Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 www.ijppp.org /ISSN:1944-8171/IJPPP1102003

Original Article Localization of gentamicin uptake in the acutely isolated inner of the rat

Katharina Schmid, Jürgen Strutz, Otto Gleich, Pingling Kwok

Department of Otolaryngology - Head and Neck Surgery, University of Regensburg, Franz-Josef-Strauß-Allee 11, D- 93042 Regensburg, Germany

Received February 25, 2011; accepted March 23 2011; Epub March 28, 2011; Published June 30, 2011

Abstract: The quantitative analysis of fluorescence in frozen sections of rat inner exposed to Texas Red conjugated gentamicin revealed distinct gradients of gentamicin fluorescence. At 500 µg/ml gentamicin fluorescence occurred in inner and outer hair cells, the interdental cell region, the below the interdental cells, the nerve fiber bundle in the spiral lamina, the inner sulcus cells and the dorsal region of the . No gentamicin fluorescence was observed in the Hensen / Claudius cells, the ventral region of the spiral ligament, the stria vascularis and the spiral ganglion. In the vestibule only the hair cell epithelium and the transitional cells of the showed gentamicin fluorescence while no gentamicin fluorescence was found in hair cell epithelia and transitional cells of and ampule, nerve fibers below hair cell epithelia of saccule, utricle and ampule and in dark cells. The gentamicin flurescence increased at higher concentrations. Gentamicin exposure led to more pronounced gentamicin fluorescence in the compared to the vestibule. Based on the predominant gentamicin fluorescence in the hair cell - limbus region of the cochlea at a low dose we propose that gentamicin may interact with the K+-flow from the inner hair cells back to the scala media.

Keywords: Gentamicin, Menière’s disease, cochlea, vestibule, rat

Introduction ture of Reissner’s membrane in the apex of the cochlea leads to loss in the lower fre- Menière’s disease was first described by Pros- quency range [6]. per Menière in 1861 [1] and comprises the symptom triad of recurrent, spontaneous ver- Therapy of Menière’s disease varies stepwise tigo, sensorineural hearing loss and tinnitus. from conservative medical treatment to radical Vertigo attacks are often accompanied by nau- surgical procedures [7,8]. sea and vomiting. Defective hearing often fluc- tuates at the beginning of the disease and nor- An innovation in treatment was the application malizes in the attack free intervals. During the of ototoxic aminoglycosides in 1957 by Schuk- following course of disease hearing loss persists necht [9]. By inducing a pharmacological commonly in the low frequencies. The most in- neurectomy, the symptoms of Menière’s dis- criminating symptom for patients is usually ver- ease could be influenced beneficially. Subse- tigo [2]. quently, it was shown that a low dose intratym- panal application of gentamicin was sufficient The pathophysiologic mechanism for Menière’s to achieve relief for the patients, without de- disease is an endolymphatic hydrops caused by stroying the complete [10- disorders of the production or re- 13]. Many studies have dealt with aminoglyco- sorption. In consequence, the Reissner`s mem- sides and have suggested an effect not only on brane ruptures [3,4] and K+ leaks from the en- hair cells but also on the stria vascularis, mar- dolymphatic into the perilymphatic space caus- ginal cells, fibrocytes [14-20] or vestibular dark ing a non-physiological activation of vestibular cells [21]. However, in these studies high con- nerve fibres with the symptom vertigo [5]. Rup- centrations of gentamicin were applied, which

Gentamicin uptake in the acutely isolated rat

destroyed not only hair cells but also a lot of dimethylformamide (Sigma, Cat. # D4254) at a other cell types in the inner ear. concentration of 2 mg/ml. For conjugation, 0.5 ml of the Texas Red solution and 3.67 ml of the Until now, the target cells for low dose gen- gentamicin solution were mixed and stored un- tamicin therapy that convey the relief from der agitation on a shaker in a cold room until Menière symptoms still remain unclear. It is the used (at least 24 hours). This stock solution aim of the present study to identify the target contains 44 mg gentamicin per ml and was di- cells in the inner ear of the rat by using short luted to the desired concentrations of 500, time exposure- and low-dose application of gen- 1250 and 2500 µg/ml in Dulbecco’s Modified tamicin. Eagle Medium (DMEM with 4mM l-glutamine, 1000mg/l d-glucose and 110 mg/l sodium py- Materials and methods ruvate; Invitrogen Cat. # 31885023). For control experiments 0.5 ml of the Texas Red solution Animals was combined with 3.67 ml 0.1M potassium carbonate solution without gentamicin and ade- Young, 2-4 month old rats were obtained from quate aliquots were added to DMEM for expo- the breeding colony of the animal facilities at sure in control experiments. In 3 experiments the medical faculty of the University of Regens- we used the lowest concentration of 500 µg/ml burg. Two offspring of black hooded (BDE) rats and additional experiments were performed initially obtained from Boehringer Ingelheim and using 1250 and 2500 µg/ml, respectively. three offspring of the ACI strain from Harlan- Winkelmann of either sex were used in the pre- Preparation sent experiments. The use of animals in this study complies with the current „German Law In order to isolate the temporal bones, the rats on the Protection of Animals“ (Tierschutzgesetz were killed by decapitation with a guillotine. §4 Satz 3). Then the skull was opened dorsally and the brain partially removed. The tympanic bullae Gentamicin were exposed and the left and right side sepa- rated from the skull and placed separately into Texas red conjugated gentamicin has been petri dishes filled with cold DMEM. Access to used to analyze aminoglycoside uptake in the the inner ear was achieved by opening the bul- inner ear [e.g. 41]. The pattern of fluorescence lae with blunt forceps. Excess tissue and bony induced by exposure to Texas Red conjugated structures were carefully removed. With the gentamicin in the inner ear was similar to that help of a small metal hook the was re- determined by immunohistochemical analysis moved from the , the following exposure to native gentamicin [e.g. was ruptured and small perforations were made 46]. In the present study we used Texas Red into the bony wall at the level of the scala tym- conjugated gentamicin to analyze the pattern of pani of the cochlear base and at the apex of the fluorescence in defined cochlear and vestibular cochlea. The isolated inner ear was then trans- structures following a short exposure of the ferred to fresh DMEM in a 24-well tissue culture acutely isolated rat cochlea (for details see be- plate and cleaned from blood by carefully flush- low). This degree of fluorescence is interpreted ing DMEM through the perilymphatic space with as an indicator of the gentamicin concentration a fine-tipped pipette at the exposed oval and in the respective cochlear and vestibular re- round windows and the apical opening. It was gions and is referred to as gentamicin fluores- then transferred to the next well with the incu- cence. bation solution that was again gently flushed through the perilymphatic spaces by a fine A stock solution of Texas Red conjugated gen- tipped pipette. The preparation of both ears tamicin was prepared according to Lyford-Pike until exposure to the incubation solution was et al. [36]. First gentamicin sulfate (Sigma, Cat. typically achieved within 7-12 minutes. In each # G1264) was dissolved in 0.1M potassium experiment one ear was exposed to gentamicin carbonate solution (K2CO3, Sigma, Cat. # while the other served as control. In two 500 P5833) with pH 9 at a concentration of 50 mg/ µg/ml experiments control ears were incubated ml. Then Texas Red-X succinimidyl ester in pure DMEM without any addition while in the (Invitrogen Cat. # T6134) was dissolved in NN- 3 other experiments corresponding amounts of

72 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Texas Red solution without gentamicin in DMEM sheim, Germany) with epifluorescence. Images were used for incubation. were digitized with a Spot RT3 Slider camera (Diagnostic instruments, Stirling Heights, Mich., Exposure USA) with the help of VisiView (Visitron Systems GmbH Puchheim, Germany). To achieve high The inner ears were incubated for 10 minutes sensitivity and resolution we used a 40x lens and then transferred to DMEM. Excess incuba- resulting in a nominal resolution of 0.19 µm per tion solution was flushed out of the perilym- pixel. For each frame, separate images were phatic spaces with a fine tipped pipette and the digitized with the Texas Red, the fluorescein and inner ears were transferred to 4% paraformalde- the DAPI fluorescence filters. The exposure time hyde in 0.1M phosphate buffer with pH 7.6 for for each channel was determined in pilot experi- fixation. Fixation in the paraformaldehyde solu- ments (2 s for Texas Red and fluorescein, 0.1 s tion was performed for 90 minutes. The solution for DAPI) and subsequently kept constant for was changed 3 times during this period and digitizing all images with a 16 bit gray level gently flushed through the perilymphatic spaces depth. Further processing of the raw images every 15 minutes to achieve a good fixation. was done using ImageJ 1.43. For each image The tissue was then transferred to 0.3% Triton we performed a background subtraction with X100 (Sigma, Cat. # X100) in 0.1M phosphate the dark background option selected and a roll- buffer for 90 minutes to make the cell mem- ing ball radius of 50 pixels. The settings for opti- branes permeable for a subsequent actin label mizing brightness and contrast for each channel with fluorescein-conjugated phalloidin. The tis- were also determined in pilot experiments and sue was transferred to the fluorescein phal- subsequently used for all images (Texas Red: loidin (Invitrogen, Cat. # F432) staining solution min 80, max 350; fluorescein: min 40, max (20 µl stock solution prepared according to the 1500; DAPI: min 100, max 1500). Following manufacturer’s instructions were diluted in 1 ml these processing steps, images were converted 0.1 M PBS with 0.3 % Triton). Incubation lasted to 8 bit gray level depth and the red, green and 90 minutes in the phalloidin solution with a gen- blue image of each frame combined to a RGB tle flushing of the perilymphatic spaces every image. To obtain overviews of a section through 15 minutes. Following the actin staining, the the cochlea including the spiral ganglion, organ cochleae were washed and perilymphatic of Corti, stria vascularis and the spiral ligament spaces flushed with decalcification solution (4 or covering complete sections through the vesti- % paraformaldehyde, 0.1 M EDTA in 0.1 M bular organs, several overlapping frames were phosphate buffer). Each ear was placed in a 50 taken and subsequently assembled to an over- ml falcon tube with decalcification solution on a view with high resolution using Photoshop CS4 shaker in the cold room at 4°C. The solution (Figure 2, 3, 4, 5). These overviews were used was changed every 2-3 days. After a decalcifica- for a qualitative visual evaluation of gentamicin tion period of 10 days the cochleae were accumulation in the inner ear. washed in phosphate buffer and placed in 30% saccharose solution under agitation at 4°C For a quantitative analysis of Texas Red fluores- overnight for cryoprotection of the tissue. Prop- cence as an indicator of gentamicin distribution erly oriented 10 µm thick frozen sections of the in the tissue we defined 11 distinct areas in the inner ear were collected on slides, dried and overviews of cochlear sections and 10 distinct covered slipped with Vectashield with DAPI areas in overviews of sections through vestibu- (Linaris, Cat. # H-1200) in order to stain nuclei. lar organs (Figure 1, Table 1). Using ImageJ Using this procedure the binding or accumula- 1.43 the regions of interest representing the tion of gentamicin was indicated by the Texas above defined cochlear or vestibular areas were Red fluorescence while the actin staining due to selected in the overviews using the polygon se- the green fluorescein and the nuclear staining lection tool and the mean gray level of the red due to the blue DAPI fluorescence provided ori- channel in each region of interest was calcu- entation and landmarks in these sections of the lated. inner ear. The software PASW statistics 17 (SPSS Inc.) Analysis was used for calculating the test statistics. First, data from each of the 11 cochlear and 10 vesti- The sections were examined under a Leica DM bular areas were assigned to one of 6 groups: RBE microscope (Leica Mikrosysteme, Ben- 1) treated with 500 µg/ml gentamicin, 2) con-

73 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Figure 1, A-D: Schematic illustration of sections showing the cochlea (A), saccule (B), utricle (C) and ampule (D). 1 = inner hair cells, 2 = outer hair cells, 3 = lower spiral limbus, 4 = interdental cells, 5 = dendrites of spiral ganglion cells, 6 = inner sulcus cells, 7 = dorsal spiral ligament, 8 = ventral spiral ligament, 9 = stria vascularis, 10 = spiral ganglion, 11 Hensen / Claudius cells, 12 = saccule hair/supporting cells, 13 = saccule transitional cells, 14 = sac- cule nerve fiber region, 15 = utricle hair/supporting cells, 16 = utricle transitional cells, 17 = utricle nerve fiber re- gion, 18 = ampule hair/supporting cells, 19 = ampule transitional cells, 20 = ampule nerve fiber region, 21 = dark cells.

Table 1. Analyzed structures Cochlea Vestibular organ 1 = Inner hair cell 12 = Saccule hair cells and supporting cells 2 = Outer hair cells 13 = Saccule transitional cells 3 = Spiral limbus below interdental cells 14 = Saccule nerve fiber region 4 = Interdental cells 15 = Utricle hair cells and supporting cells 5 = Nerve fiber bundle in the region of the Spiral lamina 16 = Utricle transitional cells 6 = Inner sulcus cells 17 = Utricle nerve fiber region 7 = Dorsal spiral ligament 18 = Ampule hair cells and supporting cells 8 = Ventral spiral ligament 19 = Ampule transitional cells 9 = Stria vascularis 20 = Ampule nerve fiber region 10 = Spiral ganglion 21 = Dark cells 11 = Hensen / Claudius cells

trol in the 500 µg/ml experiment, 3) treated formed. To identify areas where gray values with 1250 µg/ml gentamicin, 4) control in the were significantly elevated in the gentamicin 1250 µg/ml experiment, 5) treated with 2500 treated ears compared to the corresponding µg/ml gentamicin, 6) control in the 2500 µg/ml controls comparisons were performed within experiment. For each area a Kruskal Wallis test each of the 3 gentamicin doses (500 µg/ml: was performed comparing these 6 groups to group 1 versus group 2; 1250 µg/ml: group 3 determine if gray levels differed significantly versus group 4; 2500 µg/ml: group 5 versus between groups. Since these tests revealed a group 6). To identify dose dependent effects of significant effect of group for each of the 21 gentamicin, pair wise comparisons within each areas (p ≤ 0.011) subsequent pair wise com- area were performed comparing gray levels in parisons with Mann Whitney U tests were per- ears treated with 500 and 1250 µg/ml (group 1

74 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Figure 2. Overview of a section through the cochlea exposed to 500 µg/ml gentamicin (A,C) and a corresponding con- trol exposed only to DMEM (B,D). Figures A and B show composite RGB images of the three colour channels repre- senting Texas Red coupled gentamicin (red), FITC-phalloidin labelled actin (green) and DAPI stained nuclei (blue) and allow a clear identification of the different cochlear structures (e.g. spiral limbus, SL; , BM; , OC). Only the corresponding images of the red colour channel are shown in Figure 2C,D and emphasize the distinct and elevated red fluorescence in the section from a cochlea exposed to 500 µg/ml gentamicin (Figure 2C) as compared to the low degree of background fluorescence in a control (Figure 2D). The labels and arrows pointing to OC, SL and BM in the images of the red channel (Figure 2C,D) resemble those from the composite images (Figure 2A,B). The scale bar in the bottom right of Fig. 2B indicates 500 µm.

versus group 3), 1250 and 2500 µg/ml (group Cochlear structures 3 versus group 5) and 500 and 2500 µg/ml group 1 versus group 5). A corresponding analy- Visual inspection of the sections exposed to a sis was performed between the controls (group concentration of 500 µg/ml gentamicin showed 2 versus group 4, group 4 versus group 6 and elevated gentamicin fluorescence in non- group 2 versus group 6) to identify a potential cellular structures such as the basilar mem- non specific binding of the Texas Red control brane, the lining of the perilymphatic spaces solution without gentamicin. and the osseous spiral lamina compared to con- trol sections as illustrated in the overviews Results shown in Figure 2. For better orientation, the images in Figure 2A (exposed to 500 µg/ml gen- Qualitative analysis tamicin) and Figure 2B (control) show compos- ite images of the three colour channels demon- We started with a qualitative analysis of Texas strating gentamicin fluorescence in red, actin red conjugated gentamicin binding by visual labelling by phalloidin in green and nuclear inspection of the sections through the cochlea staining by DAPI in blue. In these composite and the vestibular regions. RGB images different cochlear structures can

75 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Figure 3. High magnification views of a cochlear sections exposed to 500 µg/ml gentamicin (A-D) and controls (E,F). Texas red conjugated gentamicin is visible as red fluorescence, cell nuclei are shown in blue and actin is shown by the green fluorescence. The scale bar in the bottom right of Fig. 3F indicates 100 µm.

be clearly recognized as shown by the labels background fluorescence of the red channel and arrows pointing to the Organ of Corti (OC), (e.g. Figure 2D), composite images were neces- the spiral limbus (SL) and the basilar membrane sary to unequivocally identify the cochlear and (BM). To emphasize the difference in the pat- vestibular regions defined in Figure 1 for subse- tern of red fluorescence between gentamicn quent quantitative measurements. exposed and control ears only the correspondig red channel is shown in Figure 2C and Figure With respect to cellular structures, red fluores- 2D respectively. While some unspecific red fluo- cence of gentamicin was seen in some interden- rescence is visible in the region of the spiral tal cells of the spiral limbus (Figure 2A) and in a ganglion and the stria vascularis in the control few inner hair cells. This is illustrated at higher (Figure 2B,D), most other cochlear regions were magnification in Figure 3 with red fluorescence devoid of visual red fluorescence. Figure 2D visible in a population of interdental cells illustrates that the Organ of Corti (OC), the spiral (Figure 3 A-D) and some inner hair cells (Figure ligament (SL) and the basilar membrane (BM) 3 C,D). The examples from controls (Figure 3 cannot be recognized and defined in the image E,F) demonstrate the specificity of gentamicin of the red channel despite the arrows that point fluorescence shown in Figure 3 A-D. to the identical positions illustrated in the com- posite image (Figure 2B). Thus, due to the low In the outer hair cells no gentamicin fluores-

76 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Figure 4. High magnification views of cochlear sections exposed to 1250 µg/ml gentamicin (A) and 2500 µg/ml gen- tamicin (C) and corresponding controls (B,D). Texas red conjugated gentamicin is visible as red fluorescence, cell nuclei are shown in blue and actin is shown by the green fluorescence. The scale bar in the bottom right of Fig. 3F indicates 100 µm. cence could be detected visually (Figure 3A-D) Vestibular structures at a gentamicin concentration of 500 µg/ml. The visual interpretation of the red fluorescence Following an exposure with 500 µg/ml gen- in the cells of the spiral ganglion, between the tamicin no specific gentamicin fluorescence basal and the marginal cells of the stria vascu- was seen in vestibular structures while at higher laris and the ventral spiral ligament was ham- concentrations gentamicin fluorescence be- pered by background fluorescence in controls came visible. This is exemplified in Figure 5 for (e.g. Figure 2B). the saccule. The left column shows sections from inner ears exposed to 500 (Figure 5A), At higher drug concentrations of 1250 (Figure 1250 (Figure 5C) and 2500 µg/ml (Figure 5E) 4A) and 2500 µg/ml (Figure 4C), gentamicin while corresponding controls are shown in the fluorescence appeared much more widespread right column. Over all, gentamicin fluorescence in the cochlea, especially, inner as well as outer in vestibular structures appeared lower com- hair cells, inner sulcus cells, interdental cells, pared to the cochlea (compare Figure 3-5). fibrocytes of the spiral limbus, the spiral gan- glion, stria vascularis, the ventral spiral ligament Over all, the qualitative visual analysis of the and also non-cellular structures like the basilar sections indicated only for cochlear interdental membrane, osseous spiral lamina, the lining of and inner hair cells specific gentamicin fluores- the perilymphatic spaces and the tectorial mem- cence at the lowest gentamicin concentration brane showed a clear gentamicin fluorescence tested (500 µg/ml). At the higher concentra- (Figure 4A,C). The specificity of gentamicin fluo- tions visual assessment suggested more pro- rescence is illustrated by the corresponding nounced gentamicin fluorescence in cochlear controls shown in the right column of Figure 4 compared to vestibular structures (Figure 3-5). (B,D). The images in Figure 4 A, C demonstrate a more intense red gentamicin fluorescence In addition to this qualitative visual evaluation signal as compared to the corresponding con- of gentamicin fluorescence we performed a trols shown in Figure 4 B, D. quantitative gray level analysis of the red fluo-

77 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Figure 5. Sections through the saccule exposed to 500 µg/ml gentamicin (A), 1250 µg/ml gentamicin (C) and 2500 µg/ml gentamicin (E) and corresponding controls (B,D,F). Texas red conjugated gentamicin is shown as red fluores- cence, cell nuclei are shown in blue and actin is shown by the green fluorescence. The scale bar in the bottom right of Figure 5F indicates 100 µm.

rescence for the cochlear and vestibular regions Cochlear structures defined in Figure 1 and listed in Table 1. Figure 6 compares mean gray level and stan- Quantitative analysis dard deviation obtained in eleven cochlear re- gions in controls (white bars) and following gen- Over all conditions, 1126 gray level measure- tamicin exposure at a concentration of 500µg/ ments were obtained from cochlear areas and ml (filled black bars). Because in controls and in 396 from vestibular areas. The majority of samples exposed to 500 µg/ml the red fluores- measurements was collected for the 500 µg/ml cence signal was typically low (Figure 2) the gray concentration of gentamicin (cochlear 705; ves- levels are shown on a logarithmic scale to em- tibular 207) since we were primarily interested phasize potential small differences. For most in the uptake following exposure to a low dose cochlear structures the background fluores- of gentamicin. cence in controls was low (below 10), only the ventral spiral ligament (mean 13), stria vascu- The gray level is a quantitative measure of the laris (mean 42), the spiral ganglion (mean 32) fluorescence signal. Low gray levels are associ- and the Hensen/Claudius cells (mean 14) ated with a weak fluorescence and high num- showed a higher degree of background fluores- bers are associated with a high fluorescence. cence (see also background fluorescence in Fig.

78 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

some inner hair cells that ap- peared red following gentamicin treatment, the quantitative analysis presented in Fig. 6 indi- cates that red fluorescence fol- lowing 500µg/ml gentamicin treatment significantly exceeded background fluorescence of con- trols in inner hair cells, outer hair cells, lower spiral limbus, inter- dental cells, dendrites of the spiral ganglion, inner sulcus cells and the dorsal spiral ligament. For the ventral spiral ligament, Figure 6. Comparison of the gray levels in controls (white bars) and gen- the stria vascularis, the spiral tamicin exposed (black bars) cochlear regions for the 500 µg/ml condi- ganglion and the Hensen / tion. A significant difference in gray level between exposed cochleae and Claudius cells there was no sig- controls is indicated by a star. Note the logarithmic gray level scale to nificant difference between con- visualize small differences. This quantitative analysis revealed that expo- sure to 500µg/ml gentamicin was associated with a significantly higher trols and sections from inner fluorescence in seven of the eleven analyzed cochlear structures as com- ears exposed to 500µg/ml gen- pared to background fluorescence in controls. 1 = inner hair cells, 2 = tamicin. outer hair cells, 3 = lower spiral limbus, 4 = interdental cells, 5 = den- drites of spiral ganglion cells, 6 = inner sulcus cells, 7 = dorsal spiral At a gentamicin concentration of ligament, 8 = ventral spiral ligament, 9 = stria vascularis, 10 = spiral 1250 µg/ml fluorescence in ex- ganglion, 11 = Hensen / Claudius cells. posed inner ears was generally higher compared to 500 µg/ml. In inner ears exposed to 1250 µg/ml gentamicin gray levels exceeding a value of 100 were found in outer hair cells, the stria vascularis and the spiral gan- glion. Gray levels in the range 50 -100 were found for inner hair cells, interdental cells and for the dendrites of the spiral gan- glion. The lowest mean gray level of 9 was found for inner sulcus cells but this was considerably higher than the mean gray level of 2 in inner sulcus cells of con- trols. Compared to controls, all Figure 7. Comparison of controls (white bars) and gentamicin exposed cochlear structures except for (black bars) cochlear regions for the 2500 µg/ml condition. Significantly the Hensen / Claudius cells elevated gentamicin fluorescence in comparison to controls is indicated showed a significantly elevated by a star. At this high gentamicin concentration all examined cochlear fluorescence signal following structures showed a significant elevation of gentamicin fluorescence 1250 µg/ml gentamicin expo- above background in controls. 1 = inner hair cells, 2 = outer hair cells, 3 sure. = lower spiral limbus, 4 = interdental cells, 5 = dendrites of spiral gan- glion cells, 6 = inner sulcus cells, 7 = dorsal spiral ligament, 8 = ventral spiral ligament, 9 = stria vascularis, 10 = spiral ganglion, 11 = Hensen / At a gentamicin concentration of Claudius cells. 2500 µg/ml the fluorescence signal was significantly elevated compared to background fluo- rescence in controls for all 11 2B,D, 4B,D). Although visual inspection of the cochlear regions analyzed (Figure 7). With the images revealed only some interdental and exception of inner sulcus cells, the dorsal and

79 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

cence was found for increasing gentamicin concentration from 1250 to 2500 µg/ml (p > 0.05) suggesting that gentamicin con- centration in the tissue may be saturated by doses below 2500 µg/ml in these cochlear struc- tures.

The variation of background fluo- rescence between the different cochlear regions in controls (Figure 6, 7) complicates the di- rect comparison of gentamicin Figure 8. Comparison of gray levels exceeding significantly background fluorescence in corresponding controls for the three gentamicin concen- fluorescence. trations (500, 1250, 2500 µg/ml) tested in eleven cochlear structures. Shown is the difference of the gray level in gentamicin exposed regions To minimize the contribution of and corresponding controls. White bars represent exposure to 500 mg/ background fluorescence in con- ml gentamicin, grey bars represent 1250 µg/ml gentamicin and black trols for the evaluation of gen- bars represent 2500 µg/ml gentamicin. 1 = inner hair cells, 2 = outer tamicin fluorescence, we sub- hair cells, 3 = lower spiral limbus, 4 = interdental cells, 5 = dendrites of tracted the gray level obtained in spiral ganglion cells, 6 = inner sulcus cells, 7 = dorsal spiral ligament, 8 controls from the value obtained = ventral spiral ligament, 9 = stria vascularis, 10 = spiral ganglion, 11 = in gentamicin treated inner ears. Hensen / Claudius cells. Figure 8 compares the difference obtained at the three gentamicin ventral spiral ligament and the Hensen / concentrations (500 µg/ml: white bars; 1250 Claudius cells mean gray level exceeded a value µg/ml gray bars; 2500 µg/ml: black bars) for of 100, representing a bright fluorescence. The the cochlea. Data are only shown when the dif- data in Figure 7 show that the fluorescence in ference between control and exposed tissue controls was low. was significant. This analysis confirms that the highest dose dependent increase of gentamicin The results show a dose dependent variation of fluorescence preferentially occurs in cochlear gentamicin fluorescence in cochlear structures, regions closer to the (inner and outer higher concentrations of gentamicin were asso- hair cells, spiral limbus including interdental ciated with a higher fluorescence signal. This cells and the dendrites of the spiral ganglion) was confirmed by a statistical analysis. Pair while gentamicin fluorescence appeared less wise comparisons (Mann Whitney U tests) re- pronounced in the dorsal and ventral spiral liga- vealed that rising gentamicin concentration ment, the stria vascularis and the cochlear gan- from 500 µg/ml to 1250 µg/ml led to a signifi- glion. Although the inner sulcus cells showed cant increase of fluorescence (and conse- over all a very limited gentamicin fluorescence, quantly gentamicin binding) in all 11 cochlear fluorescence was significant higher in the ex- structures analyzed (p < 0.001). posed as compared to the control at 500 µg/ml and fluorescence increased systematically by Increasing the gentamicin dose further from increasing the gentamicin dose to 1250 and 1250 to 2500 µg/ml was associated with a sig- 2500 µg/ml. The data in Fig. 8 demonstrate a nificantly increasing fluorescence in inner hair differential, region specific and dose dependent cells, spiral limbus, interdental cells, the den- degree of gentamicin fluorescence in the coch- drites of the cochlear ganglion cells, inner sul- lea. cus and dorsal spiral ligament (p ≤ 0.022), dem- onstrating a systematic dose dependent in- Vestibular structures crease of gentamicin fluorescence in these co- chlear structures for gentamicin concentrations Mean gray levels obtained with exposure to 500 varying between 500 and 2500 µg/ml. In outer µg/ml gentamicin (black bars) and correspond- hair cells, the ventral spiral ligament, the stria ing controls (white bars) are shown in Figure 9 vascularis, the spiral ganglion and the Hensen / for vestibular structures. This comparison dem- Claudius cells no significant increase of fluores- onstrates only for the hair cell / supporting cell

80 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

Comparing the mean gray levels following 500 µg/ml with those following 2500 µg/ml revealed a significant increasing fluorescence in all vestibular structures (p ≤ 0.024) except for the region of saccular afferent den- drites. Increasing the gen- tamicin dose from 1250 to 2500 µg/ml was only associ- ated with a significant in- crease of the gray level (p = 0.025) for the region of Figure 9. Comparison of controls (white bars) and gentamicin exposed utricular afferent dendrites. (black bars) vestibular regions for the 500 µg/ml condition. A significant In most vestibular regions, difference of gentamicin fluorescence between exposed and control con- significant gentamicin fluo- ditions is indicated by the star. Note the logarithmic gray level scale to rescence compared to con- visualize small differences. Only the hair cells / supporting cells and the transitional cell region of the saccule showed a specific gentamicin fluo- trols was found at a concen- rescence above background fluorescence in controls. 12 = saccule hair/ tration of 1250 µg/ml and supporting cells, 13 = saccule transitional cells, 14 = saccule nerve fiber further increasing the dose of region, 15 = utricle hair/supporting cells, 16 = utricle transitional cells, 17 gentamicin was not associ- = utricle nerve fiber region, 18 = ampule hair/supporting cells, 19 = ated with increased fluores- ampule transitional cells, 20 = ampule nerve fiber region, 21 = dark cells. cence.

The difference of gray levels and the transitional cell region of the saccule an in the presence of gentamicin and the corre- obvious increase of fluorescence above back- sponding controls is shown in Figure 11 for the ground. The statistical analysis confirmed that vestibular region. Compared to the cochlea this increased fluorescence was significant (p ≤ (Figure 8) the increase of fluorescence above 0.004; indicated by the asterisk in Figure 9). All control levels is on average lower in the vestibu- other vestibular structures (utricle, ampule and lar regions. In contrast to a number of cochlear dark cells) did not show a significant increase of regions a systematic dose dependent increase gentamicin fluorescence above background. of gentamicin from 500 to 2500 µg/ml was not seen in the vestibular system. Significant gen- At a gentamicin concentration of 1250 µg/ml all tamicin fluorescence above background in con- vestibular regions except the regions of saccular trols at 500 µg/ml was only seen in the hair afferent dendrites and the utricular transitional cells /supporting cells and the transitional cells cells showed significantly elevated fluorescence of the saccule. compared to controls (p ≤ 0.025). In conclusion, a 10 minute exposure of the The mean gray levels obtained in the vestibular freshly isolated rat cochlea to gentamicin is as- regions at a gentamicin dose of 2500 µg/ml are sociated with more pronounced gentamicin fluo- shown in Fig. 10. Compared to the cochlea rescence in the cochlea as compared to vestibu- (Figure 7) the gentamicin fluorescence at a lar organs (see Figure 3-5). At the lowest dose dose of 2500 µg/ml appeared less pronounced tested (500 µg/ml) significantly elevated gen- in the vestibular regions. For each vestibular tamicin fluorescence above background in con- region, the mean gray value in the presence of trols was observed in 7 of the eleven analyzed gentamicin was elevated compared to controls. cochlear structures (Figure 6), but only in 2 of However, most likely due to the limited sample the 10 vestibular structures (Figure 9). A sys- size, this difference was not significant for the tematically increasing gentamicin fluorescence transitional cells and the region of afferent den- for doses from 500 to 2500 µg/ml was found in drites of the utricle (3 exposed versus 5 con- 7 cochlear structures (Figure 8) but not in the trols) and the ampullar transitional cells (6 ex- vestibule (Figure 11). Thus, under these experi- posed versus 2 controls). mental conditions, gentamicin fluorescence is

81 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

tamicin. Over all, the pattern of gentamicin fluorescence that we found in the cochlear and vestibular structures analyzed shows many parallels and simi- larities with other published data (for details see discussion below) suggesting that the iso- lated cochlea is in general use- ful for analyzing the distribution of gentamicin in the inner ear. For the stria vascularis Wang and Steyger [46] reported prominent gentamicin fluores- Figure 10. Comparison of controls (white bars) and gentamicin exposed (black bars) vestibular regions for the 2500 µg/ml condition. Significantly cence following intraperitoneal elevated gentamicin fluorescence in comparison to controls is indicated application in the mouse that by a star. Significant elevation of gentamicin fluorescence above back- differs from the moderate gen- ground in corresponding controls was found in seven of the ten examined tamicin fluorescence that we vestibular structures. Compared to the cochlea (Figure 7) gentamicin fluo- observed in the stria vascularis rescence appeared less pronounced in vestibular structures. 12 = saccule following gentamicin exposure hair/supporting cells, 13 = saccule transitional cells, 14 = saccule nerve of the isolated rat cochlea. fiber region, 15 = utricle hair/supporting cells, 16 = utricle transitional While gentamicin applied sys- cells, 17 = utricle nerve fiber region, 18 = ampule hair/supporting cells, temically by intraperitoneal in- 19 = ampule transitional cells, 20 = ampule nerve fiber region, 21 = dark cells. jection enters the cochlea through the vasculature of the stria vascularis [46], gen- higher in the cochlea and lower in vestibular tamicin reaches cochlear structures without organs. passage through the stria vascularis by perfu- sion of the cochlear scalae (like in the present Discussion experiments) or by intratympanal application (that is used clinically). Thus different forms of The isolated cochlea preparation gentamicin application could explain the differ- ent pattern of gentamicin fluorescence in the In the present study we analyzed gentamicin stria vascularis. An additional potential differ- fluorescence in a freshly isolated cochlea prepa- ence to other published data may be related to ration following a 10 minute gentamicin expo- the uptake in hair cells. Most other studies re- sure. The physiological state of the isolated port the most prominent presence of gen- cochlea differs from the situation in vivo. The tamicin in inner and outer hair cells [e.g. 19] disruption of blood and oxygen supply is associ- while our qualitative observations found visible ated with a very fast decline of the endococh- gentamicin fluorescence only in interdental cells lear potential [22,23] leading to a decreased and some inner but not in outer hair cells (Fig. gain of the cochlear amplifier [24] that will af- 3) at the lowest 500 µg/ml concentration. We fect hair cell transduction [25,26]. In addition suggest that the loss of EP in the isolated coch- immersing the isolated cochlea in the cold incu- lea affects gentamicin accumulation in hair cells bation solution will reduce over all metabolic compared to the in vivo situation. activity. In summary, interpretation of the present find- Although the physiological state of the isolated ings in comparison with other studies needs to cochlea differs from the in vivo situation, iso- consider the physiological status in the isolated lated cochlea preparations have been useful for preparation as well as other methodological studying different aspects of cochlear function aspects outlined above. Bearing this in mind our [e.g. 27,28,29,30,31,32]. The advantage of the analysis provides a quantitative description of isolated preparation with perfusion of gen- the distribution of gentamicin fluorescence that tamicin solution through the scalae is that co- is in most aspects consistent with previous chlear and vestibular structures are rapidly ex- qualitative descriptions of the location of gen- posed to a well defined concentration of gen- tamicin in the inner ear. Our data allow direct

82 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

inner ear are not fully under- stood. The present study tried to attempt to identify the cells of the inner ear that accumu- late gentamicin within a short period following exposure.

Aminoglycosides have a vari- ety of adverse effects on the inner ear, such as inducing apoptosis in cochlear and vestibular hair cells [36, 37, 38, 9]. High concentrations of Figure 11. Comparison of gray levels exceeding background fluorescence 40 mg/ml gentamicin applied in corresponding controls for the three gentamicin concentrations (500, via an osmotic pump into the 1250, 2500 µg/ml) tested in 10 vestibular structures. Shown is the differ- perilymphatic space resulted ence of the gray level in gentamicin exposed regions and corresponding in a reduction of function of controls. White bars represent exposure to 500 mg/ml gentamicin, grey the cochlear and vestibular bars represent 1250 µg/ml gentamicin and black bars represent 2500 organs in guinea pigs, µg/ml gentamicin. 12 = saccule hair/supporting cells, 13 = saccule transi- whereas lower concentra- tional cells, 14 = saccule nerve fiber region, 15 = utricle hair/supporting tions (4 mg/ml gentamicin) cells, 16 = utricle transitional cells, 17 = utricle nerve fiber region, 18 = ampule hair/supporting cells, 19 = ampule transitional cells, 20 = ampule did not [40]. nerve fiber region, 21 = dark cells. Most studies have examined the changes of cochlear and vestibular structures follow- comparisons of gentamicin fluorescence for ing longer time periods (up to months) and distinct cochlear and vestibular regions (see higher gentamicin concentrations in comparison Figure 6-11). to our experiments [compare 41, 42, 43, 19, 44, 40]. We were interested in identifying those Concentration of gentamicin cells and structures within the cochlea and the vestibular organs that show elevated concentra- An innovation in the therapy of Menière`s dis- tions of gentamicin (as indicated by a higher ease was the local intratympanal application of degree of fluorescence) following acute expo- ototoxic streptomycin in 1957 [9]. The rationale sure to the lower range of concentrations behind this strategy was the aim of a chemical reached in the inner ear following intratympanic destruction of vestibular hair cells. A severe and administration of therapeutic doses. undesired side effect was the associated cochleotoxicity. Based on experience with pa- Plontke et al. developed models for the predic- tients treated between 1967 – 1977 with intra- tion of gentamicin gradients in the inner ear of tympanic aminoglycosides [33] the focus of chinchilla and guinea pig following intratym- therapy shifted from destroying vestibular hair panal application [45,43]. Their model predicts cells to interfering with the secretory epithelium a high concentration at the base of the cochlea involved in inner ear ion homeostasis, and con- (in the range of 16% of the intratympanal con- sequently avoiding hair cell loss and cochleotox- centration) that rapidly declines towards the icity. This work formed the basis for the empiri- apex reaching 1-3 % of the applied intratym- cal establishment of low dose gentamicin ther- panal concentration in the apical half of the apy that allows sufficient control of Menière cochlea (see Figure 7 in Plontke et al., 2007 attacks without destroying the complete vesti- [43]). The concentration of gentamicin solution bular system and preserving hearing applied intratympanally for the therapy of [10,34,11,12,13]. Typically 10-40 mg/ml gen- Menière`s disease is typically 40 mg/ml [e.g. tamicin were applied intratympanally [35,13]. 13]. Based on the Plontke model, we chose to Despite successful clinical use of low dose gen- expose the freshly isolated inner ears in the tamicin therapy the detailed mechanisms of present experiments to gentamicin concentra- action and the targets of gentamicin within the tions of 500, 1250 and 2500 µg/ml because

83 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

these concentrations correspond to 1-6% of the (compare Figure 11 with Figure 8). intratympanally applied 40 mg/ml solution used for the clinical treatment of Menière`s disease Our results are in many aspects consistent with [e.g. 13]. previous reports. A comparison of our data col- lected after a 10 minute exposure of the inner Accumulation of gentamicin in the inner ear ear to the gentamicin solution with short sur- vival times (up to 24 hours) reported by Ima- A number of studies have analyzed the accumu- mura and Adams [19] for the guinea pig follow- lation of gentamicin in the cochlea in some de- ing systemic or intratympanal gentamicin appli- tail [19,42,46]. Comparable data with respect cation show many parallels. Consistent with our to the vestibular system are not very detailed data is the pattern of gentamicin distribution in [19,42] or restricted to specific end organs the spiral limbus including interdental cells, in [47,48]. A direct comparison of previously pub- the dendrites of auditory nerve fibers and a lished data with our present findings is compli- more pronounced fluorescence in the dorsal as cated by methodological differences; however, a compared to the ventral spiral ligament. Our number of similarities as well as discrepancies qualitative observation of predominant gen- can be identified. tamicin fluorescence at 500 µg/ml in inner but not outer hair cells (Figure 3) contrasts with the A comparison of Figure 6 - 8 representing the predominant gentamicin uptake in outer hair cochlea with Figure 9 -11 representing the vesti- cells of the guinea pig. The observation by Ima- bular organs illustrates a more pronounced gen- mura and Adams [19] that gentamicin was ob- tamicin fluorescence in some cochlear as com- served in saccular hair cells at a shorter time pared to the vestibular regions analyzed in the following exposure compared to those of utricle present study. and ampule parallels our finding that at the low- est concentration of gentamicin significant in- Within the cochlea gentamicin fluorescence at crease of fluorescence above background was the lowest dose analyzed (500 µg/ml, Figure 6) only seen in saccule. was significantly above background fluores- cence predominantly in regions closer to the Roehm et al. report the results of intratympanal medial portion of the cochlea (both hair cells gentamicin application in the chinchilla inner types, the spiral limbus including interdental ear [42]. They found gentamicin located cells, the dendrites of the spiral ganglion cells, throughout the infused ear for survival times and the inner sulcus cells). On the lateral side of between 4 hours and 2 weeks following expo- the hair cells only the region of the dorsal spiral sure and mention the presence of gentamicin in ligament showed significantly elevated gen- cochlear and vestibular hair cells, in the spiral tamicin fluorescence above background. The ligament, in cochlear and fi- stria vacularis, the ventral spiral ligament, the bers, in the stria vascularis, in vestibular dark Hensen / region and the spiral cells and in the neurons of the spiral and Scar- ganglion did not show significant elevation of pas ganglion. These observations appear at gentamicin fluorescence at this low dose. At the least qualitatively comparable to our findings of highest gentamicin dose tested (2500µg/ml) all the distribution of gentamicin fluoresence. cochlear structures showed elevated gen- tamicin fluorescence (Figure 7). The most pro- Wang and Steyger compared the distribution of nounced systematic dose dependent increase native gentamicin (demonstrated by immuno- of gentamicin fluorescence was present in both histochemistry) with the distribution of Texas- types of hair cells, the spiral limbus including Red-conjugated gentamicin following intraperi- interdental cells and the dendrites of the audi- toneal application in the mouse cochlea [46]. In tory nerve fibers (Figure 8). contrast to our data in the rat, they describe the most prominent presence of gentamicin for the In the vestibular system we found a significant stria vascularis. In addition, and consistent with elevation of gentamicin fluorescence only for our data, they report the distribution of gen- the saccular hair cells and transitional cells at tamicin in hair cells, the spiral limbus including 500 µg/ml (Figure 9). Gentamicin fluorescence interdental cells, dendrites of auditory nerve increased with increasing dose (Figure 10), fibers, the spiral ligament and the basilar mem- however, compared to the cochlea the degree brane. The difference in the pattern of gen- of fluorescence appeared more limited tamicin in the stria vascularis between the

84 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

mouse [46] and our data in the rat could be lymph [54,55,56,57]. related to the different mode of gentamicin ap- plication. We perfused the gentamicin solution At the lowest gentamicin concentration tested directly through small holes in the bony wall of (500µg/ml) cochlear structures that showed a the cochlea at the apex and the basal turn near stronger gentamicin fluorescence and that are the round window and the round and oval win- regarded as relevant for K+ regulation were spi- dow, most likely providing direct access of gen- ral limbus including interdental cells and to a tamicin to the cochlea and the vestibular or- lower degree inner sulcus cells and the dorsal gans. In contrast, as suggested by Wang and spiral ligament (Figure 6) while vestibular dark Steyger the systemically applied gentamicin will cells showed no significant gentamicin fluores- enter the cochlea via the capillary network of cence (Figure 9). These data suggest that at a the stria vascularis. low dose gentamicin will mainly affect the recy- cling of K+ released by inner hair cells via the Steyger et al. [48] report that Texas red conju- medial pathway in the cochlea. The lateral path- gated gentamicin was found in hair cells of the way of K+ recycling via the spiral ligament and isolated bullfrog saccule while Lyford-Pike et al. the stria vascularis in the cochlea and vestibular [47] found Texas-Red-conjugated gentamicin K+ recycling via dark cells appear more resistant preferentially in Type I hair cells of the chinchilla to low concentrations of gentamicin. At higher . Our observation of gentamicin doses, gentamicin fluorescence appears more fluorescence in the hair cell epithelia at 500 µg/ generalized affecting most cell types (Figure 8, ml and above for saccule and at 1250 and 11). 2500 µg/ml for utricle and the ampule (Figure 9 -11) is consistent with these reports. However, Our data on the pattern of gentamicin fluores- since we included the cochlea as well as the cence are also consistent with previous studies vestibular organs in our quantitative analysis describing the damage and loss of cells poten- simultaneously (e.g. Figure 1) our data suggest, tially involved in the regulation of K+ following that gentamicin fluorescence is higher in the aminoglycoside treatment. Imamura and Adams cochlear compared to the vestibular regions [19] found degeneration in the spiral limbus, analyzed (Figure 8,11). the spiral ligament and the stria vascularis fol- lowing intratympanal application of gentamicin Considering the hypothesis that the therapeutic resembling previous reports showing pathologi- effect of gentamicin for treating Menière's dis- cal changes following gentamicin treatment in ease is associated with a regulation of ion ho- the spiral limbus and interdental cells [58], in meostasis [33], the most plausible targets the stria vascularis, mainly affecting the mar- would be those cells primarily involved in regu- ginal cells [16,17] and in vestibular dark cells lating the ionic composition of endolymph, espe- [21]. cially the cycling of K+ in the inner ear. Conclusions K+ is taken up from the endolymph and re- leased at the basolateral membrane of hair Following a 10 minute exposure of the acutely cells in the course of the mechano-electrical isolated rat cochlea to Texas-Red-conjugated transduction process. To maintain the high K+ gentamicin at concentrations of 500, 1250 and concentration in the endolymph, K+ released by 2500 µg/ml we found distinct and dose de- the hair cells needs to be returned to the endo- pendent patterns of gentamicin fluorescence in lymph. For the vestibular system K+ is returned the cochlear and vestibular regions analyzed to the endolymph by the dark cells [49,50]. In (Figure 8, 11). the cochlea K+ recycling involves a lateral and a medial pathway [51]. The well characterized Over all gentamicin fluorescence appeared lateral pathway involves a shift of the K+ re- more pronounced in the cochlea compared to leased by the hair cells via fibrocytes of the spi- vestibular organs, suggesting that the target of ral ligament to stria vascularis and secretion by a low dose gentamicin therapy may be the coch- the marginal cells back to endolymph [52,53]. lea rather than the vestibular organs. The less well characterized medial pathway in- volves inner sulcus cells, fibrocytes of the spiral Within the cochlea evaluation of the cells and limbus and interdental cells for the transport of regions most likely involved in K+ recycling, our K+ released by inner hair cells back to the endo- finding of significant gentamicin fluorescence

85 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

predominantly in the spiral limbus including the tamicin unter Erhalt der Kochleafunktion. HNO interdental cells and to a lesser degree in inner 2003; 51:898-902. sulcus cells and the dorsal spiral ligament [12] Hoffer ME, Kopke RD, Weisskopf P, Gottshall K, (Figure 6) suggest that gentamicin at a low dose Allen K, Wester D, Balaban C. Use of the Round Window Microcatheter in the Treatment of Me- may rather affect the recycling of K+ released by niere’s Disease. Laryngoscope 2001;111:2046 inner hair cells via the medial pathway (inner -2049. sulcus, spiral limbus, interdental cells) and to a [13] Harner SG, Driscoll CLW, Facer GW, Beatty CW, lesser degree the lateral pathway (fibrocytes of McDonald TJ. Long-Term Follow-Up of Transtym- the spiral ligament and the marginal cells of panic Gentamicin for Ménière’s Syndrome. stria vascularis). Otology&Neurotology 2001;22:210-214. [14] Anniko M. Principles in cochlear toxicity. Arch Acknowledgements Toxicol 1985;8:221-239. [15] Dumas G, Charachon R, Stoebner P. Lésions de We thank Ute Schreiter for excellent histological la strie vasculaire par la kanamycine: étude processing of the cochleae. électro-physiologique et ultra-structurale notes préliminaires. Ann Oto-Laryn 1981; 98:27-34.

[16] Forge A, Fradis M. Structural abnormalities in Address correspondence to: Dr. Katharina Schmid, the stria vascularis following chronic gentami- Department of Otolaryngology - Head and Neck Sur- cin treatment. Hear Res 1985;20:233-244. gery, University of Regensburg, D-93053 Regensburg, [17] Forge A, Wright A, Davies SJ. Analysis of structu- Germany, Tel: +49-941-944-9410, Fax: +49-941- ral changes in the stria vascularis following 944-9431, E-mail: [email protected] chronic gentamicin treatment. Hear Res regensburg.de 1987;31:253-266.

[18] Hellier WPL, Wagstaff SA, O’Leary SJ, Shepherd References RK. Functional and morphological response of

the stria vascularis following a sensorineural [1] Maladies de l’oreille interne offrant des symp- hearing loss. Hear Res 2002;172:127-136. toms de la congestion cerebral apoplectiforme . [19] Imamura S, Adams JC. Distribution of gentami- Gaz Med de Paris 1861;16:88. cin in the guinea pig inner ear after local or [2] Committee on Hearing and Equilibrium guideli- systemic application. JARO 2003a;04:176-195. nes for the diagnosis and evaluation of therapy [20] Kusunoki T, Cureoglu S, Schachern PA, Sam- in Meniere’s disease. Otolaryngol Head and paio A, Fukushima H, Oktay MF, Paparella MM. Neck Surg 1995; 113: 181-185. Effects of aminoglycoside administration on [3] Schuknecht HF, Gulya AJ. Endolymphatic Hy- cochlear elements in human temporal bones. drops: An Overview and Classification. Ann Otol Auris Nasus Larynx 2004;31:383-388. Rhinol Laryngol Suppl 1983;106:1-20. [21] Pender DJ. Gentamicin tympanoclysis: effects [4] Hallpike CS, Cairns H. Oberservation on the on the vestibular secretory cells. Am J Otolaryn- pathology of Ménière's Syndrome(Section of gol 1985;6:358-367. Otology). Proc R Soc Med 1938;31:1317-1336. [22] Konishi T, Butler RA, Fernandez C. Effect of [5] Dohlman GF. Experiments on the mechanism Anoxia on Cochlear Potentials. J Acoust Soc Am of Ménière attacks. J Otolaryngol 1976;6:135- 1961;33:349-356. 56. [23] Johnstone BM. The relation between endo- [6] Valk WL, Wit HP, Albers FW. Rupture of Reiss- lymph and the endocochlear potential during ner's membrane during acute endolymphatic anoxia. Acta Oto-Laryng 1965;60:1651-2251. hydrops in the guinea pig: a model for Méniè- [24] Schmiedt RA, Lang H, Okamura HO, Schulte BA. re's disease? Acta Otolaryngol 2006;126:1030- Effects of furosemide applied chronically to the 1035. round window: a model of metabolic presbyacu- [7] Baier G, Ott I. Die chirurgische Therapie des M. sis. J Neurosci 2002;22:9643-9650. Menière. HNO 2008; 56:553-566. [25] Leng G. The Davis theory: a review, and implica- [8] Van de Heyning PH, Verlooy J, Schatteman I, tions of recent eletrophysiological evidence. Wuyts FL. Selective vestibular neurectomy in Hear Res 1980;3:17-25. Menière’s disease: a review. Acta Otolaryngol [26] Russel IJ. Origin of the receptor potential in 1997;526:58-66. inner hair cells of the mammalian cochlea- [9] Schuknecht HF. Ablation therapy in the mana- evidence for Davis’ theory. Nature gement of Menière's disease. Acta Otolaryngol 1983;301:334-336 Suppl 1957;132:1-42. [27] Gross J, Stute K, Moller R, Fuchs J, Amarjargal [10] Hoffmann F, Beck C, Beck C, Stratulat S. Suba- N, Pohl EE, Angerstein M, Smorodchenko A, blative Gentamicintherapie bei M. Menière. Mazurek B. Expression of prestin and Gata-3,-2, HNO 1993;41:296-300. -1 mRNA in the rat organ of Corti during the [11] Lange G, Mann W, Maurer J. Intratympanale postnatal period and in culuture. Hear Res Intervalltherapie des Morbus Menière mit Gen- 2010;261:9-21.

86 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87 Gentamicin uptake in the acutely isolated rat inner ear

[28] Amarjargal N, Andreeva N, Gross J, Haupt H, 2007;117:1191-1198. Fuchs J, Szczepek AJ, Mazurek B. Differential [44] Imamura S, Adams JC. Changes in cytochemis- vulnerability of outer and inner hair cells during try of sensory and nonsensory cells in gentami- and after oxygen-glucose deprivation in organo- cin-treated . JARO 2003;04:196-218. typic cultures of newborn rats. Physiol Res [45] Plontke SKR, Wood AW, Salt AN. Analysis of 2009;58:895-902. gentamicin kinetics in fluids of the inner ear [29] Qi W, Ding D, Salvi RJ. Cytotoxic effects of dime- with round window administration. Otol Neuro- thyl sulphoxide (DMSO) on cochlear organotypic tol 2002;23:967-974. cultures. Hear Res 2008;236:52-60. [46] Wang Q, Steyger PS. Trafficking of systemic [30] He DZ, Dallos P. Mechanoelectrical transduc- fluorescent gentamicin into the cochlea and tion of adult outer hair cells studies in a gerbil Hair cells. JARO 2009;10:205-219. hemicochlea. Nature 2004; 429:766-770. [47] Lyford-Pike S, Vogelheim C, Chu E, Della Santi- [31] Tritsch NX, Bergles DE. Developmental regula- na CC, Carey JP. Gentamicin is primarily local- tion of spontaneous activity in the Mammalian ized in vestibular type I hair cells after intratym- cochlea. J Neurosci 2010;30:1539-1550. panic administration. J Assoc Res Otolaryngol [32] Beurg M, Nam JH, Crawford A, Fettiplace R. The 2007;8:497-508. actions of calcium on hair bundle mechanics in [48] Steyger PS, Peters SL, Rehling J, Hordichok A, the mammalian cochlear hair cells. Biophys J Dai CF. Uptake of gentamicin by bullfrog saccu- 2008;94:2639-2653. lar hair cells in vitro. JARO 2003;4:565-578. [33] Beck C, Schmidt CL. 10 years of experience [49] Wangemann P. Comparison of ion transport with intratympanally applied streptomycin mechanisms between vestibular dark cells and (gentamycin) in the therapy of Morbus Menière. strial marginal cells. Hear Res 1995;90:149- Arch Otorhinolaryngol 1978;221:149-52. 157. [34] Lange G. Die intratympanale Behandlung des [50] Wangemann P. Supporting sensory transduc- Morbus Menière mit ototoxischen Antibiotika. tion: cochlear fluid homeostasis and the endo- Laryng Rhinol 1977;56:409-414. cochlear potential. J Physiol 2006;576:11-21. [35] Blakley BW. Update on intratympanic gentami- [51] Couloigner V, Sterkers O, Ferrary E. What’s new cin for Menière`s disease. Laryngoscope in ion transport in the cochlea? Plugers Arch 2000;110:236-204. Eur J Physiol 2006;453:11-22. [36] Nakagawa T, Yamane H, Shibata S, Nakai Y. [52] Spicer SS, Schulte BA. The fine structure of Gentamicin ototoxicity induced apoptosis of the spiral ligament cells relates to ion return to the vestibular hair cells of guinea pigs. Eur Arch stria and varies with place-frequency. Hear Res Otorhinolaryngol 1997;254:9-14. 1996;100:80-100. [37] Nakagawa T, Yamane H, Takayama M, Sunami [53] Lang F, Vallon V, Knipper M, Wangemann P. K. Apoptosis of guinea pig cochlear hair cells Functional significance of channels and trans- following chronic aminoglycoside treatment. porters expressed in the inner ear and kidney. Eur Arch Otorhinolaryngol 1998;255:127-131. Am J Physiol Cell Physiol 2007;293:1187- [38] Nakagawa T, Yamane H, Takayama M, Sunami 1208. K, Nakai Y. Dose-dependent response of vesti- [54] Spicer SS, Schulte BA. Evidence for a medial K+ bular hair cells of guinea pigs following strepto- recycling pathway from inner hair cells. Hear mycin ototoxiation. Acta Otolaryngol Res 1998;118:1-12. 1998;118:530-533. [55] Spicer SS, Salvi RJ, Schulte BA. Ablation of in- [39] Nakagawa T, Yamane H, Takayama M, Sunami ner hair cells by carboplatin alters cells in the K, Nakai Y. Time-dependent response of vesti- medial K+ flow route and disrupts tectorial bular hair cells of guinea pigs following high- membrane. Hear Res 1999;136:139-150. dose applications of streptomycin. Acta Otola- [56] Spicer SS, Thomopoulos GN, Schulte BA. Struc- ryngol 1998;538:32-35. tural evidence for ion transport and tectorial [40] Okuda T, Sugahara K, Shimogori H, Yamashita membrane maintance in the gerbil limbus. H. Inner ear changes with intracochlear genta- Hear Res 2000;143:147-161. micin administration in guinea pigs. Laryngos- [57] Spicer SS, Salvi RJ. Ultrastructural changes in cope 2004;114:694-697. the spiral limbus associated with carboplatin- [41] Dai CF, Mangiardi D, Cotanche DA, Steyger PS. induced ablation of inner hair cells. Cell Tissue Uptake of fluorescent gentamicin by vertebrate Res 2000;302:1-10. sensory cells in vivo. Hear Res 2006;213:64- [58] Kimura RS, Nye CL, Southard RE. Normal and 78. pathological features of the limbus spiralis and [42] Roehm P, Hoffer M, Balaban CD. Gentamicin its functional significance. Am J Otolaryngol uptake in the chinchilla inner ear. Hear Res 1990;11:99-111. 2007;230: 43-52. [43] Plontke SKR, Mynatt R, Gill RM, Borgmann S, Salt AN. Concentration gradient along the scala tympani after local application of gentamicin to the round window membrane. Laryngoscope

87 Int J Physiol Pathophysiol Pharmacol 2011;3(2):71-87