Inner and outer hair cells (IHC and OHC, arrows), in an untreated control ear (left) and after application of carboplatin (5mg/ml) on the round window (right): After carboplatin treatment a great amount of IHC as well as OHC are missing. Arrow heads point to empty spaces between few remaining hair cells. Scale bar = 50μm

Expression of GAP-43 in VCN in one experimental animal surviving for 31 days (#8608) at high magnificantion: Contralaterally (A) as well as ipislaterally (B), GAP-43 is located in presynaptic endings (arrowheads point to some examples) as well as in fibers (arrows). Asterisks indicate unstained cell bodies. 31 days after carboplatin treatment, expression ipislaterally is strongly increased as compared to the contralateral side. Scale bar = 100μm

Scattergram where hair cell loss (IHC and OHC) is plotted against up-regulation of GAP-43 expression (ipsi-to-contralateral ratio) in VCN (A) and DCN (B) at 15 and 31 days after carboplatin treatment: Values were obtained separately from low-frequency regions and from high-frequency regions. A nonparametric correlation test (Spearman) showed that GAP-43 expression in VCN correlated significantly with OHC loss (P=0.0051) and total hair cell loss (OHC+IHC; P=0.019), but not with IHC loss (P=0.2424). In DCN, the pattern of GAP-43 expression did not show any correlation with hair cell loss (OHC: P=0.39; OHC+IHC: P=0.41; IHC: P=0.63).

Expression in GAP-43 in the fusiform layer (fl) in the dorsal cochlear nucleus (DCN) in a normal hearing control (A) and in an experimental animal (B, C) with unilateral application of carboplatin on the round window, 31 days after treatment (#8333): GAP-43 expression showed only little or no difference between control and experimental animal on either of the sides. In the experimental animal, expression on the ipsilateral DCN (C) showed a slightly stronger expression than contralaterally (B). Control as well as experimental DCNs showed stronger expression ventrally (arrowheads) than dorsally (arrow). Dashed line in (A) indicates outlines of DCN. fl: fusiform layer; ml: molecular layer. Scale bar = 500μm

Expression of GAP-43 in VCN (A) and DCN (B) after unilateral carboplatin treatment, as shown by the ipsilateral-to-contralateral (i:c) ratio of GAP-43 immunoreactivity. Animals surviving for 3 or 7 days showed ratios close to 1.0 in VCN as well as DCN and thus no difference between sides. At 15 and 31 days the ratio increased in VCN in all animals, demonstrating an increase of ipislateral GAP-43 expression. The difference in immuno-reactivity between sides were statistically analyzed and was found significant in all animals surviving for 15 or 31 days (grey bars; P < 0.05, paired t-test where matching ipsilateral and contralateral regions were paired) and highly significant (dark grey bars; P < 0.0001) in most of these animals. In DCN, the ratio between sides was close to 1.0 in all animals. Three animals, surviving for either 15 days or 31 days showed significant difference in GAP-43 expression (grey bars; P <0.05).

Expression of GAP-43 in the lateral superior olive (LSO) in a normal hearing control animal (A) and in an experimental animal (B, C) with unilateral carboplatin application on the round window, 31 days after treatment (#8608). For comparison, a previous study on the rat has shown that cochlear ablation results in accumulation of GAP-43 in cell bodies of lateral olivocochlear neurons in the LSO ipsilateral to lesion (Illing et al., 1999). However, in the chinchilla, we could not detect any immunopositive cell bodies in the ipsilateral LSO (C) at any survival times including 31 days. Dashed line in (A) indicates outlines of the LSO. Scale bar = 200μm

Dorsal-to-ventral (d:v) ratios of GAP-43 expression in VCN (A) and DCN (B) on the treated side (filled symbols) and the untreated control side (open symbols) in chinchillas surviving for 15 or 31 days after carboplatin treatment: In VCN, the average d:v ratio on control side was 0.978 ± 0.036 and close to the theoretical value of 1.0 with no difference between dorsal and ventral expression. The ratio on the lesioned side was 1.143 ± 0.074, showing a tendency towards a stronger up-regulation dorsally than ventrally. A paired t-test where the d:v-ratios on lesioned and control side from individual animals were coupled showed that the difference between lesioned side and control side was significant (P = 0.021). In DCN, the d:v ratio showed no difference between sides (P = 0.63). Ipsilaterally as well as contralaterally, GAP-43 expression was higher ventrally than dorsally, a pattern also observed in the untreated control.

Varying patterns and extent of GAP-43 immunoreactivity in the VCN at different survival times after unilateral carboplatin treatment of the ear; Contralateral (c) VCN to the left, VCN ipsilateral (i) to treatment to the right: Corresponding cochleograms from same animals are added for comparison. A, B: Animals surviving for 3 (A) or 7 days (B) showed no up-regulation of GAP-43 expression. There was no difference between the left and right VCN in these animals. C, D, E, F: Significant increase in GAP-43 expression ipsilaterally could be seen at 15 days (C, D) and 31 days (E, F) post-carboplatin. Some animals also showed a dorsal-to-ventral gradient of GAP-43 expression (D, F), possibly reflecting the basal-to-apical gradient of hair cell degeneration in the cochlea at present (D) or in the case of 31 days (F) possibly at an earlier time point. Arrow heads indicate region with higher expression dorsally. Scale bar = 400μm

Expression of the growth-associated protein GAP-43 in the ventral cochlear nucleus (VCN) in a normal hearing control animal and in an experimental animal with unilateral carboplatin application on the round window 31 days after treatment (#8608): In the control animal (A), GAP-43 is expressed at a modest level in fibers and presynaptic endings. In the experimental animal, the VCN GAP-43 expression on the untreated control side (B) remained at a moderate level as seen in the control. In contrast, VCN on the treated side (C) showed a strong increase of GAP-43 expression, which resulted in a darker staining. Staining dorsally was darker than ventrally (arrows), showing a high-to-low frequency gradient of expression. A small medial area (arrowhead) which is located within the 8th nerve (n8) belongs to VCN but appears isolated due to bifurcation of the fiber bundle and showed similar GAP-43 expression as the rest of the nucleus. Comparing with anatomical observations done in rat (Paxinos and Watson, 2004) this may be the region where olivocochlear fibers pass by or enter VCN. Dashed line in (A) indicates outlines of the VCN. Scale bar = 200μm

Cochleograms from carboplatin treated ears at different survival times, showing degree of hair cell degeneration along the apical-to-basal axis in percent. Solid line: inner hair cells (IHC); dashed line: outer hair cells from all three rows (OHC1, OHC2, OHC3). A, B: As early as after 3 days, severe hair cell degeneration could be observed. Basally more hair cells were missing than apically. C, D, E: Three animals surviving for 7 days showed varying degrees and patterns of hair cell degeneration. One animal had only little hair cell loss throughout the cochlea (C). A second animal showed more severe hair cell loss, where OHC degeneration was complete basally but only low apically, while IHC degeneration was more equally pronounced throughout the cochlea with strongest degeneration in the middle turn (D). A third animal also showed severe OHC loss following a strong basal to apical gradient, and almost all IHC were missing (E). F, G, H, I, J: At 15 days, hair cell loss was severe, almost complete or complete, and IHC loss was more pronounced than OHC loss: In two animals, OHC degeneration again showed a strong basal-to-apical gradient (F, G). Almost all IHC had disappeared, with remaining cells predominantly in the basal turn (F) or only apically (G). In two other animals, almost all hair cells were gone, with only few IHC and OHC remaining apically (H, I). In the fifth animal, all hair cells had disappeared (J). K, L, M: At 31 days post-carboplatin, pattern and variability in hair cell degeneration resembled observations at 15 days: one animal showed OHC degeneration following basal-to-apical gradient and an almost complete IHC degeneration (K). In a second animal, degeneration was almost complete for both IHC and OHC (L). In the third animal all hair cells were missing (M).