Investigational techniques – Examination of inflammation

1. FUNCTIONAL STUDIES:

  • Measurement of microcirculation by laser-Doppler flowmetry
  • Measurement of plasma protein extravasation by Evans blue accumulation (spectrophotometry) and 125I-labelled BSA (gamma counter)
  • Measurement of ear thickness and joint diameter with a Mitutoyo digital caliper
  • Measurement of paw oedema by plethysmometry
  • Determination of airway function by whole body plethysmography (Buxco)

    • direct measurement in anaesthesized mice and rats
    • assessment of bronchoconstriction in unrestrained, freely moving animals

2. IN VIVO IMAGING TECHNIQUES:

a.) Fluorescent and luminescent functional imaging of living, anesthetized mice/rats, excised tissue samples, and cell cultures

Analysis of the expression and distribution of various biological markers (enzyme activity, receptors, structural components, free radicals) using fluorescent contrast agents, and bioluminescent/chemiluminescent substrates.

Instruments:

  • PerkinElmer FMT 2000 – Fluroescent molecular tomography system 


  • PerkinElmer Lumina III – Luminescent/fluorescent imaging system

b.) Intravital microscopy examinations

Real time monitoring of vasodilation, plasma protein extravastion, leukocyte accumulation, etc. in murine inflammation models. Imaging and quantitative analysis of these parameters in the skin, brain surface, joints, and internal organs.

Instrument:

  • NIKON intravital microscope with camera and image analysis software 


c.) In vivo imaging of microcirculation in mice/rats, large animals and humans (skin, mucous membrane)

Measurement of microcirculation in animal models of inflammation, vasoregulatory disorders, and migraine. Noninvasive investigation of local skin blood flow in human subjects.

Instruments:

  • PeriScan PIM II laser Doppler imager with inbuilt blood flow sensor 


  • PeriFlux 5000 Laser Doppler Flowmeter – monitoring of microcirculation and PO2/PCO2. 


  • Optional application of temperature and pressure stimuli.
  • PeriCam laser speckle imager – Capable of monitoring rapid/transient changes of microcirculation

d.) In vivo micro CT

Instruments:

  • Bruker high resolution in vivo micro CT (lung, heart, bone structural imaging) 


3. CHEMICAL/ IMMUNOLOGICAL METHODS:

  • Measurement of leukocyte accumulation in tissue homogenates (skin, joint, lung) by spectrophotometric determination of the myeloperoxidase enzyme (MPO) activity
  • Measurement of inflammatory (substance P, CGRP) and anti-inflammatory sensory neuropeptides (somatostatin, PACAP) with radioimmunoassay (RIA) in tissue (skin, joint, lung), plasma and in the incubation medium obtained in the in vitro perfusion system
  • Determination of inflammatory cytokines (TNF-alpha, IL-1-beta, INF-gamma, IL-3, IL-5, etc.) and cytokine profile in the inflamed tissues and plasma by cytometric bead array (CBA) and enzyme-linked immunoassay (ELISA) techniques
  • Determination of inflammatory cell profile by flow cytometry from peripheral blood, synovial and BAL fluid and tissue homogenates
  • Western blot

4. MOLECULAR BIOLOGICAL METHODS:

  • mRNA preparation from tissues, quantitative RT-PCR
  • Receptor cDNA cloning, expression in eukariotic cell lines
  • Receptor binding and G-protein activation assays
  • Radioactive Ca45-uptake assay
  • Measurement of Ca2+ influx with fura-2 and fluo-4 microfluorimetric techniques

5. MORPHOLOGICAL TECHNIQUES: HISTOLOGY,
IMMUNOHISTOCHEMISTRY

Paraffin-embedded and cryostat sections,

  • conventional histology and semiquantitative evaluation (Olympus microscope with fluorescence setup and analysis software)
  • immunohistochemistry and immune-fluorescent techniques on animal and human tissue

Instrument:

  • PerkinElmer MANTRA automated light and fluorescent tissue analysing system 


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