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There has been recent increasing interest in the involvement of superoxide radicals (O2-) and their scavenging enzymes, the superoxide dismutases, in the patho-physiology of certain diseases. Since mitochondria are significant intracellular sources of O2- and important targets of oxidant injury, determining the intracochlear localization of mitochondrial O2- scavenging enzyme may provide important insight into the pathogenesis of injury due to cochlear oxidants. In order to locate the mitochondrial O2- scavenging enzyme, manganese superoxide dismutase (MnSOD), the authors used a modified immunoglobulin peroxidase bridge sequence method to detect MnSOD in paraffin-embedded, formalin-fixed rat cochleas. Site-specific immunolocalization of MnSOD could be demonstrated in the cochlear labyrinth, suggesting that the generation of intracochlear O2- was possibly implicated in the metabolically active sites and sites rich in vascularity. This study also provided a useful probe for detecting MnSOD immunohistochemically from ethylenediamine tetra-acetic acid-treated materials without requiring an antigen retrieval procedure.

Epithelium of the inner ear in the gerbil and mouse was examined immunocytochemically for presence of creatine kinase (CK). Marginal cells of the cochlear stria vascularis and dark cells and transitional cells of the vestibular system were found to contain an abundance of the MM isozyme (MM-CK). CK in these cells concurs with that which is coupled to Na,K-ATPase in other cells and is considered to supply ATP for the Na,K-ATPase that mediates the high KCl of endolymph. Inner hair cells revealed content of the BB isozyme and in this respect resembled the energy-transducing photoreceptor cells in retina. In addition, outer phalangeal (Deiters') cells stained for both MM- and BB-CK whereas inner phalangeal cells evidenced content of only the BB isozyme. Immunolocalization of CK appeared similar in mouse and gerbil inner ear. Specificity of the staining was affirmed by observations in agreement with those reported for CK in various cell types and by staining with antisera from more than one source.

Carbonic anhydrase activity was demonstrated ultracytochemically in the dark cells of the vestibular labyrinth and in the stria vascularis of the guinea pig. Reaction products were found both in the dark cells and in the stria vascularis. Localization of carbonic anhydrase was very similar between the dark cells and the intermediate cells, but the marginal cells had a different localization pattern. In both the dark cells and the intermediate cells, the reaction products were deposited in the cytoplasm, especially in the infoldings. In the marginal cells, carbonic anhydrase activity was observed on the lateral plasma membrane of the apical area and in a few apical vesicles, but was not detected in the infoldings of the deep portion. Reaction products were also observed in the intercellular spaces between the marginal cells. No reaction products were observed in the basal cells and transition cells. Carbonic anhydrase activity was observed in the cytoplasm of capillary endothelial cells and the fibrocytes of the spiral ligament. It is considered that carbonic anhydrase may play a major role in water and electrolyte transport in both the dark cells and stria vascularis.