Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Summary

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Abstract

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.

Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.

Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

Introduction

Homeostasis within the central nervous system (CNS) is a prerequisite for the proper communication and function of the neuronal cells. The CNS parenchyma is tightly sealed off from the periphery by the endothelial blood-brain barrier (BBB), which represents the interface between the peripheral bloodstream and the brain and plays a pivotal role in the cross-talk between these two districts^1,2. The BBB is a complex and dynamic three-dimensional structure mainly composed of specialized micro-vessel endothelial cells (ECs) linked to each other through intercellular junctional complexes - tight junctions (TJs)- and....

Protocol

All procedures on animals were approved by the IRCCS Neuromed Animal Care Review Board and by "Istituto Superiore di Sanità" (permit number: 1163/2015- PR) and were in accordance with the guidelines of the EU Directive 2010/63/EU for animal experiments.

1. Preparation of FITC-albumin Solution to be Injected into Jugular Vein

NOTE: All experiments were carried out in manifest (11-week old) HD R6/2 mice and in age and gender-matched wild-type (WT) control littermates.

1. Dissolve FITC-albumin powder (see Table of Materials) in phosphate buffered saline (PBS) at 10 mg/ml.
   ....

Results

Proper infusion of FITC-albumin into the jugular vein results in the extravasation of the green fluorescence tracer from the bloodstream into the brain parenchymawhen the BBB is compromised⁶. Under physiological conditions, distribution of infused fluorescent albumin is restricted to the inside of the brain blood vessels and no signal in the surrounding perivascular area and/or brain parenchyma is detectable (Figure 1A, micrographs on .......

Discussion

The technique we describe here is primarily useful for detecting BBB leakage under brain disease conditions. BBB dysfunction is gaining attention as a common feature of diverse neurologic disorders⁴. We previously used this approach to describe the early derangement of BBB integrity in a mouse model of a rare neurodegenerative disease like HD⁶.

This method takes advantage of the relative simplicity and effectiveness of the procedure, which provid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin-fluorescin isothiocyanate conjiugate	SIGMA	A9771-100MG
pAb anti-Laminin	Novus Biologicals	NB300-144
CY3 anti-rabbit made in goat	MILLIPORE	AP132
SUPERFROST PLUS	Thermo Scientific	J1800AMNZ
Cover Slips 24 X 50 mm	Thermo Scientific (DIAPATH)	61050
Kilik Optimal Cutting Temperature (OCT) compound	Bio Optica	05-9801
VECTASHIELD Mounting Media	VECTOR	H-1500	Mounting media with DAPI
iNSu/Light Insulin Disposible Syringe	RAYS Health & Safety	INS1ML26G13
30G 1/2"	BD Microlance	304000	Needle for Insulin disposible Syringe
Scalpel Handle	F.S.T.	91003-12
#22 Disposable Scalpel blads	F.S.T.	10022-00
Fine Iris scissors 10.5 cm	F.S.T.	14094-11
Dumont Forceps #5745 45° 0.10 x 0.06 mm	F.S.T.	11251-35
Graefe Forceps 10 cm	F.S.T.	11051-10
Dumont Forceps #5 0.1 X 0.06 mm	F.S.T.	11251-20
Medical patch	Medicalis	34788
Sterile disposable towel drape	Dispotech	TVO50VE
Stereoscopic Microscope	NIKON	SMZ 745 T
Optic Illuminator LED light (C-FLED2)	NIKON	1003167	Optic Illuminator for Stereoscopic Micrscope
Eclipse Ni-U Microscope	Nikon	932162	Epifluorescence Microscope
Microscope digital Camera	Nikon	DS-Ri2	Microscope camera
Intenslight	Nikon	C-HGFI	Microscope lamp
NIS-Elements 64 bit	Nikon	AR 4.40.00	Analysis Software
Electric Razor	Gemei	GM-3007