A re-evaluation of the retrograde axonal transport of horseradish peroxidase isoenzymes by semi-quantitative cytochemical analysis

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Retrograde axonal transport of 2 horseradish peroxidase (HRP) isoenzymes, the cationic isoenzyme C (HRP-C) and anionic isoenzyme A (HRP-A), was compared using the trigeminal innervation of the cornea in the rabbit. The effects of various fixatives, cytochemical reaction solutions and intracellular decay on the enzymatic activity and cytochemical visualization of both HRP-C and HRP-A were first evaluated quantitatively. Spectrophotometric assays showed that HRP-C and HRP-A respectively retained 92% and 84% of their initial activity after exposure to a fixative containing 1% paraformaldehyde and 0.5% glutaraldehyde for 1 h at 20°. The specific activity of HRP-A was 26% that of HRP-C when assayed in a solution containing 0.5 mg/ml diaminobenzidine and 0.01% H 2O 2 in 0.15 m citrate buffer, pH 5.1. By killing the rabbits at different times following injection of HRP into the corneas, the intracellular half-life of HRP-C was estimated to be 40–52 h and HRP-A 8–12 h. After taking all of these differences into consideration, corrected concentrations of the 2 isoenzymes were compared for retrograde axonal transport. Results showed that both label intensity (scored according to 4 classes) and total number of labeled cell profiles in animals injected with HRP-C was significantly greater than in animals injected with HRP-A. These data are consistent with evidence from previous in vivo and in vitro studies demonstrating a certain selectivity of both uptake and retrograde axonal transport of HRP isoenzyme C that does not appear to be simple ‘fluid-phase’ internalization. The methodology and precautionary steps taken in this study in obtaining a semi-quantitative evaluation of HRP cytochemical visualization should be of general use in a variety of histochemical studies.