Fields of research:

Methods of research:

Stereotaxic operation

Gerbils or rats are anaesthetised with pentobarbital  and fixed in a stereotaxic device. A cannulae (diameter 0.6/0.8 mm) can be  inserted in the lateral ventricle to overcome the blood brain barrier.

Transient global cerebral ischemia in gerbils (2-VO)

Male Mongolian gerbils (Meriones unguiculatus; 60-80 g) are obtained from Charles-River (Tumblebrook farm strain).  Anesthesia is induced with 4% halothane in a mixture of nitric oxid/oxygen and maintained with halothane during the following procedures. Both common carotid arteries are exposed by a ventral midline incision and separated carefully from the adjacent veins and nerves. The vessels are transiently clamped by surgical clips and halothane is reduced to 0.75%. After removal of the clips restoration of blood flow is visually confirmed. The operation is performed on a heating mat and rectal temperature is kept at 37-38°C. The animals are kept under a heating lamp at 30°C environmental temperature until they regain consciousness. Under standard conditions brains are removed after a survival period of 7 days.
This model was verified in our laboratory using NBQX ( ip) as a reference substance.
   

Transient global cerebral ischemia in rats

Male Wistar rats (250-300 g) are used to induce transient forebrain ischemia under normothermic conditions with two-vessel occlusion combined with systemic hypotension according to the method of Dirnagl et al., 1993. After anaesthesia with 3-4% halothane, the rats are intubated and connected to a small animal ventilator that deliveres 1% halothane in a nitrous oxide/oxygen mixture. Both common carotid arteries are isolated and the tail artery is cannulated (tube rinsed with Heparin) for measurement of blood gases and blood pressure. The lower body of the animal (excluding the thorax) is housed in a negative pressure chamber, which is connected to a vacuum pump. Ischemia is induced by clamping both common carotid arteries and reducing the mean arterial blood pressure to 40-45 mmHg by controlled negative pressure (venous pooling). After 10 min, the carotid clamps are removed and normal blood pressure is restored. The animals are removed from the respirator as soon as they regain spontaneous respiration and kept for 90 min under 30°C environmental temperature. Under standard conditions the brains are removed after 7 days. Brains are fixed by immersion in a mixture of formaldehyde, ethanol and glacial acetic acid, dehydrated with alcohol, transfered into chloroform and embedded in paraffin. Hippocampal slices (10 µm) are cut on a microtome and life/death staining is performed with toluidine blue/fuchsin acid. Healthy neurons are counted on a microscope within a 500 µm area of the hippocampal CA1-region. This model was verified in our laboratory using NBQX ( ip) as a reference substance With the rat model of transient forebrain ischemia we have the possibility to monitor blood pressure, blood gases (pH, pCO2, pO2) and blood glucose. The brain of ischaemic rats suitable for further investigations using antibodies (Immunohistochemistry, Western blotting), Primer (PCR) or RNA-probes (in-situ-hybridisierung). (Histochemistry) 

4VO - Global cerebral ischemia in rats

Cerebral ischemia in rats is induced by four-vessel-occlusion  in male Wistar rats. Both vertebral arteries are occluded by electrocauterization in pentobarbital anesthesia. The animals are allowed to recover for 24 hours. The next day the carotid arteries are occluded for 10 min using microvascular claps. Subsequently, both clamps are removed and both arteries are inspected for immediate reperfusion. During the operation and the following 3 hours normothermia of the animals (37.5 °C) is maintained by using a thermostatically controlled heating blanket connected to a rectal thermometer. For control, in sham-operated animals both vertebral arteries are cauterised and both common carotid arteries are exposed but not clamped.

After a survival time of 7 days after ischemia, twenty-micrometer cryostat sections comprising the hippocampal formation were Nissl stained with toluidine blue or NeuroTrace fluorescence. The mean number of morphologically intact neurons per 400 mm length is calculated in CA1 region for each group. Cell counting is performed in 3-5 serial sections per animal and 6 times 400 mm CA1 area per section using a light microscope equipped with a 20 x objective. Morphological intact hippocampal CA1 neurons were characterized by Nissl staining (toluidine blue and NeuroTrace) with the following criteria: clear shape of a neuronal perikarya, large nucleus with a positive labeled nucleolus, a small cytoplasm zone around the nucleus with positive Nissl staining, indicating the intact rough endoplasmic reticulum with ribosomes and therefore the intact protein synthesis machinery.
10 min of global ischemia (mild ischemia) and a survival time of 7 days leads to a neurodegeneration of pyramidal cells selectively in hippocampal CA1 region (see diagram).  Approximately  60% of CA1 neurons die after 10 min of global ischemia. The number of neurons in the animal group of ischemia and injection of vehicle is comparable to that of the ischemia group alone. Protection control with NBQX (30 mg/kg) (disodium salt, because more water soluble) shows a significant  neuroprotection in CA1 pyramidal cells compared to the ischemia group.
   

For further information please contact:

Dr. Petra Henrich-Noack
phone +49(391)6263794 or 6117204
noack@ifn-magdeburg.de 

Recent publications: