Jihye Rhee; Dong Woo Nam; Sung Joong Moon; Ja-Won Koo

doi:10.21790/rvs.2025.012

Articles

Page Path: HOME > Res Vestib Sci > Volume 24(2); 2025 > Article

Original Article
Inner ear uptake of fluorescent gentamicin in neonatal mice: an experimental animal study: Jihye Rhee^1,2,*, Dong Woo Nam^3,*, Sung Joong Moon¹, Ja-Won Koo^1,3; Research in Vestibular Science 2025;24(2):98-106.
DOI: https://doi.org/10.21790/rvs.2025.012
Published online: June 15, 2025

¹Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Korea

²Department of Otorhinolaryngology-Head and Neck Surgery, Veterans Hospital Service Medical Center, Seoul, Korea

³Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Bundang Hospital, Seongnam, Korea

Corresponding author: Ja-Won Koo Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Bundang Hospital, 82 Gumi-ro 173 beon-gil, Bundang-gu, Seongnam 13620, Korea. E-mail: jwkoo99@snu.ac.kr

*These authors contributed equally to this work as co-first authors.

• Received: May 21, 2025 • Revised: June 3, 2025 • Accepted: June 5, 2025

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

1,484 Views
22 Download

prev next

Full Article

Download PDF

Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ARTICLE INFORMATION
REFERENCES

Abstract

Objectives
This study aimed to investigate whether systemic administration of fluorescently labeled gentamicin (gentamicin-Texas Red, GTTR) results in differential inner ear uptake during postnatal maturation of the blood-labyrinth barrier (BLB) in neonatal mice.
Methods
Neonatal C57BL/6 mice were intraperitoneally injected with GTTR at postnatal day 7 (P7), 21 (P21), or 35 (P35). Cochlear and renal tissues were harvested 60 minutes after injection and examined via confocal microscopy. Fluorescence intensity of GTTR in cochlear structures—including marginal cells, strial tissues, basal cells, spiral ligament, and hair cells—was quantified using ImageJ. Renal proximal tubules served as systemic uptake controls.
Results
GTTR uptake was significantly higher in cochlear lateral wall structures and hair cells of P7 mice compared to P35 mice (p<0.01). No significant differences in fluorescence were observed in the renal proximal tubules across age groups. Mice injected with unconjugated Texas Red showed negligible fluorescence in all tissues, confirming specific uptake of GTTR.
Conclusions
Inner ear uptake of aminoglycosides is markedly increased prior to full maturation of the BLB in neonatal mice. These findings suggest that immature cochlear barriers in early postnatal stages may enhance aminoglycoside entry into sensory hair cells, highlighting the need for cautious use of these antibiotics in premature infants.
Keywords: Ototoxicity; Aminoglycosides; Newborn; Mice; Premature birth

INTRODUCTION

Ototoxicity in neonates remains poorly characterized [1], with reported incidence in the general population varying widely from 0% to 47% [2,3]. The mechanisms underlying aminoglycoside-induced ototoxicity are complex and multifactorial, but the toxicity is generally dose-dependent [4]. Although aminoglycosides are widely used, particularly in the prophylactic treatment of infections in premature infants, the precise routes by which these drugs reach cochlear hair cells remain incompletely understood.

Previous studies have suggested that aminoglycosides primarily enter cochlear hair cells across their apical membranes via endolymph in vivo [5-8]. Indirect evidence also indicates that systemic aminoglycosides may traverse from strial capillaries across the stria vascularis into the endolymph [5,9]. This trafficking appears to be regulated by tight junctions at both the strial endothelial cell layer and the interface between marginal and intermediate cells [9], which together form the cochlear blood-labyrinth barrier (BLB)—a structure functionally analogous to the blood-brain barrier. Disruption or immaturity of the BLB may lead to increased permeability and enhanced cochlear uptake of circulating substances such as gentamicin [10].

Despite their known ototoxic potential, aminoglycosides remain commonly prescribed in neonatal care. In premature infants, developmental immaturity of the inner ear and BLB may increase the risk of cochlear drug entry and subsequent toxicity. To model this susceptibility, neonatal mice offer a valuable experimental system, as their cochlear structures—including the BLB—continue to mature during the early postnatal period. Tight junctions between marginal and endothelial cells are established around 2 weeks after birth. The average life span of C57BL/6 mice is approximately 9 to 12 months, and in rodent models, neonatal life is generally considered to encompass the first 10 postnatal days, during which critical maturation processes occur in the inner ear. This dynamic postnatal maturation provides a useful model for examining how ototoxic agents penetrate the immature cochlear barrier.

In the present study, we investigated whether systemic administration of fluorescently labeled gentamicin (gentamicin-Texas Red, GTTR) results in increased cochlear uptake in neonatal mice at various postnatal stages.

METHODS

Ethics Statement

All experimental protocols were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (No. BA1207-108/053-01).

Preparation of Gentamicin-Texas Red Conjugate

GTTR was prepared as previously described [11]. Briefly, gentamicin sulfate (in 100 mM K₂CO₃, pH 10) was mixed with succinimidyl esters of Texas Red (10 mg/mL in dimethylformamide, Invitrogen) at a high molar ratio of gentamicin to Texas Red. The reaction mixture was gently agitated at 4 °C for several days to ensure conjugation of a single Texas Red molecule per gentamicin moiety. The GTTR conjugate was purified using reversed-phase chromatography on C-18 columns (Grace) to remove unreacted gentamicin and free Texas Red [12].

Experimental Animals and Drug Administration

C57BL/6 mice were divided into experimental and control groups according to postnatal age: postnatal day 7 (P7) (n=6), P21 (n=3), and P35 (n=6) for experimental groups; and P7, P21, and P35 (n=2 each) for control groups. Experimental animals received intraperitoneal injections of GTTR at 2 mg/kg. Sixty minutes after injection, animals were anesthetized and euthanized.

To evaluate the possibility of nonspecific uptake of Texas Red, control mice received intraperitoneal injections of an equivalent molar amount of unconjugated Texas Red.

Tissue Preparation

Euthanasia was performed via transcardiac perfusion with 1 mL of Dulbecco’s phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The cochlea and kidneys were dissected and post-fixed in 4% paraformaldehyde for 15 minutes. The lateral wall was dissected, and cochlear coils including the organ of Corti were isolated and whole-mounted. Kidney tissues were embedded and sectioned at 4-µm thickness using a microtome.

Tissues were permeabilized with 0.5% Triton X-100 for 45 minutes, washed with PBS, and stained with Alexa Fluor 488-conjugated phalloidin (Molecular Probes) to visualize filamentous actin. Samples were then post-fixed in 4% paraformaldehyde for an additional 15 minutes [5].

Immunohistochemistry and Frozen Sections

To obtain broad anatomical views, selected cochlear tissues were subjected to cryosectioning. Tissues were fixed in 4% paraformaldehyde for 24 hours at 4 °C, decalcified in 0.135 M ethylenediaminetetraacetic acid for 3 days, washed in PBS, and embedded in optimal cutting temperature compound. Sections were cut at 5 µm and dried at 37 °C before immunostaining.

Sections were blocked with buffer (1% bovine serum albumin, 0.1% Triton X-100 in PBS) for 60 minutes. Primary antibody against calretinin (MAB1568, 1:250; Millipore) was applied overnight at 4 °C. After washing, sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (A11017, 1:200; Molecular Probes) for 1 to 2 hours at room temperature in the dark. Slides were mounted using VECTASHIELD mounting medium (Vector Laboratories).

Confocal Imaging

Apical and basal cochlear turns were mounted on slides with VECTASHIELD and imaged using a Zeiss LSM 510 META confocal microscope. All red-channel images (GTTR fluorescence) were acquired using identical laser intensity and gain settings for consistency across experimental groups [9]. Imaging conditions were separately optimized for kidney sections.

Image Analysis

Regions of interest including marginal cells, intrastrial tissues, basal cells, spiral ligament, and outer hair cells were defined using the green phalloidin channel. Red-channel fluorescence intensity (GTTR signal) was manually segmented and measured using ImageJ software (NIH). To normalize between experiments, signal intensities were standardized against marginal cell intensity in control specimens.

Statistical Analysis

All data are presented as mean ± standard error of the mean. Statistical analyses were performed using PASW Statistics ver. 18.0 (IBM Corp.). Group comparisons among P7, P21, and P35 were initially evaluated using the Kruskal-Wallis test. When significant differences were detected, post hoc pairwise comparisons were conducted using the Mann-Whitney U-test. Among the three possible pairwise comparisons (P7 vs. P21, P7 vs. P35, and P21 vs. P35), only two comparisons (P7 vs. P21 and P7 vs. P35) were of primary interest and tested. Therefore, Bonferroni correction was applied for two comparisons, adjusting the significance threshold from p<0.05 to p<0.025.

RESULTS

Increased Gentamicin-Texas Red Uptake in Neonatal Cochlea

At P7, GTTR fluorescence was markedly increased in both the cochlear lateral wall and outer hair cell regions compared to later developmental stages. In contrast, mice at P21 and P35 exhibited substantially weaker and more restricted GTTR signals within the lateral wall (Fig. 1).

Absence of Uptake in the Texas Red-Only Control

In animals injected with unconjugated Texas Red, no significant fluorescence was observed in cochlear marginal cells or renal proximal tubules, confirming that the observed uptake in experimental groups was specific to the GTTR conjugate (Fig. 2).

Quantitative Analysis of Gentamicin-Texas Red Uptake in the Cochlear Lateral Wall

Kruskal-Wallis analysis revealed significant differences in GTTR fluorescence intensity among postnatal ages (P7, P21, P35) in several cochlear regions, including the marginal cells, intrastrial tissues, basal cells, and spiral ligament (p<0.05) (Table 1 and Fig. 3). Subsequent pairwise comparisons using the Mann-Whitney U-test demonstrated that P7 mice had significantly higher fluorescence intensity than P35 mice in most regions of the basal turn (Bonferroni-corrected p<0.025; data not shown).

As summarized in Table 1, Kruskal-Wallis tests revealed statistically significant differences in GTTR fluorescence among the three postnatal age groups across nearly all regions of the cochlear lateral wall and outer hair cells. In the basal turn, the strongest group differences were observed in the spiral ligament (H=10.225, p=0.006) and outer hair cells (H=11.025, p=0.004). Similarly, in the apicomiddle turn, the hair cells (H=10.522, p=0.006) and spiral ligament (H=10.381, p=0.005) showed the most prominent differences. These findings highlight the regional specificity of age-related differences in aminoglycoside uptake.

Quantitative Analysis of Gentamicin-Texas Red Uptake in Outer Hair Cells

Outer hair cells also showed a significant difference among age groups by Kruskal-Wallis test (p<0.05) (Table 1), and post hoc analysis confirmed that GTTR uptake in P7 mice was significantly higher than in P35 mice (Bonferroni-corrected p<0.025) (Figs. 4 and 5).

Gentamicin-Texas Red Uptake in Renal Proximal Tubules

To assess age-dependent variability in systemic uptake, GTTR fluorescence intensity was also measured in renal proximal tubules. No significant differences were observed among age groups (P7, P21, and P35), indicating that systemic exposure to GTTR was comparable across groups and did not account for cochlear differences (Fig. 6).

DISCUSSION

This study demonstrates that cochlear uptake of GTTR is significantly modulated by postnatal age in mice, with increased accumulation observed in the cochlear lateral wall and hair cells of neonatal mice (P7). In contrast, uptake in renal proximal tubules did not differ across age groups, indicating that the enhanced cochlear uptake is not due to systemic differences in drug exposure but rather to developmental immaturity of the cochlear barrier.

Importantly, mice injected with unconjugated Texas Red showed negligible fluorescence in both cochlear and renal tissues, confirming that the observed uptake was specific to the gentamicin moiety. These findings eliminate the possibility of nonspecific uptake of the fluorescent dye and support the validity of GTTR as a marker of aminoglycoside trafficking into target tissues.

Developmental Considerations and Model Selection

The neonatal mouse is an established model for studying ototoxicity during cochlear development [13], as auditory function in mice matures postnatally [14,15]. Previous reports indicate a critical window of susceptibility to aminoglycoside-induced hair cell damage between P7 and P10 [16], during which systemic administration of aminoglycosides such as neomycin or gentamicin in P2 to P5 mice has been shown to induce severe and permanent auditory threshold shifts as measured by auditory brainstem responses (ABR) [17,18]. During this period, the structural and functional maturation of the BLB is still incomplete, especially in the stria vascularis, where tight junctions between marginal and endothelial cells continue to form. Our findings are consistent with this timeline and extend it by providing anatomical evidence of drug trafficking that supports functional studies showing permanent ABR threshold elevation following neonatal aminoglycoside exposure. This further suggests that immature BLB function—including underdeveloped tight junctions and elevated transcytosis in the strial microvasculature—may contribute significantly to enhanced cochlear drug permeability [19].

While previous studies have primarily assessed functional outcomes using methods such as distortion-product otoacoustic emissions [20], ABR [21-24], or electrocochleography [25], our study focused on the anatomical basis of aminoglycoside accumulation. Notably, prior ABR-based studies have demonstrated that even a single neonatal exposure to aminoglycosides can lead to profound and irreversible hearing loss in mice, consistent with our anatomical observations of increased GTTR uptake during this period [17]. These functional outcomes support the notion that elevated cochlear drug penetration in early postnatal stages has clinically relevant consequences. By directly visualizing GTTR uptake, we provide structural evidence that supports the hypothesis that BLB immaturity facilitates drug entry into the developing cochlea.

Clinical Implications

These findings may have clinical relevance for premature infants, whose cochlear structures, including the BLB, may be similarly immature. Increased permeability in the developing ear could enhance aminoglycoside accumulation in sensory hair cells, elevating the risk of ototoxicity. Therefore, careful dosing and vigilant monitoring of aminoglycoside use in neonates—particularly those born prematurely—are warranted.

To our knowledge, this is one of the first studies to provide direct imaging-based evidence linking cochlear barrier immaturity with increased aminoglycoside uptake. These results emphasize the importance of considering developmental stage when assessing ototoxic risk and support further research into age-dependent pharmacokinetics in the inner ear.

One limitation of this study is the absence of functional assessments, such as ABR, which prevents direct correlation between histological uptake and hearing outcomes. Furthermore, because the analysis was confined to cochlear structures, without detailed evaluation of the vestibular apparatus, the generalizability of our findings to the entire inner ear remains limited. Future studies incorporating both functional hearing tests and comprehensive assessment of vestibular structures are warranted to more fully characterize aminoglycoside uptake and its implications.

ARTICLE INFORMATION

Funding/Support

This study was supported in part by Seoul National University Bundang Hospital (grant number 13-2020-0006).

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Availability of Data and Materials

The datasets are not publicly available but are available from the corresponding author upon reasonable request.

Authors’ Contributions

Conceptualization, Funding acquisition, Project administration: JWK; Data curation: JR, DWN; Formal analysis: SJM; Methodology: JR; Visualization: DWN, SJM; Writing–original draft: JR; Writing–review & editing: All authors.

All authors read and approved the final manuscript.

Fig. 1.

Postnatal age-dependent cochlear gentamicin-Texas Red (GTTR) uptake. Confocal micrographs of cochlear sections from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. Sections are stained with phalloidin (cyan, for F-actin) and show GTTR uptake (red). At P7, strong GTTR fluorescence is evident in both the stria vascularis (asterisks) on the lateral wall and the organ of Corti (arrows). In contrast, GTTR uptake is markedly diminished by P21 and P35, with only weak, restricted signal in the stria vascularis and substantially reduced or absent fluorescence in the organ of Corti.

Fig. 2.

Comparison of gentamicin-Texas Red (GTTR) and unconjugated Texas Red (TR, green) localization in cochlear marginal cells (MC) and renal cortex (RC). Micrographs show cochlear MCs (A) and RC tubules (B) from postnatal day 7 (P7) and P35 mice. (A) In MCs, strong intracellular GTTR (red) uptake is evident at P7, diminishing to minimal by P35. (B) GTTR uptake is also observed in RC tubules at both P7 and P35. In control animals injected with unconjugated TR, negligible fluorescence was detected in either MCs or RC tubules at P7 or P35, confirming GTTR-specific uptake.

Fig. 3.

Age-dependent gentamicin-Texas Red (GTTR) uptake in cochlear lateral wall layers. Confocal micrographs show GTTR fluorescence (red, presented in grayscale) in the marginal cell (MC), intrastrial cell (IC), basal cell (BC), and spiral ligament (SL) layers of cochleae from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. GTTR signal is most intense across all layers at P7, then progressively decreases at P21 and P35. By P35, uptake is minimal in the MC, BC, and SL layers. Notably, intrastrial blood vessels (asterisk within IC) retain prominent GTTR signal through P35, while uptake in the surrounding IC stroma diminishes with age.

Fig. 4.

Relative gentamicin-Texas Red fluorescence intensity in cochlear marginal cells, intrastrial tissue, basal cell, and hair cells at the apical and basal turns. Fluorescence intensity was significantly higher in postnatal day 7 (P7) mice compared to P35 mice in both marginal cell layers and outer hair cells. ^*p<0.025.

Fig. 5.

Age-dependent gentamicin-Texas Red (GTTR) uptake in cochlear outer hair cells (OHCs). Confocal micrographs show GTTR signal (gray scale) in OHCs (brackets) within apical and basal turns of cochleae from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. At P7, OHCs in both apical and basal turns exhibit prominent GTTR uptake. This signal is markedly reduced by P21 and becomes minimal to largely absent in OHCs of both cochlear regions by P35.

Fig. 6.

Gentamicin-Texas Red (GTTR) uptake in renal proximal tubules. (A) GTTR fluorescence in the renal proximal tubules at postnatal day 7 (P7), P21, and P35. (B) Kruskal-Wallis test revealed no significant differences in fluorescence intensity among age groups (p>0.05).

Table 1.

Kruskal-Wallis test results comparing gentamicin-Texas Red uptake across postnatal ages

Cochlear structure	Apicomiddle turn		Basal turn
Cochlear structure	H (df=2)	p-value	H (df=2)	p-value
Marginal cells	6.866	0.032^*	9.458	0.009^*
Intrastrial cells	6.240	0.044^*	10.150	0.006^*
Basal cells	5.950	0.051	10.350	0.006^*
Spiral ligament	10.381	0.005^*	10.225	0.006^*
Hair cells	10.522	0.006^*	11.025	0.004^*

H, Kruskal-Wallis test statistic; df, degrees of freedom.

^*p<0.05.

REFERENCES

1. McCracken GH Jr, Nelson JD. Antimicrobial therapy for newborns: practical application of pharmacology to clinical usage. Grune & Stratton; 1977.
2. Kahlmeter G, Dahlager JI. Aminoglycoside toxicity: a review of clinical studies published between 1975 and 1982. J Antimicrob Chemother 1984;13 Suppl A:9–22. Article PubMed
3. Barclay ML, Kirkpatrick CM, Begg EJ. Once daily aminoglycoside therapy: is it less toxic than multiple daily doses and how should it be monitored? Clin Pharmacokinet 1999;36:89–98. Article PubMed
4. Forge A, Schacht J. Aminoglycoside antibiotics. Audiol Neurootol 2000;5:3–22. Article PubMed PDF
5. Dai CF, Steyger PS. A systemic gentamicin pathway across the stria vascularis. Hear Res 2008;235:114–124. Article PubMed
6. Dai CF, Mangiardi D, Cotanche DA, Steyger PS. Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo. Hear Res 2006;213:64–78. Article PubMed PMC
7. Marcotti W, van Netten SM, Kros CJ. The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels. J Physiol 2005;567(Pt 2):505–521. Article PubMed PMC
8. Hashino E, Shero M. Endocytosis of aminoglycoside antibiotics in sensory hair cells. Brain Res 1995;704:135–140. Article PubMed
9. Wang Q, Steyger PS. Trafficking of systemic fluorescent gentamicin into the cochlea and hair cells. J Assoc Res Otolaryngol 2009;10:205–219. Article PubMed PMC PDF
10. Koo JW, Wang Q, Steyger PS. Infection-mediated vasoactive peptides modulate cochlear uptake of fluorescent gentamicin. Audiol Neurootol 2011;16:347–358. Article PubMed PDF
11. Sandoval R, Leiser J, Molitoris BA. Aminoglycoside antibiotics traffic to the Golgi complex in LLC-PK1 cells. J Am Soc Nephrol 1998;9:167–174. Article PubMed
12. Myrdal SE, Johnson KC, Steyger PS. Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis. Hear Res 2005;204:156–169. Article PubMed PMC
13. Alford BR, Ruben RJ. Physiological, behavioral and anatomical correlates of the development of hearing in the mouse. Ann Otol Rhinol Laryngol 1963;72:237–247. Article PubMed PDF
14. Prieve BA, Yanz JL. Age-dependent changes in susceptibility to ototoxic hearing loss. Acta Otolaryngol 1984;98:428–438. Article PubMed
15. Henley CM, Rybak LP. Ototoxicity in developing mammals. Brain Res Brain Res Rev 1995;20:68–90. Article PubMed
16. Chen CS, Saunders JC. The sensitive period for ototoxicity of kanamycin in mice: morphological evidence. Arch Otorhinolaryngol 1983;238:217–223. Article PubMed PDF
17. Cutri RM, Lin J, Nguyen NV, Shakya D, Shibata SB. Neomycin-induced deafness in neonatal mice. J Neurosci Methods 2023;391:109852. Article PubMed
18. Tao L, Segil N. CDK2 regulates aminoglycoside-induced hair cell death through modulating c-Jun activity: inhibiting CDK2 to preserve hearing. Front Mol Neurosci 2022;15:1013383. Article PubMed PMC
19. Li G, Gao Y, Wu H, Zhao T. Gentamicin administration leads to synaptic dysfunction in inner hair cells. Toxicol Lett 2024;391:86–99. Article PubMed
20. Berenholz LP, Rossi DL, Lippy WH, Burkey JM. Is there an ototoxicity risk from Cortisporin and comparable otic suspensions?: distortion-product otoacoustic emission findings. Ear Nose Throat J 2012;91:106–135. Article PubMed PDF
21. Bernard PA, Péchère JC, Hébert R. Altered objective audiometry in aminoglycosides-treated human neonates. Arch Otorhinolaryngol 1980;228:205–210. Article PubMed PDF
22. Bernard PA. Freedom from ototoxicity in aminoglycoside treated neonates: a mistaken notion. Laryngoscope 1981;91:1985–1994. Article PubMed
23. Parini R, Rusconi F, Cavanna G, Vigliani E, Cornacchia L, Assael BM. Evaluation of the renal and auditory function of neonates treated with amikacin. Dev Pharmacol Ther 1982;5:33–46. Article PubMed PDF
24. McCracken GH. Aminoglycoside toxicity in infants and children. Am J Med 1986;80:172–178. Article PubMed
25. Henry KR, Chole RA, McGinn MD, Frush DP. Increased ototoxicity in both young and old mice. Arch Otolaryngol 1981;107:92–95. Article PubMed

Figure & Data

References

Citations

Citations to this article as recorded by

PubReader
ePub Link

Cite

Cite: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

Inner ear uptake of fluorescent gentamicin in neonatal mice: an experimental animal study

Res Vestib Sci. 2025;24(2):98-106. Published online June 15, 2025

DOI: https://doi.org/10.21790/rvs.2025.012

XML Download

Figure

Inner ear uptake of fluorescent gentamicin in neonatal mice: an experimental animal study

Fig. 1. Postnatal age-dependent cochlear gentamicin-Texas Red (GTTR) uptake. Confocal micrographs of cochlear sections from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. Sections are stained with phalloidin (cyan, for F-actin) and show GTTR uptake (red). At P7, strong GTTR fluorescence is evident in both the stria vascularis (asterisks) on the lateral wall and the organ of Corti (arrows). In contrast, GTTR uptake is markedly diminished by P21 and P35, with only weak, restricted signal in the stria vascularis and substantially reduced or absent fluorescence in the organ of Corti.

Fig. 2. Comparison of gentamicin-Texas Red (GTTR) and unconjugated Texas Red (TR, green) localization in cochlear marginal cells (MC) and renal cortex (RC). Micrographs show cochlear MCs (A) and RC tubules (B) from postnatal day 7 (P7) and P35 mice. (A) In MCs, strong intracellular GTTR (red) uptake is evident at P7, diminishing to minimal by P35. (B) GTTR uptake is also observed in RC tubules at both P7 and P35. In control animals injected with unconjugated TR, negligible fluorescence was detected in either MCs or RC tubules at P7 or P35, confirming GTTR-specific uptake.

Fig. 3. Age-dependent gentamicin-Texas Red (GTTR) uptake in cochlear lateral wall layers. Confocal micrographs show GTTR fluorescence (red, presented in grayscale) in the marginal cell (MC), intrastrial cell (IC), basal cell (BC), and spiral ligament (SL) layers of cochleae from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. GTTR signal is most intense across all layers at P7, then progressively decreases at P21 and P35. By P35, uptake is minimal in the MC, BC, and SL layers. Notably, intrastrial blood vessels (asterisk within IC) retain prominent GTTR signal through P35, while uptake in the surrounding IC stroma diminishes with age.

Fig. 4. Relative gentamicin-Texas Red fluorescence intensity in cochlear marginal cells, intrastrial tissue, basal cell, and hair cells at the apical and basal turns. Fluorescence intensity was significantly higher in postnatal day 7 (P7) mice compared to P35 mice in both marginal cell layers and outer hair cells. *p<0.025.

Fig. 5. Age-dependent gentamicin-Texas Red (GTTR) uptake in cochlear outer hair cells (OHCs). Confocal micrographs show GTTR signal (gray scale) in OHCs (brackets) within apical and basal turns of cochleae from (A) postnatal day 7 (P7), (B) P21, and (C) P35 mice. At P7, OHCs in both apical and basal turns exhibit prominent GTTR uptake. This signal is markedly reduced by P21 and becomes minimal to largely absent in OHCs of both cochlear regions by P35.

Fig. 6. Gentamicin-Texas Red (GTTR) uptake in renal proximal tubules. (A) GTTR fluorescence in the renal proximal tubules at postnatal day 7 (P7), P21, and P35. (B) Kruskal-Wallis test revealed no significant differences in fluorescence intensity among age groups (p>0.05).

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Inner ear uptake of fluorescent gentamicin in neonatal mice: an experimental animal study

Cochlear structure	Apicomiddle turn		Basal turn
Cochlear structure	H (df=2)	p-value	H (df=2)	p-value
Marginal cells	6.866	0.032^*	9.458	0.009^*
Intrastrial cells	6.240	0.044^*	10.150	0.006^*
Basal cells	5.950	0.051	10.350	0.006^*
Spiral ligament	10.381	0.005^*	10.225	0.006^*
Hair cells	10.522	0.006^*	11.025	0.004^*

Table 1. Kruskal-Wallis test results comparing gentamicin-Texas Red uptake across postnatal ages

H, Kruskal-Wallis test statistic; df, degrees of freedom.

p<0.05.