Histochemical Journal, 11 (1979), 337-344
Horseradish peroxidase as a label of injured cells GI~INTHER G E Y E R , H A N S - P E T E R SCHMIDT and MANFRED BIEDERMANN Institute of Anatomy and Institute of Physiology, Friedrich Schiller University of Jena, Jena, East Germany
Received 13 September 1978 and in revised form 27 November 1978
Synopsis. The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H2 02 medium for the shortest possible period. Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.
Introduction
Recently, we claimed to have shown, from two sources of evidence, that exclusion of horseradish peroxidase (HRP) is a suitable test of cell viabifity. The first hint arose from work on the organ of Corti. In the guinea-pig inner ear which has previously been exposed to high-energy impulse noise and perfused perilymphatically with HRP-Ringer solution, single sensory cells exhibit an intensely-stained cytoplasm due to penetration of the cells by the peroxidase (Geyer et al., 1978; Schmidt et al., 1978). Additional evidence had been gained from a study on murine peritoneal ceils. Following an incubation in buffered saline devoid of metabolites, the number of dead macrophages 9 1979 Chapman and Hall Ltd. Printed in Great Britain.
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and lymphocytes increased with time. Consistently, the peroxidase test revealed an increasing number of cells whose membrane had become permeable to HRP (K6hler & Geyer, 1978). Although these findings are in accord with exclusion tests employing Trypan Blue or Ruthenium Red (PapperLheimer, 1971; Bahai, 1977; Mickwitz et al., 1977), the apparent demonstration that HRP may exert a cytotoxic activity (Ross et al., 1977) would seem to invalidate its use in a test for cell viability. Surprisingly, in spite of the widespread use of this protein as an extracellular and axonal tracer, toxic effects have rarely been reported. According to Cotran & Karnovsky (1967), HRP induces vascular leakage in the rat and guinea-pig, but not in mice. The effect was shown to be caused by histamine and serotonin liberated by emiocytosis of mast cell granules, a responce the authors considered indicative of cellular damage. Penetration of HRP into single Sertoli cells and type B spermatogonia was shown by Willson et aL (1973) to be associated with the breakdown of the testicular-tubular barrier in guinea-pigs previously treated with Freund's complete adjuvant. However, no definite conclusion could be reached about the real cause(s) of this finding nor of the HRP staining of various types of other cells (Hugon et al., 1968; Brightman et al., 1970; Olsson et al., 1970; Hugon, 1971; B6ck 1972;Garrett & Parsons, 1976), although artificial effects have been considered as a possible source of the cytoplasmic tracer infiltration of single cells. The discrepancies prompted us to carry out the present study. Our findings lack support for the idea that HRP causes acute toxicity, but show that methodical errors may have an effect. Materials and methods
A group of six young healthy guinea-pigs was anaesthetized with urethane and diethyl ether prior to the injection into the cerebello-medullary cistern of 0.2 ml 6% HRPRinger solution (pH 7.6, 277 -+3 mosmol; HRP purchased from VEB Arzneimittelwerk Dresden, RZ