A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis
Introduction
Dr. Barnett Rosenberg's experiments on bacteria motility in 1965 (Rosenberg et al., 1965), eventually led to anti-tumor studies using cisplatin (cis-diamminedichloroplatinum II; CDDP). By 1969, limited human studies were conducted and today CDDP is widely used as an effective and potent anti-neoplastic drug (Rosenberg et al., 1969; Rozencweig et al., 1977). CDDP has resulted in remissions of otherwise resistant solid tumors, especially of genito-urinary origin (Merrin, 1979; Wiltshaw and Kroner, 1976; Yagoda et al., 1976). It is also efficient against cancers of the head, neck and lung (Peppard et al., 1980; Gralla et al., 1979).
Several clinical trials, as well as in vitro models, have demonstrated a steep dose-response relationship for a variety of tumor types but numerous toxic side effects have been reported limiting its use (Gandara et al., 1989; review by Hacker, 1991). Nephrotoxicity was originally a major dose limiting side effect. However, different therapeutic regimens, including the use of chemoprotective agents, have allowed CDDP administration at increasingly higher doses (Tognella, 1990; Schweitzer, 1993). Ozols et al. (1984), utilizing the nephroprotective protocols of hypertonic saline and forced hydration, was able to use CDDP dosages at higher levels. Remission rates were low and nephrotoxicity was minimal, but ototoxicity was a dose limiting factor.
Ototoxicity remains a major problem because of a high correlation between the degree of hearing loss and CDDP dosage (Hoeve et al., 1988; Waters et al., 1991). Hydration protocols and most nephroprotectives have not been shown to be effective in preventing ototoxicity, and hearing loss appears to be permanent (Vermorken et al., 1983). Based on an in vitro study comparing inner ear concentrations of other known ototoxic drugs, CDDP was shown to be the most toxic (Anniko and Sobin, 1986).
The mechanisms of CDDP's anti-tumor effects are relatively well understood (Eastman, 1986), but the cellular and molecular activities which manifest themselves as toxic side effects are more elusive. Within a cell's cytoplasm, CDDP becomes a very reactive complex, allowing possible interactions with cytosolic proteins especially if they have amino acid side chains that contain sulphur (Rosenberg, 1979; Gonias et al., 1988). In vitro reactions of CDDP, in solutions with methionine and cysteine, resulted in the formation of chelate products (Appleton et al., 1988). Two hours after administration, in vivo, 90% of CDDP is protein bound (review by Schweitzer, 1993).
CDDP-induced hearing loss begins in the high frequency range then progresses to lower ranges. The hearing loss may be moderate to severe and is almost invariably irreversible (Piel et al., 1974; Kopelman et al., 1988; Pollera et al., 1988).
Most experimental studies on the anatomical effects of CDDP ototoxicity, focus on the outer hair cells (OHC) of the organ of Corti. Hair cell loss is common in the basal turn, correlating well with physiological measures of high frequency hearing loss and with dosage (Fleischman et al., 1975; Hoeve et al., 1988; Campbell et al., 1996).
Only a few studies have reported morphological changes to the stria vascularis secondary to CDDP exposure (Kohn et al., 1988; Tange and Vuzevski, 1984; Nakai et al., 1982; Bonheim and Bichler, 1985). However, observations vary, with Bonheim and Bichler (1985)finding no changes to the stria while Nakai et al. (1982)found minor changes. Kohn et al. (1988), as well as Tange and Vuzevski (1984), both reported extensive damage. All three strial cell types are affected, but marginal cells seem to be especially sensitive with effects ranging from no change, to cystic degeneration with protrusions into the endolymphatic space, followed by a loss of the cells (Kohn et al., 1988; Tange and Vuzevski, 1984). There appears to be no pattern or uniform distribution to the damage, with reports of normal cells found adjacent to degenerating ones (Kohn et al., 1988; Tange and Vuzevski, 1984). Under light microscopy, translucent sites which appeared in the strial tissue were found to be degenerated areas when examined by transmission electron microscopy (Kohn et al., 1988). Apparently these lesions were secondary to the depletion of organized cytoplasmic organelles. Tange and Vuzevski (1984)did not report `edema', a common effect secondary to other drug-induced toxicities, but did report `swelling and protrusion of the marginal cells into the endolymphatic space'. Kohn et al. (1988)also reported strial swelling in at least one animal.
The lack of a standard animal methodology may account for the variable results found in these studies. All used guinea pigs as animal models but with various dosing regimens and different methods of delivery; subcutaneous, intraperitoneal and intramuscular injections. Cumulative doses ranged from 10–42 mg/kg CDDP administered over 5–20 days, with the standard dose at 1.5–2.0 mg/kg/day (Kohn et al., 1988; Tange and Vuzevski, 1984; Nakai et al., 1982; Bonheim and Bichler, 1985). Mortality rates varied; Kohn et al. (1988)reported a 90% loss, while Bonheim and Bichler (1985)lost 33% of their animals. In some cases mortality was not clear. Tange and Vuzevski (1984)only lost 19% of their guinea pigs but mentioned that several animals did not finish the dosing regimen because of the animal's poor condition. No study provided semiquantitative analysis rendering comparisons across studies difficult.
Later, Ravi et al. (1995), developed a model in which rats were given a single 16 mg/kg dose of CDDP by infusion pump over a 30 min period, which 3 days later produced consistent hearing loss with low mortality. At this dosage level, high-peak platinum levels resulted in marked ototoxicity. This level corresponds well to the cumulative doses received in recent common clinical protocols for ovarian cancer which frequently range from 413 mg/m2 (11 mg/kg) for low dose regimens, to 726 mg/m2 (20 mg/kg) for high dose regimens, over a period of 6–12 months (Waters et al., 1991; Gandara et al., 1989). We based these conversions from meter squared to kilograms on an assumed average value in human adult females of 1.73 m2 and 63.5 kg.
Because of its importance in cochlear function and hearing, we wanted to determine the toxic effects of high dose CDDP on the stria vascularis in the rat. Previous reports present conflicting data on the effects of CDDP on the stria, which need to be resolved. Furthermore, the lack of semiquantitative analysis in the previous studies renders it difficult to sufficiently determine CDDP's deleterious effects. Also, the search for promising otoprotective agents for CDDP toxicity, their effects on strial tissue, and objective, quantitative analysis techniques, are needed for careful comparisons. We developed a standard model assessing the damaging effects of CDDP on the cellular architecture of the stria, which can be used to compare the effects of various otoprotective agents for CDDP ototoxicity. We used the rat model, because of its high ototoxicity and low mortality when exposed to CDDP. The focus of the study was to observe changes in the stria vascularis as a whole, and intracellular changes in the marginal cells. Also, parallels between the CDDP destructive processes in the organ of Corti and in the stria vascularis were analyzed and then correlated to hearing loss.
Section snippets
Material and methods
Fifteen male Wistar rats (250–400 g) were used in a previous collateral study which provided the strial tissue for the focus of this study (Campbell et al., 1996). Ten animals received an injection of CDDP to induce ototoxicity. Another group of five controls received an equivalent volume of saline. Five of the CDDP treated animals died, yielding five animals available for study in that group. Auditory brainstem responses (ABRs) were recorded from both groups to determine auditory thresholds at
Survival
We used a total of 15 animals in this experiment, five in the control group and ten in the experimental group. All of the control animals survived. Of the ten animals used in the CDDP treated group, five did not survive until the end of the experiment and were not further included in the analysis.
Light microscopy
Light microscopy imparted a full view of the stria vascularis cross-sectional profile in the tissue from the normal control animals taken from the basal turn. The border of marginal cells lining the
Discussion
In agreement with Kohn et al. (1988)and Tange and Vuzevski (1984), our light microscopy and low magnification TEM examination of the strial tissue showed lesions and cystic deformations. Of greater interest however, was our higher magnification TEM ultrastructural examination of the marginal cells in this study. Kohn et al. (1988)identified lesioned areas using light microscopy and then, using low magnification TEM, described depletion of cytoplasmic organelles in those areas. However they also
Acknowledgements
The authors wish to thank David M. Davenport and Debbie Larsen for their assistance with data collection and analysis, and Donna Wagahoff for her technical assistance with research imaging. This study was funded by SIU School of Medicine Central Research Council.
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