Methods to Assess Neuroinflammation

Florianne Monnet‐Tschudi1, Antoinette Defaux1, Olivier Braissant2, Laurène Cagnon2, Marie‐Gabrielle Zurich1

1 Swiss Centre for Applied Human Toxicology (SCAHT), Lausanne, Switzerland, 2 Clinical Chemistry Laboratory, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.19
DOI:  10.1002/0471140856.tx1219s50
Online Posting Date:  November, 2011
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Abstract

Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO‐synthase (i‐NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell‐to‐cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three‐dimensional cell culture system provides optimal cell‐to‐cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT‐PCR to measure the mRNA expression of cytokines (TNF‐α, IL‐1β, IL‐6, IL‐1ra, TGF‐β, IL‐15, IFN‐γ), chemokines (ccl5, cxcl1, cxcl2), and i‐NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases). Curr. Protoc. Toxicol. 50:12.19.1‐12.19.20. © 2011 by John Wiley & Sons, Inc.

Keywords: neuroinflammation; microglia; astrocyte; cytokine; chemokine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Evaluation of Microglial Reactivity, Astrogliosis, and Cytokine Expression by a Morphological Approach
  • Basic Protocol 2: Gene Expression of Inflammatory Markers Measured by Quantitative RT‐PCR
  • Basic Protocol 3: Measurement of the MAP Kinase Phosphorylation Levels by Immunoblotting
  • Reagents and Solution
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of Microglial Reactivity, Astrogliosis, and Cytokine Expression by a Morphological Approach

  Materials
  • Aggregated neural cell cultures (unit 12.9)
  • 0.1 M phosphate‐buffer saline (PBS), pH 7.4 ( appendix 2A)
  • Number 00 gelatin capsules (Feton International, Brussels; http://www.brusselslifetech.com/)
  • Cryoform (Cryomatric, Shandon Scientific, http://www.thermoscientific.com/)
  • Isopentane, cooled to −80°C with liquid nitrogen
  • Dry ice
  • Fixative: 4% (w/v) paraformaldehyde in PBS; heat until dissolved and filter on Schleicher & Schuell filter paper; cool down before use; prepare fresh
  • 0.1 M Tris‐buffered saline (TBS), pH 7.4 ( appendix 2A) containing 1% (v/v) Triton X‐100
  • 0.1 M Tris‐buffered saline (TBS), pH 7.4 ( appendix 2A)
  • Image iT‐FX signal enhancer (Invitrogen)
  • Isolectin B4 (IB4; see Table 12.19.1)
  • Normal donkey serum (see Table 12.19.2)
  • Monoclonal antibodies against OX‐42 and ED1 (Table 12.19.1)
  • Monoclonal antibody against GFAP (Table 12.19.1)
  • Normal horse serum (see Table 12.19.2)
  • Normal goat serum (see Table 12.19.2)
  • Normal rabbit serum (see Table 12.19.2)
  • Horse anti‐mouse biotinylated IgG (see Table 12.19.2)
  • Goat anti‐rabbit IgG (see Table 12.19.2)
  • Rabbit anti‐goat IgG (see Table 12.19.2)
  • Avidin coupled to either FITC or Texas Red (see Table 12.19.2)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Cryostat, or other cooled sectioning machine
  • Glass microscope slides (Superfrost Plus; Thermo Scientific)
  • Dako Pen (Dako)
  • Moisture chamber: a box with a lid, in which the bottom paper is soaked with water, with a support to dispose the slides horizontally
  • 0.22‐µm syringe filters
  • ProLong Gold antifade reagent (Invitrogen)

Basic Protocol 2: Gene Expression of Inflammatory Markers Measured by Quantitative RT‐PCR

  Materials
  • Aggregated neural cell cultures (unit 12.9) expressing inflammatory markers
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • QIAshredder kit (Qiagen, cat. no. 79656)
  • 2‐mercaptoethanol
  • 70% ethanol
  • RNeasy Protect Mini Kit (Qiagen, cat.no. 74126)
  • High Capacity cDNA Reverse Transcription kit (Applied Biosystems, cat. no. 4368814)
  • SYBR Green PCR master mix (Applied Biosystems, cat. no. 4312704)
  • Primers (Microsynth; see Table 12.19.3)
  • TaqMan Universal PCR master mix (Applied Biosystems, cat. no. 4324020)
  • TaqMan Gene Expression Assays (Applied Biosystems; see Table 12.19.5)
  • 15‐ml sterile plastic tubes (e.g., BD Falcon)
  • Centrifuge capable of spinning microtiter plates (e.g., Heraeus Multifuge 3S‐R)
  • Spectrophotometer
  • PCR strip tubes (Axygen, cat.no. PCR‐0208‐C) and PCR strip caps (Axygen, cat.no. PCR‐02CP‐C)
  • Thermal cycler: 7900HT (Applied Biosystems) or equivalent qRT‐PCR thermal cycler
  • Safe‐Lock microtubes, 1.5‐ml (Eppendorf), 0.5‐ml microtubes (Eppendorf), and suitable racks
  • MicroAmp optical 96‐well reaction plate (Applied Biosystem, cat. no. N801‐0560)
  • MicroAmp optical adhesive film (Applied Biosystems, cat. no. 4311971)

Basic Protocol 3: Measurement of the MAP Kinase Phosphorylation Levels by Immunoblotting

  Materials
  • Aggregated neural cell cultures (unit 12.9) expressing MAP kinase
  • Western Blot lysis buffer (see recipe)
  • Protease inhibitors (Complete EDTA‐free, Roche, cat.no. 04693132001)
  • Phosphatase inhibitors (phosphatase inhibitor cocktail 2, Sigma, cat. no. P5726; or sodium orthovanadate, LC Laboratories, cat. no S‐8507; http://www.lclabs.com/)
  • BCA Protein Assay kit (Thermo Scientific, cat.no. 23227)
  • Ultrapure H 2O
  • 4× NuPage LDS Sample Buffer (Invitrogen, NP0008)
  • 10× NuPAge Sample Reducing Agent (Invitrogen, cat. no. NP0009)
  • 20× NuPage SDS Running Buffer (Invitrogen, cat. no. NP001)
  • 1× NuPage Antioxidant (Invitrogen, cat. no. NP005)
  • NuPage 12% Bis‐Tris minigel, 10 well, 1 mm (Invitrogen, cat. no. NP0341BOX)
  • Protein standards selected according to mol. wt. of protein of interest
  • PVDF Immobilon‐P Transfer Membrane (Millipore, cat. no. IPVH0010)
  • Methanol
  • Bjerrum buffer (see recipe)
  • TBS‐t (see recipe)
  • Ponceau solution: 1.31 mM Ponceau S (Acros Organics, cat. no. 161470250) prepared in 5% (v/v) acetic acid
  • Blotting‐Grade Blocker non‐fat dry milk (BioRad, cat. no. 170‐6404)
  • Primary antibodies (Table 12.19.6) against phosphorylated p38 or p44/42 MAPK
  • Secondary antibody (Table 12.19.6)
  • Pierce ECL Western Blotting Substrate (Thermo Scientific, cat. no. 32106)
  • Re‐Blot Plus Mild antibody Stripping Solution (Chemicon, cat. no. 2502)
  • Antibody against p38 MAPK total or p44/42 MAPK total (see Table 12.19.6)
  • Centrifuge
  • Probe sonicator
  • Xcell SureLock Mini‐cell (Invitrogen, cat.no. EI001)
  • PowerPac HC Power Supply (BioRad, cat. no. 164‐5052)
  • Extra Thick Blot Paper, Protean XL size (BioRad, cat. no. 170‐3969)
  • Trans‐Blot Semi‐Dry Cell (BioRad, cat. no. 170‐3939)
  • Amersham Hyperfilm ECL (GE Healthcare, cat. no. 28906837)
  • Sealable plastic bags
  • Curix 60 Processor (AGFA Healthcare, http://www.agfa.com)
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Figures

Videos

Literature Cited

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