Focal Ischemia - for the evaluation of anti-stroke compounds

Testing neuroprotective drugs in transgenic animals

Neuroprotective effects of metabotropic glutamate receptor ligands

Agonists and antagonists of protease activated receptors

Neuroprotection by Immunophillins

Focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCA) via intracerebral microinjection of endothelin 1 in rats. Male Spargue-Dawley rats (250-280 g) are housed with a 12 h light-dark cycle and given food and water ad libitum. At the beginning of surgical preparation anaesthesia is induced with halothane in a mixture of nitrous oxide/oxygen (70:30) and maintained with 2-3% halothane during the following procedures. The rats are then placed in a stereotaxic frame. To induce occlusion of the middle cerebral artery the rats receive an injection via a cannula 7.5 mm below the dura of 60 pmol endothelin 1 over a time period of 5 min. The operation is performed on a heating towel and rectal temperature is kept at 37-38°C.

After a survival time of 7 days after ischemia, the animals are perfusion fixed transcardially with 4% paraformaldehyde. 1 mm coronal brain slices are cut with a razor blade at 14 predetermined anterior-posterior levels. After cryoprotection in sucrose, the slices are rapidly frozen in isopentane. Four to five cryostat sections (30 mm) from each brain slice are cut in a cryo-microtome and histological stained with Toluidine blue (Nissl staining).
   

Histopathological verification of ischemic brain damage induced by intracerebral injection of endothelin 1, endothelin 1 and i.p. injection of vehicle (NaCl), endothelin 1 and multiple i.p. injection of FK506 (10mg/kg, Fujisawa, Japan) and brain damage induced by the needle tract alone. Anterior - Posterior level of the sections A - B between 1.00 mm to 0.20 mm.

The extent of the infarct area at each level can annotated onto diagrams of the brain and the volume of the infarction calculated by integrating the area of damage at each stereotactic level and the distances between the various levels.

Ischemic brain damage induced by intracerebral microinjection of endothelin 1 close to the middle cerebral artery causes infarction in the lateral parts of the frontal cortex extending through parietal and insular cortex rostrally and through temporal and into occipital cortex caudally. In addition, there is evidence of infarction within dorsolateral portions of the caudate nucleus. T FK506 proved to be a powerful neuroprotective agent. The neuroprotective effect was apparent in the cortex at each of the stereotactic levels examined. Cortical damage was reduced by 51.83% after i.p. administration of FK506 as compared with vehicle controls, whereas the striatal damage was unaltered.

P. Henrich-Noack, J.H.M. Prehn, J. Krieglstein
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Henshall, D. C., Butcher, S. P.and J. Sharkey. A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion. Brain Res. 843 :105-111, 1999.