Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

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 function^1,2,3. For example, diabetes increases the risk of cognitive decline and neurodegenerative disorders, including Alzheimer's disease^3,4. Moreover, people with diabetes have defective glucose sensing in the brain^5,6. 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 infusing glucose into the lateral ventricle, they were single housed to prevent any damage to the implants from other mice.

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.......

Materials

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