300 microliters of sterile saline subcutaneously before removing the mouse from the apparatus. Give postoperative care to the animals until it is time to remove the sutures, about 7 to 10 days after surgery.CSF collected 10 days after the surgery showed about a sixfold increase in glucose level in mice, infused with 50%glucose compared to that in mice infused with CSF. However, the blood glucose levels were not different between the groups. While lowering the cannula in the brain, retract it back and check if CSF comes out of the hole.It is also vital to correctly identify cisterna magna and prevent contamination of CSF with blood. This technique can also be used to deliver any drug that is unable to cross the blood-brain barrier to test its effect on brain function. There is a very limited number of animal models of diabetes, and they disturb glucose homeostasis in the peripheral environment.This model, however, helps investigate brain specific changes in glucose regulation on the neural function.

Research

JoVE Journal

Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...