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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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 ....

Materials

NameCompanyCatalog NumberComments
0.22 µm syringe filterMembrane solutionsSFPES030022S
1 mL sterile Syringe (Luer-lok tip)BD309628
1 mL TB syringeBD309659
100 mL Glass beakerFisher N/a
100% Ethanol (Koptec)DLIUN170Use 70% dilution to clean the surgery area
50 mL conical tubeFisher N/A
Allignment indicatorKOPF1905
Alzet brain infusion kitDURECTKit # 3; 0008851Cut tubing in the kit to 1 inch length
Alzet osmotic pumpDURECT2004Flow rate 0.25 µL/h
Anesthesia systemKent ScientificSomnoSuite
Betadine solutionAvrio HealthN/A
CaCl2 . 2H2OFisher C79-500
Cannula holderKOPF1966
Centering scopeKOPF1915
Dental Cement LiquidLang DentalREF1404
Dental cement PowderLang DentalREF1220-C
D-glucose  SigmaG8270
Electric drillKOPF1911While drilling a hole avoid rupturing dura mater
Eye lubricant (Optixcare)CLC MedicaN/A
Glass Bead sterilizer (Germinator 500)VWR101326-488Place instruments in sterile water to let them cool before surgery
Glucose Assay KitCayman chemical10009582
H2O2SigmaH1009-500mlApply 3% H2O2 on skull surface to make the cranial sutures visible.
Hair ClipperWAHLN/A
heating padHeatpax19520483
HemostatN/AN/A
Isoflurane (Fluriso)ZoetisNDC1385-046-60
KClVWR0395-500g
Magnetic standWPIM1
Magnifying desk lampBrightechLightView Pro Flex 2
Metal SpatulaN/AN/A
MgCl2 . 6H2OFisher BP214-500
Micromanipulator (Right handed)WPIM3301R
Micromanipulator with digital displayKOPF1940
Na2HPO4 . 7H2OFisher S373-500
NaClSigmaS7653-5Kg
NaH2PO4 . H2OFisher S369-500
NeosporinJohnson & JohnsonN/AApply topical oinment to prevent infection
ParafilmBemisDM-999
Rimadyl (Carprofen) 50mg/mlZoetisN/A5 mg/kg, subcutaneous, for analgesia
ScalpelN/AN/A
Stereotaxic allignment systemKOPF1900
Sterile 27 gauge needleBD305109
Sterile cotton tip applicators (Solon)AMD Medicom56200
Sterile nylon sutures (5.0)OasisMV-661Use non-absorable suture for closing the wound
Sterile sharp scissors N/AN/A
Sterile surgical bladesVWR55411-050
Surgical gloves (Nitrile)AmmexN/AChange gloves if there is suspision of contamination
TrayN/AN/A

References

  1. Moheet, A., Mangia, S., Seaquist, E. R. Impact of diabetes on cognitive function and brain structure. Annals of the New York Academy of Sciences. 1353, 60-71 (2015).
  2. Takeda, S., et al.

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Osmotic Minipump ImplantationCSF Glucose LevelsMouse BrainCognitive DysfunctionNeurodegenerative DisordersStereotaxic SurgeryBregmaDura MaterCannulaDental Cement

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