Kanamycin Damages Early Postnatal, but Not Adult Spiral Ganglion Neurons

Abstract

Although aminoglycoside antibiotics such as kanamycin are widely used clinically to treat life-threatening bacterial infections, ototoxicity remains a significant dose-limiting side effect. The prevailing view is that the hair cells are the primary ototoxic target of aminoglycosides and that spiral ganglion neurons begin to degenerate weeks or months after the hair cells have died due to lack of neurotrophic support. To test the early developmental aspects of this issue, we compared kanamycin-induced hair cell and spiral ganglion pathology in rat postnatal day 3 cochlear organotypic cultures with adult whole cochlear explants. In both adult and postnatal day 3 cultures, hair cell damage began at the base of the cochleae and progressed toward the apex in a dose-dependent manner. In postnatal day 3 cultures, spiral ganglion neurons were rapidly destroyed by kanamycin prior to hair cell loss. In contrast, adult spiral ganglion neurons were resistant to kanamycin damage even at the highest concentration, consistent with in vivo models of delayed SGN degeneration. In postnatal day 3 cultures, kanamycin preferentially damaged type I spiral ganglion neurons, whereas type II neurons were resistant. Spiral ganglion degeneration of postnatal day 3 neurons was associated with upregulation of the superoxide radical and caspase-3-mediated cell death. These results show for the first time that kanamycin is toxic to postnatal day 3 spiral ganglion neurons, but not adult neurons.

Keywords: Auditory nerve fibers; Caspase-3; Hair cells; Kanamycin; Ototoxicity; Spiral ganglion neurons.

Fig. 1

Representative confocal images from the upper basal turn of the cochlea of P3 organotypic cultures treated with 0 (control), 0.5 or 1 mM KM for 24 h. Specimens double labeled with neurofilament 200 kDa (red) and phalloidin-Alexa-488 (green). (A) In control cultures (0 mM KM), outer hair cells (OHC, bracket) and inner hair cells (IHC, white arrowhead) were arranged in orderly rows. Numerous thick fascicles of auditory nerve fibers (ANF, yellow arrow) project radially from the round spiral ganglion neurons (SGN, white arrows) toward the hair cells. Nerve fibers terminate as a dense network as they approach the IHC (white star). (B) After 24 h treatment with 0.5 mM KM, considerable thinning of auditory nerve fiber fascicles (yellow lightning bolt) occurred along with shrinkage of SGN somata (v). Note complete loss of the dense fiber network near the IHC (compare to stars in panel A). There was little evidence of hair cell loss. (C) After 24 h treatment with 1 mM KM, there was significant hair cells loss, considerable thinning and loss of auditory nerve fascicles (yellow lightning bolt), complete loss of the dense fiber network near the IHC (compare to star in panel A), and shrinkage of SGN somata (v).

Fig. 2

Organotypic cultures from basal turn of adult (top two rows) and P3 (bottom row) rats treated for 24 h with different doses of KM. Phalloidin label of stereocilia and cuticular plate shown in red and TO-PRO-3 label of nuclei shown in green. Second row shows representative photomicrographs of surface of organ of Corti from adult rats; first row shows the Z-plane image through a section of the surface preparation. Bottom row shows representative surface preparation view from P3 rat organ cultures. (A, a) OHC and IHC in adult rats after 24 h in culture without KM (control). OHC and IHC arranged in orderly rows separated by tunnel of Corti. Note strong phalloidin labeling of the stereocilia (St) and cuticular plate. Large, round nuclei present in OHC and IHC; note homogeneous labeling of nuclei with To-Pro-3. (B, b) Treatment with 0.1 mM KM resulted in considerable OHC loss. Most OHC nuclei condensed and severely shrunken (yellow arrows). Stereocilia present on most IHC, but IHC nuclei slightly shrunken and irregularly labeled with To-Pro-3 (white arrowheads). (C, c; D, d; E, e) OHC and IHC losses increased as KM dose increased from 0.2 to 1 mM; nearly all hair cell missing with 0.5 mM KM. (F) Three rows of OHC and single row of IHC in P3 control groups; strong, homogeneous To-Pro-3 labeling evident in IHC. (G) OHC and IHC present after 0.1 mM KM treatment, but To-Pro-3 nuclear labeling was irregular (white arrowheads). (H) Most OHC and IHC present after 0.2 mM KM treatment, but To-Pro-3 labeling was heterogeneous, nuclear profiles were irregular and condensed. (I, i; J, j). Nearly all OHC and IHC destroyed by 0.5 and 1 mM KM

Fig. 3

Photomicrographs of SGN from basal turn of adult (top row) and P3 rats treated for 24 h with 0 (control) to 1 mM KM. Specimens stained with neurofilament 200 kDa (red) and To-Pro-3 (green). (A) Adult SGN in 0 mM control group characterized by large round somas labeled by neurofilament stain and round, centrally located To-Pro-3-labeled nucleus. (B–E) Soma and nuclear morphology of adult SGN treated with 0.1 to 1 mM KM were similar to 0 mM control. (F) P3 SGN in 0 mM control group characterized by large, round To-Pro-3-labeled nucleus located in the center of the SGN; cytoplasm of SGN soma heavily labeled with neurofilament. (G–J) As KM dose increased, soma size decreased and many SGN had condensed and/or fragmented nuclei (white arrowhead). (H) Mean percentage (+/− SEM) of apoptotic SGN versus KM dose; Control = 0 mM KM. (# indicates significant differences between doses in the P3 group)

Fig. 4

Representative photomicrographs from organ of Corti (A–E) and SGN (F–J) in the basal turn of P3 rats treated with 0 (control) or 1 mM KM for 3, 6, 12 or 24 h. Dihydroethidium (DHE) used to detect superoxide (red). TO-PRO-3 used to label nuclei (blue) in the organ of Corti (A–E) and neurofilament 200 kDa (green) used to label the SGN (F–J)

Fig. 5

P3 cultures of organ of Corti (A–E, surface view; a–e, Z-plane section) from basal turn treated with 0 (control) or 1 mM KM for 3, 6, 12 or 24 h. Organ of Corti stained with caspase-3 (Cas3, red), TO-PRO-3 (blue) and phalloidin-Alexa-488 (green). Many hair cells missing after 12 h and 24 h KM treatment; many caspase-3 labeled cells (yellow arrows) present at this time (D–E). P3 SGN cultures (F–J) from the basal turn treated with 0 (control) or 1 mM KM for 3, 6, 12 or 24 h. Specimens labeled with neurofilament 200 kDa (green) and caspase-3 (Cas3, red). Note increase in caspace-3 SGN between 3 and 24 h of kanamycin treatment. Histograms showing percentages of casapse-3 positive hair cells (K) and caspase-3 positive SGN (L). Horizontal lines indicate significant (p<0.05) between group differences (see text for details).

Fig. 6

Basal turn P3 organotypic control cultures (A, D, G) and cultures treated with 1 mM KM for 24 h (B, E, H) or 48 h (C, F, I). Specimens labeled with neurofilament-200 kDa (green), expressed in both type I and type II SGN, and peripherin (red), expressed only in type II SGN. Note large loss of neurofilament positive SGN soma and radiating fibers (A, B, C), but major retention of peripherin-positive type II SGN and fibers (D, E, F). Panels in left and middle column merged (yellow) in right column

Fig. 7

P3 rat cochlear organotypic culture treated with 0.5 mM gentamicin for 24 h. Only a few OHC were still present (white arrow) and a few IHC were missing (white arrowhead). Auditory nerve fibers (ANF, yellow arrow) and spiral ganglion neurons (SGN, green arrow) intact.