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 FTC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic midd le cerebral artery (MCA) occlusion (n = 9) and to mice that were not operat ed on (n = 3); after 1 minute of circulation, brains were removed, immersio n-fixed, and processed for LSCM, In some of these animals (n = 5), the volu me of endogenous microtubule-associated protein-2 (MAP2) fluorescence was a lso determined using immunohistochemical staining. For mice that were not o perated on, this methodology yielded highly localized volumes of (1) microv ascular plasma, which agree with those determined for rodents by other tech niques, and (2) MAP2 expression, which appears physiologically acid morphol ogically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of dis tribution were less than 10% of normal in the ipsilateral hemisphere in whi ch plasma perfusion essentially ceased. In conclusion, precise colocalizati on and quantitation of early ischemic neuronal damage and cerebral plasma p erfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.