In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes

Summary

In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.

Abstract

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

Introduction

ISF comprises 15-20% of total brain volume and offers a microenvironment critical for signal transduction, substrate transport and waste clearance¹. Therefore, the ability of collecting ISF from living animals will provide greater implications for various biological processes as well as disease mechanism. In vivo microdialysis is one of the few methods that sample and quantify extracellular molecules from ISF from awake, freely moving animals and thereby serves as a useful tool in neuroscience research field^2,3. In this method, microdialysis probes with semipermeable membranes ....

Protocol

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Graduate School of Medicine at the University of Tokyo.

1. Pre-surgical Procedure

1. Before starting surgery, wipe everything with 70% ethanol to maintain sterile conditions. Thermal support using a heating pad is recommended.
2. Anesthetize the mice by intraperitoneal injection of chloral hydrate (400 mg/kg). Confirm anesthetization by performing a toe pinch. Use of meloxicam SR at induction and buprenorphine upon recovery BID for at least 24 h is recommended.
3. Shave the hair with a surgical clippe....

Results

To stimulate or inhibit neuronal activity in reverse microdialysis^11,12,13, picrotoxin, GABA_A receptor antagonist or tetrodotoxin, Na⁺ channel blocker have been used. It has been shown that tau release is stimulated by increase of neuronal activity^13,14. Consistent with these previous observations, when 50 µM picroto.......

Discussion

Microdialysis with high molecular weight cut off membranes has to be operated by a push-pull mode, thus it is critical that the flow rate is accurate and constant. The inaccuracy in the flow rates can be the cause of air bubble generation and inconsistency in the sample concentration. If the flow is inconsistent, check all connections for leakage. If the problem still persists, it may be necessary to re-start with new probes and tubings.

Microdilaysis probes are continuously perfused by the pe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
The Univentor 820 Microsampler	Univentor	8303002	Refrigerated fraction collector
Syringe pump	KD scientific	KDS-101
Roller pump	Eicom microdialysis	ERP-10
Raturn Stand-Alone System	BASi	MD-1409	Free-moving system
Dual species cage kit	BASi	CX-1600
AtmosLM Microdialysis probe (shaft length 8 mm, membrane length 2 mm)	Eicom microdialysis	PEP-8-02	Shaft length for a probe, a guide, a dumy probe and a stereotaxic adaptor should be identical.
Microdialysis guide (shaft length 8 mm)	Eicom microdialysis	PEG-8
Microdialysis dummy probe (shaft length 8 mm)	Eicom microdialysis	PED-8
Bone screw	BASi	MD-1310
Super bond C&B set	Sunmedical		Dental cement
Small animal Stereotaxic Instrument with digital display console	Kopf	Model 940	Stereotaxic apparatus
Mouse and neonatal rat adaptor	Stoelting	51625
Standard Ear Bars and Rubber Tips for Mouse Stereotaxic	Stoelting	51648
Albumin solution from bovine serum	Sigma	A7284-50ML	30% BSA solution
FEP tubing (70 cm)	Eicom microdialysis	JF-10-70	Internal volume = 0.5 µL/cm
Teflon tubing (50 cm)	Eicom microdialysis	JT-10-50	Internal volume = 0.08 µL/cm
Byton tube	Eicom microdialysis	JB-30
Intramedic luer stab adaptor 23G	BD	427565	Blunt end needle
Roller tube	Eicom microdialysis	RT-5S	Internal volume = 4 µL
Cap nut	Eicom microdialysis	AC-5
0.25 mL microcentrifuge tube with cap	QSP	503-Q	Tubes for fraction collector
Sterotaxic adaptor (shaft length 8 mm)	Eicom microdialysis	PESG-8
Connection needle	Eicom microdialysis	RTJ
Mouse animal collar	BASi	MD-1365
High Speed Rotary Micromotor kit	FOREDOM	K.1070	Drill
Picrotoxin	Sigma	P1675
Screw driver for bone screws
Scalpel
Cotton swab
Surgical clipper