Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images o f the distribution of one or two fluorescent markers in mouse brain specime ns were generated by LSCM and processed by a system developed for morphomet ric analysis of fixed and stained serial brain histologic samples. To deter mine the volume of perfused cerebral capillaries, one of two fluorescent pl asma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blu e, was intravenously administered to mice subjected to 1 hour of embolic mi ddle cerebral artery (MCA) occlusion (n = 9) and to mice that were not oper ated on (n = 3); after 1 minute of circulation, brains were removed, immers ion-fixed, and processed for LSCM. In some of these animals (n = 5), the vo lume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) micr ovascular plasma, which agree with those determined for rodents by other te chniques, and (2) MAP2 expression, which appears physiologically and morpho logically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of di stribution were less than 10% of normal in the ipsilateral hemisphere in wh ich plasma perfusion essentially ceased. In conclusion, precise colocalizat ion and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.