Modified Labeling Technique for in vivo Visualization of Platelets in the Cerebral Microcirculation of Mongolian Gerbils

• Published in: Journal of cerebral blood flow and metabolism Vol. 27; no. 2; pp. 327 - 333
• Main Authors: Waldner, Maximilian, Hutter, Joerg, Uhl, Eberhard, Baethmann, Alexander, Lehmberg, Jens
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.02.2007; Lippincott Williams & Wilkins; Sage Publications Ltd
• Subjects: Adenosine Diphosphate - pharmacology; Animals; Biological and medical sciences; Blood Platelets - physiology; Cerebral circulation. Blood-brain barrier. Choroid plexus. Cerebrospinal fluid. Circumventricular organ. Meninges; Cerebrovascular Circulation - physiology; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gerbillinae; Headache. Facial pains. Syncopes. Epilepsia. Intracranial hypertension. Brain oedema. Cerebral palsy; Male; Medical sciences; Meriones unguiculatus; Microcirculation - physiology; Microscopy, Fluorescence; Microscopy, Video; Nervous system (semeiology, syndromes); Neurology; Platelet Aggregation - drug effects; Stereotaxic Techniques; Vascular diseases and vascular malformations of the nervous system; Vertebrates: nervous system and sense organs; pial vasculature; thrombosis; animal model; fluorescence microscopy; Gerbil; microcirculation; Animal model; Visualization; Nervous system diseases; Rodentia; Cardiovascular disease; Thrombosis; Cerebral disorder; Vascular disease; Microcirculation; Vertebrata; Platelet; Mammalia; Central nervous system disease; Cerebrovascular disease; Fluorescence microscopy
• ISSN: 0271-678X; 1559-7016
• DOI: 10.1038/sj.jcbfm.9600341

Activation of platelets induces interactions with platelets, endothelial cells, and leukocytes. In vivo observation of these interactions in the cerebral microcirculation is rare. The purpose of the present study was to develop a model enabling the in vivo observation of platelet kinetics in the cerebral microcirculation. Intravital fluorescence microscopy was performed in the Mongolian gerbil. Platelets of a donor were labeled ex vivo with carboxyfluorescein diacetat-succinimidylester (CFDA-SE), providing long-term fluorescence. Platelet function was tested ex vivo by flow cytometric analysis and in vivo by analyzing platelet-endothelium interactions. Labeled platelets stimulated with adenosine diphosphate ADP (200 μmol/L) or thrombin (1000 U/L) showed aggregation in flow cytrometric analysis, whereas unstimulated platelets were not aggregated. Irradiation of the brain surface after intravenous injection of the photosensitizing dye Photosan first induced rolling and firm adherence of platelets on arteriolar and venular endothelium, followed by the formation of a thrombus obstructing the vessel. Quantitative analysis (nx100 μm−1 min−1) before and after 6 mins of irradiation showed 2.6±3.2 versus 29.0±28.9 rolling, and 0.0±0.0 versus 1.7±2.3 firm adherent platelets in arterioles, and 3.9±3.3 versus 36.6±20.9 rolling and 0.0±0.0 versus 13.6±8.9 firm adherent platelets in venules. Thus, we conclude that ex vivo labeling of platelets with CFDA-SE does not activate platelets. Platelet aggregation and adhesion was achieved by platelet-specific stimulation such as ADP, thrombin or irradiation. In vivo assessment of physiologic and pathophysiologic mechanisms of platelets in the cerebral microcirculation can be achieved in this model.