Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

Summary

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans. 

Abstract

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Introduction

The central nervous system (CNS) coordinates all body and mental functions in vertebrates, and CNS homeostasis is essential for a proper communication of neurons. CNS homeostasis is warranted by the neurovascular unit (NVU), which protects the CNS from the changing milieu of the blood stream. The NVU is composed of CNS microvascular endothelial cells, which are biochemically unique and establish the blood-brain barrier (BBB) in continuous crosstalk with pericytes, astrocytes, neurons, and extracellular matrix (ECM) components, establishing two distinct basement membranes¹. The endothelial basement membrane that ensheathes the abluminal aspect o....

Protocol

All studies were conducted under the guidelines according to the Swiss legislation on the protection of animals and were approved by the Veterinary Office of the Canton of Bern, Switzerland (permission numbers: BE 31/17 and BE 77/18).

1. Housing of C57BL/6 mice in specific pathogen free (SPF) conditions

1. House mice in individual ventilated cages with a 12/12 h light-dark cycle. Provide food and water ad libidum. To supervise microbiological quality of the mice, the experimental.......

Discussion

Here, we present a protocol to induce and monitor EAE in female C57BL/6 mice. Females are preferentially chosen, and there is a incidence of women : men of 3:1 in MS. To assess the severity of EAE, we made use of a 3-point scoring sheet. EAE severity is generally scored with respect to the severity of motor dysfunctions. Mice with advanced stages of EAE, i.e. exhibiting a score above 2 should be sacrificed to avoid unnecessary suffering of the animals. Thus, it is recommended to score the mice at close intervals e.g. twi.......

Materials

Name	Company	Catalog Number	Comments
AMCA anti-rabbit antibody	Jackson ImmunoResearch	111-156-045	Store at 4 °C; protect from light
Anti-CD45 Antibody (30F11)	Pharmingen	07-1401	Store at 4 °C
Anti-Laminin Antibody	DAKO	Z0097	Store at 4 °C
Breeding food	e.g. PROVIMI KLIBA SA	3336
Individually ventilated cages, Blue line Type II or III	e.g. Tecniplast	1145T, 1285L
BSA fraction V	Applichem	A1391	Store at 4 °C
Cold gelatine	Sigma-Aldrich	G 9391
Coplin jar + rack	e.g. Carl Roth GmbH + Co. KG	H554.1; H552.1
Cy3 anti-rat antibody	Jackson ImmunoResearch	111-156-144	Store at 4 °C; protect from light
Cover slips 24 x 40 mm # 1	e.g. Thermo Scientific	85-0186-00
Dextran Alexa Fluor 488 (10,000 MW)	e.g. Molecular probes	D22910	Store at -20 °C; protect from light
Dextran Texas Red (3000 MW)	Invitrogen	D3328	Store at -20 °C; protect from light
EnzChek Gelatinase/Collagenase Assay Kit	Thermo Fisher Scientific; EnzCheck	E12055	Store at -20 °C; protect form light
Female C57BL/6J mice (8-12 weeks)	e.g. Janvier Labs		Females, 8-12 weeks
Freezing box for histology slides	e.g. Carl Roth GmbH + Co. KG	2285.1
18G x 1½’’ (1.2mm x 40mm) injection needle	e.g. BD, BD Microlance 3	304622
27G x ¾’’ – Nr. 20 (0.4mm x 19mm) injection needle	e.g. BD, BD Microlance 3	302200
30G x ½’’ (0.3 mm x 13 mm) injection needle	e.g. BD, BD Microlance 3	304000
Incomplete Freund’s adjuvant (IFA)	e.g. Santa Cruz Biotechnology	sc-24648	Store at 4°C
Maintenance food	e.g. PROVIMI KLIBA SA	3436
MOGaa35-55 peptide	e.g. GenScript		Store at -80 °C
microscope slides (Superfrost Plus )	Thermo Scientific	J1800AMNZ
Mycobacterium tuberculosis H37RA	e.g. BD	231141	Store at 4 °C
NaCl 0.9 %	B. Braun	3535789
O.C.T. compound (Tissue-Tek )	Sakura	4583
Omnican 50 30G x ½’’	B. Braun	9151125S
Paraformaldehyde	Merck	30525-89-4
Pertussis toxin	e.g. List biological laboratories, Inc.	180	Store at 4 °C
poly(vinyl alcohole) (Mowiol 4-88)	Sigma-Aldrich	81381
Protease Inhibitor EDTA free (Roche)	Sigma-Aldrich	4693132001	Store at 4 °C
repelling pen e.g. DAKO Pen	e.g. DAKO	S2002
sealing film e.g. Parafilm M	e.g Sigma-Aldrich	P7793
Silica gel	e.g. Carl Roth GmbH + Co. KG	9351.1
Stitch scissor	F.S.T	15012-12
syringe 1 ml	e.g. PRIMO	62.1002
syringe 10 ml	e.g. CODAN Medical ApS	2022-05
vaporizer system Univentor 400	UNO.BV