PI-"death staining"  - evaluation of delayed cell death and anti-ischaemic compounds

Fields of research:

Detection of neuroprotective effects after hypoxia / hypoglycemia

Detection of lethal compound actions on brain tissue in vitro (neurotoxicity testing)

Role of ion channels and ion exchangers in apoptosis/necrosis

Influence of ligands of transmitter and protease activated  receptors on ischemic damage

Development and standardization of brain tissue cultures for reduction of animal experimentation

In collaboration with Keyneurotek AG 

Methods of research:

"Life-Death" staining in organotypic hippocampal and cortical slice cultures of rat and mice alone or in combination with electrophysiological recordings (in vitro hypoxia)

Examples: Transmission images (a,c) and propidium iodide (PI) - fluorescence images (b,d) of control organotypic slice cultures (a,b) and cultures 24 h after 40 minutes of  oxygen glucose deprivation (OGD) (c,d). The insult reduces transmission and leads to a pronounced fluorescence signal after PI staining predominantly in area CA1, but also in area CA3 and in the dentate gyrus of the damaged culture.

Procedure:  To induce OGD, the slices are transferred into another 6-well plate containing 1 ml Ringer-mannitol/well and incubated under 95% N2/5% CO2 at 36°C for 5 min (sublethal OGD) or 40 min (lethal OGD). After OGD, the organotypic slices were cultured in the presence of glucose and oxygen for another 24 h. Subsequently, slices can be  used either for quantification of cell death or for RT-PCR analysis . The fluorescence of propidium iodide (PI), which has been shown to enter exclusively damaged cells (Macklis, 1990) is used to determine neuronal cell death. For PI staining, the slices are incubated in the culture medium containing additionally 0.1 mg/ml PI for 2 h. Then, slices are transferred into Ringer buffer (pH 7.4) containing 0.05 mg/ml PI. The PI fluorescence is monitored using an inverted fluorescence microscope. Excitation and emission wavelengths were 545 nm and 610 nm, respectively. Both transmission and fluorescence images are captured using a CCD camera. The fluorescence images are analyzed using the Lucia M software package. The neuronal damage within each organotypic slice is calculated as the area of PI fluorescence in the hippocampal CA1 and CA4/CA3 region in relation to the total slice area.
   

For further information please contact:

Dr. Frank Striggow
phone: +49(391)6117200 
frank.striggow@keyneurotek.de 

Dr. Ulrich Schröder
phone: +49(391)6263781 or 6117208
schroede@ifn-magdeburg.de  

Recent Publications:

Sabelhaus CF, Schröder UH, Breder J, Henrich-Noack P, Reymann KG. (2000) Neuroprotection against hypoxic/hypoglycaemic injury after the insult by the group III metabotropic glutamate receptor agonist (R, S)-4-phosphonophenylglycine Br J Pharmacol 131:655-658.

Breder J, Sabelhaus CF, Opitz T, Reymann KG, Schröder, UH (2000) Inhibition of different pathways influencing Na+ homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury. Neuropharmacology 39: 1779-1787

Macklis, J.D. & Madison, R.D. (1990) Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. J. Neurosci. Methods, 31, 43-46

Striggow, F., Riek, M., Breder, J., Henrich-Noack, P., Reymann, K.G. and Reiser, G.: The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc. Natl. Acd. Sci. 97, 2000, 2264-2269

Breder J., Sabelhaus, C.F., Opitz, T., Reymann, K.G., and Schröder, U.H.: Inhibition of different pathways influncing Na+ homeostasis protects organotypic hippocampal slice cultures from hypoxia/hypoglycemia. Neuropharmacol. 39, 2000, 1779-1787