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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 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 µL/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's disease and/or other neurodegenerative disorders associated with diabetes.
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'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.
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
2. Surgery to implant osmotic pumps
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.
4. CSF collection procedure
5. Glucose assay
6. Blood glucose assay
Male mice were implanted with a cannula assembled to an osmotic minipump (Figure 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...
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 will be useful in delineating the effects of long-term hyperglycorrhachia on cognition, systemic glucose metabolism, and energy balance and for better understanding the pathogenesis of diabetes complications.
Chronic diabetes causes brain damage that inte...
The authors declare that they have no conflict of interests.
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.
Name | Company | Catalog Number | Comments |
0.22 µm syringe filter | Membrane solutions | SFPES030022S | |
1 mL sterile Syringe (Luer-lok tip) | BD | 309628 | |
1 mL TB syringe | BD | 309659 | |
100 mL Glass beaker | Fisher | N/a | |
100% Ethanol (Koptec) | DLI | UN170 | Use 70% dilution to clean the surgery area |
50 mL conical tube | Fisher | N/A | |
Allignment indicator | KOPF | 1905 | |
Alzet brain infusion kit | DURECT | Kit # 3; 0008851 | Cut tubing in the kit to 1 inch length |
Alzet osmotic pump | DURECT | 2004 | Flow rate 0.25 µL/h |
Anesthesia system | Kent Scientific | SomnoSuite | |
Betadine solution | Avrio Health | N/A | |
CaCl2 . 2H2O | Fisher | C79-500 | |
Cannula holder | KOPF | 1966 | |
Centering scope | KOPF | 1915 | |
Dental Cement Liquid | Lang Dental | REF1404 | |
Dental cement Powder | Lang Dental | REF1220-C | |
D-glucose | Sigma | G8270 | |
Electric drill | KOPF | 1911 | While drilling a hole avoid rupturing dura mater |
Eye lubricant (Optixcare) | CLC Medica | N/A | |
Glass Bead sterilizer (Germinator 500) | VWR | 101326-488 | Place instruments in sterile water to let them cool before surgery |
Glucose Assay Kit | Cayman chemical | 10009582 | |
H2O2 | Sigma | H1009-500ml | Apply 3% H2O2 on skull surface to make the cranial sutures visible. |
Hair Clipper | WAHL | N/A | |
heating pad | Heatpax | 19520483 | |
Hemostat | N/A | N/A | |
Isoflurane (Fluriso) | Zoetis | NDC1385-046-60 | |
KCl | VWR | 0395-500g | |
Magnetic stand | WPI | M1 | |
Magnifying desk lamp | Brightech | LightView Pro Flex 2 | |
Metal Spatula | N/A | N/A | |
MgCl2 . 6H2O | Fisher | BP214-500 | |
Micromanipulator (Right handed) | WPI | M3301R | |
Micromanipulator with digital display | KOPF | 1940 | |
Na2HPO4 . 7H2O | Fisher | S373-500 | |
NaCl | Sigma | S7653-5Kg | |
NaH2PO4 . H2O | Fisher | S369-500 | |
Neosporin | Johnson & Johnson | N/A | Apply topical oinment to prevent infection |
Parafilm | Bemis | DM-999 | |
Rimadyl (Carprofen) 50mg/ml | Zoetis | N/A | 5 mg/kg, subcutaneous, for analgesia |
Scalpel | N/A | N/A | |
Stereotaxic allignment system | KOPF | 1900 | |
Sterile 27 gauge needle | BD | 305109 | |
Sterile cotton tip applicators (Solon) | AMD Medicom | 56200 | |
Sterile nylon sutures (5.0) | Oasis | MV-661 | Use non-absorable suture for closing the wound |
Sterile sharp scissors | N/A | N/A | |
Sterile surgical blades | VWR | 55411-050 | |
Surgical gloves (Nitrile) | Ammex | N/A | Change gloves if there is suspision of contamination |
Tray | N/A | N/A |
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