Mitochondrial defects have been implicated in the degeneration of axons in a number of CNS disorders, including multiple sclerosis. Uniquely, mitochondria harbor the only non-nuclear DNA (mitochondrial DNA or mtDNA), which encodes functionally important subunits of the respiratory chain. The pattern of mitochondrial respiratory chain subunit expression provides important clues to the underlying mechanism of mitochondrial injury. In snap frozen tissue mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity may be determined using a well-established histochemical technique, COX histochemistry. Lack of COX activity may be the result of mtDNA mutations, degradation of transcripts of subunits, modification of subunits or inhibition of complexes. Mitochondria lacking complex IV activity, however, have not been further explored within axons in CNS disorders. By combining COX histochemistry with immunofluorescent labeling of mitochondrial proteins we describe a method to identify mitochondria lacking complex IV activity in CNS tissue and locate inactive mitochondria to axons using confocal microscopy. Inactive axonal mitochondria may then be further investigated using confocal microscopy to define the pattern of mitochondrial respiratory chain complex subunit expression. Our technique may be used to gain important clues to the underlying mechanisms of mitochondrial injury within axons in a number of CNS disorders and relevant animal models.
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