A semiquantitative analysis of the effects of cisplatin on the rat stria vascularis

Hearing Research 124 (1998) 44^59

A semiquantitative analysis of the e¡ects of cisplatin on the rat stria vascularis Robert P. Meech, Kathleen C.M. Campbell *, Larry P. Hughes, Leonard P. Rybak Department of Surgery, SIU School of Medicine, P.O. Box 19230, Spring¢eld, IL 62794-1618, USA Received 24 December 1997; revised 27 April 1998; accepted 16 June 1998

Abstract Cisplatin (CDDP) is a very effective chemotherapeutic agent but is highly ototoxic. Most studies have focused on the effects of CDDP on the outer hair cells. The purpose of this study was to examine changes in the stria vascularis in cisplatin treated male Wistar rats and to provide semiquantitative analysis of the results. We removed a section of the stria vascularis from the basal turn of five control and five CDDP (16 mg/kg) treated rats. Using transmission electron microscopy (TEM) we analyzed: (1) changes to the strial tissue as a whole; and (2) intracellular changes in the marginal cells. We also subjected the samples to semiquantitative analysis using the MCID, focusing on three aspects of strial profile abnormalities ; the number of abnormal marginal cells in CDDP treated tissue, intracellular strial edema and densitometry. Controls appeared normal, but many pathologic changes were apparent in the experimental group. Results from the semiquantitative analysis indicate cisplatin has a deleterious effect on the stria vascularis including strial edema; bulging, rupture and/or compression of the marginal cells and depletion of the cytoplasmic organelles. z 1998 Elsevier Science B.V. All rights reserved. Key words: Cisplatin; Stria vascularis; Ototoxicity; Semiquantitative analysis

1. 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 e¡ective 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 e¤cient 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 e¡ects have been reported limiting its use (Gandara et al., * Corresponding author. Tel.: +1 (217) 524-4419; Fax: +1 (217) 524-0253.

1989 ; review by Hacker, 1991). Nephrotoxicity was originally a major dose limiting side e¡ect. However, di¡erent 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 e¡ective 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).

0378-5955 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 1 6 - 6

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The mechanisms of CDDP's anti-tumor e¡ects are relatively well understood (Eastman, 1986), but the cellular and molecular activities which manifest themselves as toxic side e¡ects 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 e¡ects 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) ¢nding 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 a¡ected, but marginal cells seem to be especially sensitive with e¡ects 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 e¡ect 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

45

dosing regimens and di¡erent 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 ¢nish the dosing regimen because of the animal's poor condition. No study provided semiquantitative analysis rendering comparisons across studies di¤cult. 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 e¡ects of high dose CDDP on the stria vascularis in the rat. Previous reports present con£icting data on the e¡ects of CDDP on the stria, which need to be resolved. Furthermore, the lack of semiquantitative analysis in the previous studies renders it di¤cult to su¤ciently determine CDDP's deleterious e¡ects. 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 e¡ects 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. 2. Material and methods Fifteen male Wistar rats (250^400 g) were used in a previous collateral study which provided the strial tissue

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for the focus of this study (Campbell et al., 1996). Ten animals received an injection of CDDP to induce ototoxicity. Another group of ¢ve controls received an equivalent volume of saline. Five of the CDDP treated animals died, yielding ¢ve animals available for study in that group. Auditory brainstem responses (ABRs) were recorded from both groups to determine auditory thresholds at the beginning of the experiment and again 3 days later. After the ¢nal testing, the animals were killed and cochleae were removed and preserved for histological studies. Scanning electron microscopy (SEM) was used to observe OHCs in the basal turn and for semiquantitative analysis of damaged OHCs, as described in Campbell et al. (1996). Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe cross-sectional pro¢les of the stria vascularis from the basal turn. Semiquantitative analysis of strial pro¢les was performed with the Microcomputer Imaging Device (MCID) (Imaging Analysis Inc.), an image analysis instrument capable of spatial and densitometry measures. Strial samples from the basal turn were used to correlate with the area of greatest hearing loss and outer hair cell loss secondary to CDDP ototoxicity. Sections taken from the basal turn of the cochlea were dehydrated in EtOH (30%, 50%, 70%, 85%, 2U95% and 3U100%), rotated three times (10 min each) in propylene oxide and in¢ltrated, three-fold, with Poly/Bed 812 (Polysciences Inc.) resin for 1^2 h each. Once in¢ltrated, the tissue was rotated in 100% polybed overnight, placed in £at molds with labels, covered with fresh polybed and allowed to harden in an oven. Resulting blocks were thick sectioned 1 micron thick and placed on glass slides, then stained with toluidine blue for light microscopy. Thin sections, 90 nm thick, were sectioned on an RMC MT-7 ultra-microtome and placed on grids. Thin sections were stained with 2% uranyl acetate in 50% ethyl alcohol, and Reynold's lead citrate before viewing under a Hitachi H-7000 electron microscope using an accelerating voltage of 75 kV. Changes, secondary to CDDP treatment, to the stria vascularis harvested from the basal turn were observed and recorded. Two approaches were used: (1) changes to the strial tissue as a whole; and (2) changes in the ultrastructure of the marginal cells. Glass slides and electron micrographs of cross-sections from the stria vascularis were subjected to semi-

quantitative analysis using the MCID. The analysis focused on three aspects of strial pro¢le abnormalities; the number of abnormal marginal cells in CDDP treated tissue, intracellular strial edema, and densitometry. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. 2.1. Marginal cell abnormalities A grading system was used to semiquantify marginal cell abnormalities observed bordering the lumen of the scala media in the basal turn. However, it was ¢rst necessary to determine the number of cells in these cross-sectional strial pro¢les. Apical tight junctions of marginal cells were con¢rmed with TEM. The electron micrographs were marked using the MCID and the number of cells determined for each subject. Each marginal cell, at the region of the lumen, was rated by two independent judges according to whether it was: (1) normal ; (2) bulging (into the lumen) or ruptured; (3) compressed, as frequently occurs between two bulging cells ; or (4) bulging and compressed. To facilitate the counting process LM slides were digitized on the MCID and marked to approximate the location of tight junctions and thus the borders between marginal cells. This allowed a complete cross-sectional view of the stria vascularis and marginal cells. 2.2. Intracellular edema To measure the intracellular strial edema noted in CDDP treated stria, ¢rst light micrographs of crosssections through the stria vascularis from the basal region of the cochlea were digitized on the MCID at a standard magni¢cation of 160U. Four sections of the stria vascularis from each control animal and each CDDP treated animal were digitized and used in this study, for a total of 40 strial images. Two judges, familiar with strial tissue, outlined the boundary of each cross-section except for the luminal edge as described by Santi et al. (1983). Images were previously coded and presented in random order so that the judges were blinded regarding whether a strial image was from a treated or control animal. The areas within the boundaries were measured by the computer and compared between groups. The outlined images were

C Fig. 1. Two light micrographs showing cross-sections through the basal coil of the stria vascularis: a: from a normal control showing the normal appearance of marginal cells which line the endolymphatic space (arrows); b: from a cisplatin treated animal, which shows the characteristic abnormal bulging and protrusions of the apical regions of the marginal cells into the endolymphatic space (arrows), as well as translucent sites (*). Notice the changes in the ICs and BCs (arrowheads). (Original magni¢cation U100.) As discussed in the text, the separation of the strial tissue from the spiral ligament can occur as a preparation artifact but occurred rarely in the untreated controls and in every tissue sample from CDDP treated animals.

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2.3. Densitometry

Fig. 2. A graph comparing the means and standard errors for crosssectional areas (Wm2 ) from strial pro¢les of control and the experimental groups. Four sections of the stria were taken from each control and CDDP treated animal for a total of 40 strial images. Mean area was then determined from four separate trials (A1, A2, A3, A4) by two independent judges and found to be signi¢cantly greater in the CDDP treated group (P 9 0.01).

subjected to spatial measurements using the MCID, calibrated in microns, to a known linear scale. This method allowed determination of the cross-sectional areas from the strial pro¢les.

Semiquantitative densitometry measurements were obtained using the MCID by measuring the relative optical density (ROD) of target tissues. RODs were measured in randomly selected marginal cells (MCs) and adjacent intermediate cells (ICs). Cell density ratios were then determined for control and CDDP treated tissues. Measurement conditions were tightly controlled in the following manner: (a) Cross-sectional pro¢les, of the stria vascularis, were divided into thirds based on the number of marginal cells bordering the scala media lumen. This allowed sampling from three regions along the strial cross-section : the Reissner's membrane end, the spiral prominence end, and the middle. A sample of marginal cells was selected from each region, using a table of random numbers. (b) Tissue preparation was standardized, so that all strial tissue was prepared in the same manner from the initial ¢xative to embedding in polybed resin. (c) Thin sections were taken on a RMC MT-7 ultramicrotome, set at 90 nm thickness, and placed on un-

Fig. 3. TEM micrograph of the stria vascularis from a normal control animal showing normal characteristics such as the consistently marked di¡erences in electron density between darker marginal cells (MCs) and the lighter intermediate cells (ICs), regular spacing and smooth luminal surface of MCs, oval shaped nuclei (Nu) found apical in the MC, and a highly compartmentalized system of processes and infoldings in the subnuclear region of the MCs (P), which interdigitate with ICs and basal cells. (Original magni¢cation U3714.)

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Fig. 4. TEM micrograph of a cisplatin treated stria showing several degenerating marginal cells (MCs); abnormalities include inconsistent variations in electron density di¡erences between MCs and intermediate cells (*), MCs compressed between adjacent cells (arrows) which were also observed in serial sections, bulging apical surfaces of MCs (arrowheads), large, round, centrally located nuclei containing little heterochromatin (Nu) and a break down in the compartmentalization of the subnuclear processes. Unlike normal cells, note the presence of large vacuoles (V) and apical gatherings of mitochondria (m). (Original magni¢cation U3033.)

coated #200 or #300 mesh grids. Sections were stained for 7 min each in uranyl acetate and lead citrate. (d) Electron micrographs were taken on a Hitachi 7000 electron microscope, using a 75 kV accelerating voltage, at a magni¢cation of 2000U with a spot size of 5. Image recording was controlled to produce negatives with the same optimum photographic density by timing exposures for 2 s and manually adjusting the light density setting (LDS) to 310.25. (e) The ¢lm exposure size was set at one-half to allow two exposures per ¢lm. In this way, the ¢rst exposure was a blank area of the screen. The second half of the negative was an exposure centered on the marginal cell of interest with adjacent intermediate cells and a portion of plain polybed resin visible. The LDS for the second exposure was the same as the blank and was not reset. All exposures were taken in as short a time period as possible to minimize atmospheric di¡erences, which may a¡ect scope operation, and all negatives were developed simultaneously. (f) Negatives were used for MCID analysis. Distance from the light stand platform to the camera holder was

84 cm, the camera lens was set at f-16, and illumination was corrected for shading error. (g) All blanks were digitized to a predetermined Northern Light (NL) setting, which produced the best overall specimen image, and the RODs recorded. Averaged density readings served as a bench mark, to which NL settings for each blank were adjusted. This standardized the light setting for each specimen exposure. (h) Sampling size was standardized for the marginal cells and intermediate cells, and the RODs of twenty targets, from each cell type (marginal and intermediate), were averaged and recorded. Sampling tools, which can be altered as to size and shape, facilitate the recording of RODs for speci¢c tissue structural di¡erences. Because of the interdigitations common in ICs, we used a small circle at a standard diameter (20 pixels) for sampling MCs and ICs. Each target was an average grey level value of all the pixels in the sampling tool window (circle). For resins, as much of the resin as possible was targeted and RODs averaged. In blanks, the ROD from one target of the complete screen was recorded.

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50 Table 1 Classi¢cation of damage in marginal cells Control

Total # MC

#1

%

#2

%

#3

%

#4

%

1 2 3 4 5

23 21 26 23 23

23 21 26 23 23

100 100 100 100 100

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

Average

23.2

23.2

100

0

0

0

0

0

0

Experimental

Total # MC

#1

%

#2

%

#3

%

#4

%

1 2 3 7 10

27 21 23 26 24

11 4 12 14 7

40.7 19.0 52.2 53.8 29.2

9 12 5 4 15

33.3 57.1 21.7 15.4 62.5

5 2 5 7 1

18.5 9.5 21.7 26.9 4.2

2 3 1 1 1

7.4 14.3 4.3 3.8 4.2

Average

23.8

9.6

39.0

9

38.0

4

16.2

16

6.8

The upper and lower parts showing the total number of marginal cells in cross-sectional pro¢les from control and experimental groups. Upper part: Controls showed no distortions and 100% fell in the normal category. Lower part: The experimental group showed a number of abnormalities. The table lists the number of cells and the percentages of the total number which fell into each category. The di¡erences between groups were found to be signi¢cant (P 9 0.01).

2.4. Statistical analysis Analysis of covariance (ANCOVA) between and within subject variables was used on the collected semi-

quantitative data from marginal cell abnormalities and mean areas of strial cross-sectional pro¢les. Post-hoc analysis utilized the Tukey HSD test. In the densitometry experiment, analysis of variance between mean

Fig. 5. TEM micrograph from a normal control of the supranuclear region in a marginal cell showing the normal array of cytoplasmic organelles such as rough endoplasmic reticulum (rER), large numbers of vesicles (v), with some indication of endocytosis or exocytosis (arrows) and a prominent nucleus (Nu) with a well-de¢ned nuclear envelope (arrowheads). Only a few mitochondria are present in this region (m). (Original magni¢cation U15 080.)

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Fig. 6. TEM micrograph from a cisplatin treated animal, of the supranuclear region in a clearly degenerating marginal cell, showing a lack of normal organelles and vesicles, and a small nucleus appearing to be degenerating and lacking a nuclear envelope (Nu). Also note the congregation of mitochondria (m); many are damaged and associated with vacuoles (arrows). L, lumen. (Original magni¢cation U13 000.)

ROD ratios of IC/MC was used, co-varying with blank and resin measures. A Pearson correlation matrix was used to compare the marginal cell damage to the previously collected hair cell data and hearing threshold data from our collateral study (Campbell et al., 1996). For all analyses, a statistical signi¢cance criteria of (P 9 0.01) was used. 3. Results 3.1. Survival We used a total of 15 animals in this experiment, ¢ve 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, ¢ve did not survive until the end of the experiment and were not further included in the analysis. 3.2. Light microscopy Light microscopy imparted a full view of the stria vascularis cross-sectional pro¢le in the tissue from the

normal control animals taken from the basal turn. The border of marginal cells lining the endolymphatic space appeared smooth with no bulging. Marginal cells and intermediate/basal cells showed the characteristic di¡erences in density. In a few of the thick sections, the strial tissue separated from the underlying spiral ligament. This ¢nding can occur as a preparation artifact but occurred in all of the samples from experimental animals but rarely in the samples from the control animals (2/5). The apical regions of marginal cells from experimental group tissues showed bulging and protrusions into the endolymphatic space. Translucent areas, described in previous reports, were evident (Kohn et al., 1988). The intermediate and basal cells appeared abnormally large, with large round, rather than oval shaped nuclei. Also, in our light micrographs of the CDDP treated tissue, stria from all of the experimental animals were separated from the spiral ligament and appeared swollen (Fig. 1). Using semiquantitative analysis of strial pro¢les for edema, the area measurements from the experimental group were signi¢cantly larger than the controls (P 9 0.01). Mean areas for the control group and experimental group were 7229.107 Wm2 and

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Fig. 7. TEM micrograph of the subnuclear region in a marginal cell (MC), from a normal control showing the abundance of normal mitochondria of di¡erent sizes and shapes (m) and the highly organized system of processes and infoldings. IC, intermediate cell. (Original magni¢cation U13 000.)

8899.514 Wm2 respectively. Interjudge agreement was excellent revealing no signi¢cant di¡erence between judges (P = 0.799) (Fig. 2). 3.3. TEM survey In the control tissues, by TEM examination, the stria vascularis appeared normal, as indicated by the characteristic di¡erences in electron density between the darker marginal cells with their ¢nger-like processes, and the lighter intermediate and basal cells. Tissue lining the endolymphatic space appeared smooth with few minor bulges, and cells appeared to be normally spaced. Overall, cell nuclei size and shape appeared normal, with nucleoli and proportional amounts of euchromatin and denser heterochromatin (Fig. 3). In the CDDP treated tissues, the stria did not appear normal in any of the samples. All of the strial tissues had several medium to minor bulges along the endolymphatic space. Often, a major bulge into the lumen appeared as a membranous sac into which a cell had spilled its contents. The plasma membrane of marginal cells along the endolymphatic space occasionally showed small ruptures and some spilling of cell contents directly into the lumen. The appearance of marginal

cells along the lumen often were not evenly spaced. There were several occurrences of cells appearing squeezed between two adjacent bulging cells. This ¢nding was con¢rmed in analyzing serial sections of MCs compressed between adjacent cells. In some tissue samples, this damage was not as severe. Also most nuclei did not appear normal, especially in the marginal cells, being large and round rather than oval in shape, and often with no distinguishable nucleolus. These nuclei, which were very light, appeared to have very small amounts of heterochromatin present. Other nuclei appeared small, compact and irregular in shape, as well as very dense. Some cells did not have a nucleolus present at all. In the latter two cases, these anomalies may have been due to the sectioning plane of the thin sections, but there was a higher incidence in the CDDP group (Fig. 4). The number of marginal cells bordering the lumen of the scala media ranged from 23.2^23.8 on average for the two groups, which was consistent with previous reports in the literature for the rat basal turn (Lohuis et al., 1990). Separate rankings, carried out by two individual judges on 237 marginal cells, were 99.6% in agreement. In controls, 100% of the cells were ranked in the #1 (normal) category, whereas in the experimen-

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Fig. 8. TEM micrograph of the subnuclear region in a marginal cell (MC), from a cisplatin treated animal showing reduced numbers of mitochondria (m), damaged mitochondria (*), and general disorganization. IC, intermediate cell. (Original magni¢cation U13 000.)

tal group, only 39.0% fell into this category. Also in the experimental group, 38.0% fell in category #2 (bulging or ruptured), 16.2% in category #3 (compressed) and 6.8% in category #4 (bulging and compressed). The frequency of abnormalities in the marginal cells from the experimental group as a whole, when compared to the controls, was signi¢cant (P90.01). A signi¢cant difference was also found between the controls, and each category of the experimental group (P90.01) (Table 1). Ultrastructural examination (high magni¢cation) of the stria vascularis from control animals revealed highly compartmentalized marginal cells. In the supranuclear region large numbers of vesicles and small vacuoles were present. Invaginations of the luminal membrane were sometimes apparent. Arrays of rough endoplasmic reticulum (ER) appeared as short, sometimes isolated, rough surfaced cisterns. Free ribosomes were found, as well as smooth ER, which appeared as networks of ¢ne tubules. Only a few mitochondria appeared scattered in the supranuclear region (Fig. 5). Ultrastructural examination of marginal cells from all of the CDDP treated tissue samples examined at high magni¢cation showed abnormalities. The nuclear envelopes surrounding the nuclei of many cells often could not be clearly delineated. In some instances, leak-

age of nucleoplasm was apparent. The characteristic compartmentalization of marginal cells was often in disarray. Supranuclear regions of many cells were found to be devoid of vesicles, rough ER, and smooth ER. Mitochondria, many severely damaged, seemed to congregate in this area unlike normal tissue. Invaginations of the plasma membrane were not as apparent. Larger than normal vacuoles and membranous remnants were plentiful. Many vacuoles were found associated with damaged mitochondria (Fig. 6). In controls at high magni¢cation, an orderly system of processes and infoldings from marginal cells interdigitated with intermediate and basal cells below the nucleus. Mitochondria and smooth ER were prevalent in this area and occurred in a highly organized manner. A few mitochondria appeared damaged, but the majority remained intact. Small lipid droplets and glycogen granules were frequently found throughout the cells (Fig. 7). In the CDDP treated tissues, the processes below the marginal cell nuclei appeared disorganized. Mitochondria were reduced in numbers, and damage was common (Fig. 8). Marginal cells from CDDP treated tissue, which appeared normal at lower magni¢cation, showed subtle

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Fig. 9. TEM micrograph from a cisplatin treated animal, of the supranuclear region in a marginal cell appearing normal at low magni¢cation but at high magni¢cation abnormalities are apparent. Abnormalities include less rough endoplasmic reticulum (rER), fewer vesicles (v), an absence of invaginations, interruptions to the nuclear envelope (arrowheads) surrounding the nucleus (Nu), the appearance of more mitochondria (m) in this region, and the presence of a large vacuole (Vac). (Original magni¢cation U13 000.)

abnormalities at high magni¢cation, which included changes to nuclei, a lack of normal organelles, the appearance of vacuoles and increased numbers of mitochondria (Fig. 9). Mean ROD ratios of ICs to MCs were determined for three regions. Based on averages from the marginal cell counts described above, each region of the stria vascularis was assigned marginal cells in the following manner: (1) the spiral prominence and Reissner's membrane regions, seven cells each; (2) all remaining cells were assigned to the middle region. This facilitated randomly choosing marginal cells from each region for densitometry measures as described in Section 2. There was a signi¢cant di¡erence in ROD ratios between groups in both the spiral prominence and middle regions (P 9 0.01). In the Reissner's membrane region, the di¡erence in ROD ratios were marginally signi¢cant between groups (P = 0.08). The di¡erence in IC/MC ROD ratios for all three regions combined, were signi¢cant (P 9 0.0001) (Figs. 10 and 11). In all analyses, the ROD ratios were larger for the control than for the experimental group. The morphologic changes to the strial marginal cells in this study correlated well with the physiologic and

morphologic data from our previous collateral study (Campbell et al., 1996). All of the CDDP treated animals experienced hearing loss, with mean ABR threshold changes as follows: 30.0 dB, clicks; 24.0 dB, 1 kHz; 34.0 dB, 4 kHz ; 26.0 dB, 8 kHz; and 52.0 dB, 14 kHz. We assessed the strial and OHC damage from tissue removed from the basal turn. This area is the high frequency region of the cochlea which showed the greatest ABR threshold shifts. Strial damage and OHC damage were 30.5% and 17.6%, respectively. All of the correlations between threshold elevations, strial damage and OHC damage were signi¢cant at P 9 0.01, except for the clicks, 1 kHz and 4 kHz, which correlated to OHC damage at the P 9 0.05 level but not P 9 0.01. The largest mean ABR threshold change was at 14 kHz (Table 2). 4. Discussion In agreement with Kohn et al. (1988) and Tange and Vuzevski (1984), our light microscopy and low magni¢cation TEM examination of the strial tissue showed lesions and cystic deformations. Of greater interest

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Fig. 11. Analysis of all regions combined found IC/MC ROD ratios signi¢cantly higher in controls than experimental groups (P 9 0.001).

Fig. 10. Graphs showing the relative optical density (ROD) ratios of IC/MC, from three regions in the cross-sectional strial pro¢les of the control (CON) and experimental (EXP) groups. a: In the spiral prominence region (SP), IC/MC ROD ratios were found to be signi¢cantly higher in control than in experimental groups (P 9 0.01). b: In the middle region (MID), IC/MC ROD ratios were found to be signi¢cantly higher in control than in experimental groups (P 9 0.01). c: In Reissner's membrane region (RM), IC/MC ROD ratios were higher in control than in experimental groups but did not reach statistical signi¢cance (P = 0.08).

however, was our higher magni¢cation TEM ultrastructural examination of the marginal cells in this study. Kohn et al. (1988) identi¢ed lesioned areas using light microscopy and then, using low magni¢cation TEM, described depletion of cytoplasmic organelles in those areas. However they also described normal appearing regions using low magni¢cation TEM. However, using TEM at high magni¢cation (v 10 000U), with a thorough ultrastructural examination of the marginal cells

from CDDP treated rats, we consistently found subtle but obvious changes to the cellular architecture in marginal cells that appeared normal at lower TEM magni¢cation. Kohn et al. (1988) also reported that CDDP damage to marginal cells was intermittent with abnormal cells next to normally appearing cells. We also found areas of intermittent damage but in most strial samples from experimental animals, we also consistently found regions of major, consistent marginal cell damage along the lumen. Our semiquantitative analysis of marginal cell damage classi¢cation revealed a signi¢cant number of abnormal marginal cells in the CDDP treated animals, with 61.0% classi¢ed in abnormal categories. Previous descriptive assessments of the strial pro¢le, including marginal, intermediate, and basal cells, estimated that only one-third was degenerated by CDDP toxicity, with marginal cells being the most frequently damaged. We only analyzed marginal cells because, with their extensive basal-lateral projections, they constitute a major part of the stria vascularis, and thus are representative of the strial pro¢le as a whole (Friedmann and Ballantyne, 1984). Our nonquantitative observations of intermediate and basal cells in our samples suggested abnormalities in those areas as well, in keeping with our marginal cell analysis. Our semiquantitative analysis of strial cross-sectional pro¢les revealed a statistically signi¢cant incidence of intracellular edema secondary to CDDP toxicity. In previous studies, edema was not reported by Tange and Vuzevski (1984), or by Kohn et al. (1988) but they did not conduct a formal analysis. However, Tange and Vuzevski (1984) reported swelling in marginal cells and Kohn et al. (1988) reported swelling in the strial tissue from one animal. Edema can refer to swelling or an excessive accumulation of £uid in either intraor extracellular areas (Stedman's Medical Dictionary, 1995). Our results were similar to the e¡ects of ototoxic diuretic drugs, such as furosemide, bumetanide and

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56

Table 2 Correlations between strial damage, hair cell damage/loss, and ABR threshold shifts Strial damage HC damage CLK DIFF 1 K DIFF 4 K DIFF 8 K DIFF 14 K DIFF

Strial damage

HC damage

CLK DIFF

1 K DIFF

4 K DIFF

8 K DIFF

14 K DIFF

1.000 0.864* 0.808* 0.832* 0.916* 0.972* 0.982*

1.000 0.888* 0.714** 0.760* 0.819* 0.841*

1.000 0.672** 0.784* 0.839* 0.844*

1.000 0.632** 0.845* 0.784*

1.000 0.897* 0.959*

1.000 0.977*

1.000

The table shows the correlation between damage to the stria vascularis, OHC damage and hearing loss (P 9 0.01). *, signi¢cant at the P 9 0.01 level. **, signi¢cant to P 9 0.05 level. HC, hair cell; CLK, clicks; DIFF, di¡erence; K, kHz.

ethacrynic acid, in which tissue swelling, bulging marginal cells and edema is a characteristic ¢nding (Santi and Duvall, 1979; Rybak et al., 1992; Bosher, 1980 ; Brummett et al., 1977) although we did not ¢nd an accumulation of extracellular £uid secondary to CDDP administration as was found secondary to diuretic administration. Electron density ratios, between randomly selected marginal cells (MCs) and adjacent intermediate cells (ICs) in controls, were found to be signi¢cantly di¡erent from the ratios of the experimental group in this study (P 9 0.01). In normal controls, electron density was markedly greater in marginal cells as compared to intermediate/basal cells. We found that the ratios of relative optical densities (RODs) between cell types (IC/MC) were reduced in the CDDP treated tissues, indicating a depletion of cytoplasmic organelles and inclusions in the marginal cells. Normal density di¡erences between marginal cells and intermediate/basal cells were seen at both the LM and TEM level, and were attributed to the amount of cytoplasmic organelles intrinsic to the functions of these di¡erent cell types. The high metabolic activity of the marginal cells would require large and diverse numbers of organelles and inclusions. Our observations agree with Kohn et al. (1988), who previously found that the translucent sites appearing in the strial tissue, and observed under LM, were actually degenerated areas when examined by TEM. The statistical results of our semiquantitative analysis suggest an overall depletion of cytoplasmic organelles and inclusions in the marginal cells of the experimental group. We also found strong, statistically signi¢cant correlation among marginal cell damage, hair cell damage and hearing loss. In previous studies, when comparisons were made, the reports varied. Tange and Vuzevski (1984) did not examine OHC damage, but reported hearing loss after the 8th day of CDDP treatment, with a corresponding degeneration of the stria vascularis. Kohn et al. (1988) found characteristic OHC damage secondary to CDDP toxicity but variable hearing loss, ranging from no change in hearing thresholds (20 dB SPL þ 5 dB) to elevations of 30 dB SPL, with no

clear correlation in the degree of strial damage. Nakai et al. (1982) reported hearing loss and OHC loss, but only minor strial damage. Variability in hearing loss (Kohn et al., 1988) may be partially attributed to the stimuli of only 100 Ws clicks which may not be a sensitive measure of high frequency hearing loss in rodents. We measured ABR thresholds for click (100 Ws) and also tone burst stimuli centered at 1, 4, 8, and 14 kHz and in an intensity series starting at 100 dB peSPL (SPL for tone bursts) down to 0 dB peSPL (Campbell et al., 1996). Correlations between strial damage and hearing loss were stronger as the frequency of the tone burst increased in our study. The strial damage which we found may be explained, at least in part, by the results reported in the other studies. In a platinum distribution study, guinea pigs were administered CDDP labeled with radioactive 195m platinum. There was a two- to three-fold increase in platinum uptake in the stria vascularis, compared to the organ of Corti, indicating the preferable localization of CDDP in this tissue (Schweitzer et al., 1986). Although uptake into the ear is slow, 90% of CDDP is protein bound within 2 h of administration, and is possibly binding to cytosolic proteins and enzymes associated with strial tissue (review by Schweitzer, 1993; Gonias et al., 1988; Appleton et al., 1988). BaggerSjoëbaëck et al. (1980) reported that CDDP signi¢cantly inhibits adenylate cyclase, a membrane bound enzyme highly active in the stria vascularis, which mediates the formation of cyclic AMP, a second messenger responsible for a wide variety of cellular responses. It is commonly held that the stria vascularis is the tissue responsible for EP generation, so pathologic damage, secondary to CDDP toxicity, should have an e¡ect on this potential. Ravi et al. (1995), who developed the animal model and dosing protocol we used, had similar results in CDDP treated rats, but also reported a decrease in the EP, which suggested strial damage. However, there is a large body of con£icting data regarding this matter. Other studies of CDDP's e¡ect on the EP in guinea pigs, indicate that hearing loss is not necessarily accompanied by a reduction of the EP (McAlpine and Johnstone, 1990). Laurell et al.

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(1995) observed a decrease in K‡ concentration in the endolymph, but no change in the EP, when rats were treated with CDDP. Konishi et al. (1983)'s recordings of the EP, in CDDP treated guinea pigs, found it signi¢cantly suppressed but not markedly, and alterations in the electrolyte concentrations of the endolymph were not signi¢cant. Finally, Barron and Daigneault (1987) reported hearing loss and hair cell damage in CDDP treated guinea pigs, but little e¡ect on Na,K-ATPase activity in the lateral wall. Na,K-ATPase is believed to be responsible for maintaining the ionic composition of the endolymph (Kuijpers, 1969). It would appear that the responsible active mechanisms, in the stria vascularis, are apparently not a¡ected, at least not to an extent that would make a perceptible change in the EP. Decreases in K‡ concentration have been attributed to CDDP's interaction with the passive transport system in the stria vascularis, and not with active transport (Laurell et al., 1995). This experiment demonstrates that CDDP has a major, deleterious e¡ect on the stria vascularis, generally supporting previous experiments by Kohn et al. (1988) and Tange and Vuzevski (1984). However, di¡erences in animal models may have contributed to discrete differences among our results and the results of the previous studies. To produce acute ototoxicity we used the rat as an animal model, a single large dose of CDDP (16 mg/kg), and our method of delivery was a 30 min intraperitoneal infusion. This protocol resulted in consistent hearing loss with low mortality. The other studies used similar cumulative doses of CDDP, but in guinea pigs, and their dosing regimens called for daily doses over several days, with di¡erent methods of delivery. For example, Laurell and Engstroëm (1989) found that multiple low doses of CDDP caused hearing loss in guinea pigs without a change in the EP, but a single high dose caused a simultaneous change in hearing threshold and EP, indicating a two-fold a¡ect of CDDP on the inner ear (Laurell and Engstroëm, 1989). In our study, and Ravi et al. (1995)'s, a single large dose of CDDP was given to rats, which produced hearing loss, OHC damage, strial damage, and a decrease in EP. Investigation into the e¡ects that our protocol has on the ionic composition of the endolymph, Na,K-ATPase activity, and adenylate cyclase in the stria vascularis, would be worthwhile. Also, further studies of the ototoxic e¡ects of CDDP, on the intermediate and basal cells, are warranted because they may be involved in ionic transport. Further, we have developed new methods for semiquantitative analysis of CDDP strial damage that have enabled us to elucidate and de¢ne changes more clearly than in the previous studies. Previous studies have developed quantitative methods to assess strial damage (Santi et al., 1983, 1985). Three studies analyzed volume densities of di¡erent cellular components in the strial

57

tissue of animals treated with bumetanide, an ototoxic loop diuretic, Mannitol, a hyperosmotic agent, and in normal controls. Tissue from bumetanide or Mannitol treated animals showed increased volume density in intercellular spaces and ICs but decreased volume density in MCs (Santi et al., 1983 ; Santi and Lakhani, 1983; Santi et al., 1985). Like the previous studies for other agents, we also found overall increases in strial area secondary to CDDP. However our methodology was di¡erent by using the MCID computer analysis for overall strial areal measurement as opposed to a point counting stereological method. The MCID methodology, which only recently became available, is substantially faster and easier to use. Additionally, we classi¢ed the type of marginal cell damage observed secondary to CDDP ototoxicity as bulging, compressed, or bulging and compressed, as opposed to normals which provides a semiquantitative method for statistical comparison between groups. Although earlier studies examining the e¡ects of diuretics quantitatively analyzed intercellular edema, intercellular edema was not observed secondary to CDDP administration in our study although intracellular edema was observed. We also observed depletion of the cytoplasmic organelles in the marginal cells of the CDDP treated strial tissues and provided semiquantitative analysis of these changes by comparing the relative optical densities of the marginal versus the intermediate cells. Further studies on the e¡ects of CDDP on the volume densities of strial components may help determine the mechanisms involved in intracellular edema. In summary, the tissue surveys and semiquantitative analysis from this study indicate severe damage to the rat stria vascularis, secondary to CDDP treatment and provide some additional semiquantitative techniques for analyzing these changes. A signi¢cant number of abnormal marginal cells in cross-sectional strial pro¢les and the presence of intracellular edema were found. At high magni¢cation, an insidious alteration of the ultrastructural architecture in marginal cells was discovered. Differences in densitometry ratios, of IC/MC, were signi¢cant between experimental and control tissues. Finally, a strong correlation between marginal cell abnormalities, OHC damage, and hearing loss, was indicated. This experiment serves well as a standard model for high dose CDDP in which comparisons can be made, especially as the search for otoprotective agents for high dose CDDP therapy continues. While CDDP toxicity is believed to primarily a¡ect the outer hair cells in the organ of Corti, this study reinforces evidence that the stria vascularis is also a major site of CDDP ototoxicity. The important role that the stria vascularis plays in cochlear dynamics cannot be overlooked in future studies on CDDP ototoxicity and the search for otoprotective agents. This experiment provides a standard model

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in which the e¡ects of CDDP on the stria vascularis and the e¡ects of otoprotective agents, can be compared. Acknowledgments The authors wish to thank David M. Davenport and Debbie Larsen for their assistance with data collection and analysis, and Donna Wagaho¡ for her technical assistance with research imaging. This study was funded by SIU School of Medicine Central Research Council. References Anniko, M., Sobin, A., 1986. Cisplatin: evaluation of its ototoxic potential. Am. J. Otol. 7, 276^293. Appleton, T.G., Conner, J.W., Hall, J.R., 1988. S,O- versus S,N-chelation in the reactions of cis-diamminediaquaplatinum(II) cation with methionine and S-methylcysteine. Inorg. Chem. 27, 130^137. Bagger-Sjoëbaëck, D., Filipek, C.S., Schacht, J., 1980. Characteristics and drug responses of cochlear and vestibular adenylate cyclase. Arch. Otorhinolaryngol. 228, 217^222. Barron, S.E., Daigneault, E.A., 1987. E¡ect of cisplatin on hair cell morphology and lateral wall Na,K-ATPase activity. Hear. Res. 26, 131^137. Bonheim, K., Bichler, E., 1985. Cisplatin-induced ototoxicity: Audiometric ¢ndings and experimental cochlear pathology. Arch. Otorhinolaryngol. 242, 1^6. Bosher, S.K., 1980. The nature of the ototoxic actions of ethacrynic acid upon the mammalian endolymph system. Acta Otolaryngol. 90, 40^54. Brummett, R., Smith, C.A., Ueno, Y., Cameron, S., Richter, R., 1977. The delayed e¡ects of ethacrynic acid on the stria vascularis of the guinea pig. Acta Otolaryngol. 83, 98^112. Campbell, K.C.M., Rybak, L.P., Meech, R.P., Hughs, L., 1996. D-Methionine provides excellent protection from cisplatin ototoxicity in the rat. Hear. Res. 102, 90^98. Eastman, A., 1986. Reevaluation of interaction of cis-dichloro(ethylene-diamine)platinum (II) with DNA. Biochemistry 25, 3912^ 3915. Fleischman, R.W., Stadnicki, S.W., Ethier, M.F., Schaeppi, U., 1975. Ototoxicity of cis-dichlorodiammine platinum (II) in the guinea pig. Toxicol. Appl. Pharmacol. 33, 320^332. Friedmann, I. and Ballantyne, J. (Eds.) (1984) Ultrastructural Atlas of the Inner Ear. Butterworth and Co. Ltd., Boston, MA. Gandara, D.R., Perez, E.A., Phillips, W.A., Lawrence, H.J., DeGregorio, M., 1989. Evaluation of cisplatin dose intensity: current status and future prospects. Anticancer Res. 9, 1121^1128. Gonias, S.L., Swaim, M.W., Massey, M.F., Pizzo, S.V., 1988. cisDichlorodiammine-platinum(II) as a selective modi¢er of the oxidation-sensitive reactive-center methionine in K1 -antitrypsin. J. Biol. Chem. 263, 393^397. Gralla, R.J., Cvitkovic, E., Golbey, R.B., 1979. cis-Dichlorodiammineplatinum(II) in non-small cell carcinoma of the lung. Cancer Treat. Rep. 63, 1585^1588. Hacker, M.P. (1991) Toxicity of platinum-based anticancer drugs. In: Powis, G. and Hacker, M.P. (Eds.), The Toxicity of Anticancer Drugs. Pergamon Press, Oxford, pp. 82^105. Hoeve, L.J., Mertens zur Borg, I.R.A.M., Rodenburg, M., Brocaar, M.P., Groen, B.G.S., 1988. Correlations between cis-platinum dosage and toxicity in a guinea pig model. Arch. Otorhinolaryngol. 245, 98^102.

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