[Figures will be inserted soon!]
In focal cerebral ischemia, the extent of parenchymal tissue damage is closely related to the degree and duration of blood flow reduction during the ischemic interval. During the reperfusion period after ischemia, the restored perfusion is suggested to stop the ischemic injury process by reassessment of the principal functions of tissue perfusion: oxygen delivery, provision of substrates for metabolism and clearance of metabolic wastes. But on the other side, blood supply to the damaged tissue may lead to further cell injury for example due to oxygen free radical formation, impaired nitric oxide production and leukocyte activation.
Leukocytes, in particular the polymorphonuclear granulocyte, may play an important role in the pathogenesis of tissue damage both due to microvascular obstructions and release of vasoactive and cytotoxic mediators, among which the most important are: oxygen free radicals, in particular superoxide anion and nitric oxide (respiratory burst), arachidonic acid metabolites, cytokines (TNF() and PAF.
Nevertheless the main question concerning leukocyte activation during and after focal ischemia seems to be: Do leukocytes cause cell damage or respond to it?
Until now the involvement of leukocytes in ischemia and reperfusion has been studied mainly with histologic methods ex vivo.
Therefore the aim of this study is to examine the leukocyte-endothelium interaction in the early reperfusion period after focal cerebral ischemia online in vivo.
As an experimental model of focal cerebral ischemia and reperfusion in the rat we use the so called thread model, in which the middle cerbral artery (MCA) is occluded by a nylon suture introduced via the common carotid artery in the cervical internal artery to the origin of the middle cerebral artery (fig.1).
Schematic drawing of the Circle of Willis and the position of the occlusive thread within the internal carotid artery during ischemia (A) and reperfusion (B). CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; ACA, anterior cerebral artery; MCA, middle cerebral artery; PCA, posterior cerebral artery. (Nagasawa and Kogure, 1989)
Leukocyte-endothelium interaction is studied with confocal laser scanning microscopy (CLSM) through a closed cranial window implanted over the parietal cortex supplied by the MCA (for detail see article: Lindauer U, Villringer A, Dirnagl U: Capillary recruitment in the microcirculation of the brain: confocal laser scanning microscopy in vivo. in this issue). In fig.2 the position of the closed cranial window and the ischemic area within the anatomic region is shown.
To visualize leukocytes in the pial circulation, the fluorescent dye Rhodamine 6G is injected intravenously (200µg/ml 0.9% saline, 1ml as a bolus injection, followed by continuous infusion with an infusion rate of 1 ml/h). After intravital injection of Rhodamine 6G, circulating leukocytes (polymorphnuclear leukocytes, lymphocytes and momocytes) and platelets are labelled, whereas endothelial cells and red blood cells remain unstained.
Anatomic region of ischemic area (shaded) and position of the closed cranial window (modified after Nagasawa and Kogure, 1989)
Leukocyte-endothelium interaction in small pial arteries and veins is studied as numbers of rolling and sticking leukocytes per 1 minute and 100µm vessel length before the ischemic period and 1 to 6 hours during the reperfusion period after 1 hour of focal ischemia. In fig.3 a typical exampel of a sticking leukocyte in a pial venule is shown.
Leukocyte labeled withRhodamine 6G (arrow) sticking firmly to the endothelium of a pial vein: no change in position in B (image taken 10 seconds after A). Scale bar is 30µm
At the end of each experiment, the brains are perfusion fixated for pathohistological examination to detect early tissue injury.
In conclusion, the aim of this study is to find out whether there is a correlation between the degree of leukocyte activation during the early reperfusion period after focal ischemia and the extend of early damage of neurons and glial cells in the histological sections.