Methods & Models

Fully equipped laboratories (140 square meter) are located in the Biomedical Research Center Seltersberg (BFS) Schubertstrasse 81, BFS, 35392 Giessen, Germany.

We use different in vitro and in vivo models to investigate the effects  of  food and bioactive food ingredients as well as pharmacological substances on physiological and pathological parameters reflecting the situation in the aging brain and in neurodegeneration. In cooperation with partners from nutritional science and medicine, we aim to evaluate our data in human studies. Standard analytical methods are used to determine the composition of food and food ingredients. Molecular, cellular and biochemical methods together with special techniques are used to characterize the biofunctional or pharmacological effects of test substrates.  

  • Food and food ingredients to be used in our in vitro and in vivo models are analysed by classical analytical methods. Selection of methods used in our lab: 

    total water (gravimetric) total dietary fiber (enzymatically)
    total carbohydrates (phenol-sulfuric-acid-method) reducable sugars (according Luff-Schorl)
    glucose (enzymatically) total polyphenols (folin reagent - phototmetry)
    total protein (according Kjeldahl) total phosphate (photometry)
    total fat (according Weibull-Stoldt) fat parameters (VZ, SZ, IZ, OHZ, EZ; titrimetric)
    fatty acids (GC-MS) cholesterol (enzymatically)
    phospholipids (HPLC-UV/VIS, HPTLC) isoprenoids (HPLC-FD)
  • Basic lab methods include:

    qRT-PCR, gelelektrophoreses, Western blot, Radio-immunoassays, ELISA, HPLC-UV/Vis und HPLC-FD, DC, GC-MS, GC-FID, photometry, fluorescence spectroscopy and –microscopy, receptor binding assays, isolation and characterisation of synaptosomal and mitochondrial membranes, isolation and characterisation of lipid rafts, polarisation-fluorescence spectrometric measurement of membrane fluidity (DPH, TMA-DPH, Pyren), preparation of liposomes, determination of intracellular reactive oxygen species (FACS), malondialdehyde, hydroxynonenal, protein carbonyls, anti-oxidative enzymes (SOD, catalase, GPx, GR), hydroperoxides, 8-OHDG, isoprostanes.  

  • Mitochondrial dysfunction is assessed by a whole range of methods:

    Methods Mito
    • Isolation of dissociated brain cells and metabolic active mitochondria
    • Isolation and purification of mitochondrial membranes
    • High Resolution Respirometry (Oxygrapgh-2k, Oroboros), activity of complexes (I-IV)
    • Mitochondrial membrane potential (ΔΨm) [Rhodamin123, TMRE]
    • ATP levels
    • Mitochondrial mass markers (Citrate synthase activity, Mitotracker green, cardiolipin content)
    • Gene expression of relevant genes (quantitative RT-PCR), e.g. mRNA levels of PGC1-alpha, PPARgamma, CREB, NRF-1, TFAM, Sirt1
    • Levels of mitochondrial proteins (Western blot) (a-h)
    • Membrane fluidity
    • Mitochondrial swelling (mPTP opening)
    • ROS production (FACS), lipid peroxidation products (protein carbonyls, malondialdehyde)
    • Antioxidant enzymes (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase


    more methods are available due to our manifold cooperations.

  • Synaptic dysfunciton is assessed using the following methods:

    Methods Mevalonate
    • Isolation and purification of synaptosomal plasma-membranes and cytosolic fractions from mouse brains
    • Isolation and purification of lipid rafts (detergent and detergent free)
    • Isolation of plasmamembranes and cytosolic fractions from cells
    • WesternBlot analysis of 
      - small GTPases (Rac1, Cdc42, RhoA, Rab3A, Rab11B, H- Ras) in cytosolic and membrane fractions
      - synaptic markers (GAP43, synaptophysin)
      - enzymes of the mevalonate pathway (HMGR, FDPS)
      - transferases (GGTaseI, FTase)
      - GEF-proteins
    • Determination of GGTase activity
    • HPLC-FD analysis of isoprenoids (FPP, GGPP)
    • Cholesterol assays
    • High-performace thin layer chromatography for phospholipids

      more methods are available due to our manifold cooperations.
  • Stroke is induced experimentally and effects of thereof are measured by different methods:

    Methods stroke
    • Experimental stroke model via middle cerebral artery occlusion (MCAO)*:
    • Monofilament is inserted to occlude the middle cerebral artery
    • Ischemia is induced permanently or transiently
    • Neurological Severity Score (NSS)
    • Brain slices are stained using TTC to indicate ischemic damage in the brain and to determine stroke volume
    • Measurement of mitochondrial function (MMP, ATP, respiration, complex I-IV activity)
    • Determination of inflammation markers (iNOS, COX, TNFa, PGE2, Ig-6, Ig-10)
    • GC-MS analysis of fatty acids and metabolites

      more methods are available due to our manifold cooperations.

    *in close cooperation with Dr. Jochen Klein, Frankfurt

Models

Models invivo

in vitro 

Alzheimer:

  • SH-SY5Y cells
  • SY5Y-Mock cells
  • SY5Y-APP (695) wt cells
  • PC12neo cells
  • PC12sw cells

in vivo

aging:   

  • aged NMRI mice
  • aged C57/BL6 mice
  • SAMP8 and SAMR1 mice

Alzheimer‘s disease:

  • Thy1-APP751 SL mice

Lipid-reserach:

  • Dunkin Hartley guinea pigs

Ischemic Stroke:

  • MCAO model using CD-1 mice (in close cooperation with Dr. Jochen Klein)

Behaviour tests:

  • Open field
  • Passive avoidance
  • Social recognition
  • Object recognition
  • Y-maze
  • Rotarod
Nutritional Neuroscience opt.jpg


© Prof. Dr. Gunter P. Eckert 2017