An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Summary

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Abstract

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer's disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.

We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

Introduction

The blood-brain barrier (BBB) consists of the microvascular endothelial cells (ECs) supported by closely associated pericytes (PCs), which are ensheathed in the basal lamina, and astrocytes (ACs) that envelop the basement membrane with their end-feet^1,2. ECs interact with several cell types that support and regulate the barrier function, primarily ACs and PCs, and also neurons and microglia, all of which together form the neurovascular unit (NVU). The NVU is critical for the function of the BBB, which limits the transport of blood-borne toxins and pathogens from entering the brain. This function is a result of....

Protocol

All animals were handled with utmost care minimizing pain or discomfort during the procedure. This procedure follows the animal care guidelines of our institution and has been approved by the local committee (Regierungspraesidium Darmstadt, approval number FK/1044).

A schematic of the work steps for in vivo permeability assay in mice is shown in Figure 1. The details of each step are described below.

1. Animal Handling

1. Preparation and administration of tracers and anesthetics
   1. Prepare labeled tubes and molds for tissue embedding at lea....

Results

We have recently shown that angiopoietin-2 (Ang-2) gain-of-function (GOF) mice have higher brain vascular permeability than control mice in healthy conditions¹⁰. In stroke-induced mice, it was also shows that the GOF mice had bigger infarct sizes and greater permeability than the control littermates. These results show a critical role of Ang-2 in permeability at the BBB. The protocol therefore utilized the GOF mice and compared them to control littermates to descri.......

Discussion

Blood-brain barrier dysfunction is associated with a number of neurological disorders, including primary and secondary brain tumors or stroke. BBB breakdown is often associated with life-threatening CNS edema. The elucidation of the molecular mechanisms that trigger the opening or closure of the BBB is therefore of therapeutic significance in neurological disorders and commonly investigated by researchers. However, methods to investigate BBB permeability in vivo reported in the literature, are often associated w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tetramethyl Rhodamine (TMR) dextran 3kD	Thermosfisher	D3308
Fluorescein isothiocyanate (FITC) dextran 3kD	Thermosfisher	D3306
Ketamine (Ketavet)	Zoetis
Xylazine (Rompun)	Bayer
0.9% Saline	Fresenius Kabi Deutschland GmbH
1X PBS	Gibco	10010-015
Tissue-tek O.C.T compound	Sakura Finetek	4583
37% Formaldhehyde solution	Sigma	252549-1L	prepare a 4% solution
Bovine Serum Albumin, fraction V	Roth	8076.3
Triton X-100	Sigma	T8787
rat anti CD31 antibody, clone MEC 13.3	BD Pharmingen	553370
goat anti rat alexa 568	Molecular Probes	A-11077
goat anti rat alexa 488	Molecular Probes	A-11006
DAPI	Molecular Probes	D1306
Aqua polymount	Polyscience Inc	18606
21-gauge butterfly needle	BD	387455
serum collection tube	Sarstedt	41.1500.005
2mL eppendorf tubes	Sarstedt	72.695.500
Kimtech precision wipes tissue wipers	Kimberley-Clark Professional	05511
384-well black plate	Greiner	781086
slides superfrost plus	Thermoscientific	J1800AMNZ
PTFE pestle	Wheaton	358029
electric overhead stirrer	VWR	VWR VOS 14
plate reader	Tecan	Infinite M200
Cryostat	Microm GmbH	HM 550
Nikon C1 Spectral Imaging confocal Laser Scanning Microscope System	Nikon
peristaltic perfusion system	BVK Ismatec
microcentrifuge	eppendorf	5415R