The expression changes of high-mobility group box 1 (HMGB1) in the mouse cochlea have recently been implicated in noise-induced hearing loss, suggesting that HMGB1 participates in regulating cochlear function. However, the precise role of HMGB1 in the auditory system remains largely unclear. This study aimed to investigate its function in the developing mouse cochlea by examining the expression pattern of HMGB1 in the mouse cochlea from embryonic day (E) 18.5 to postnatal day (P) 28 using double immunofluorescence on frozen sections. Our findings revealed that HMGB1 was extensively expressed in the cell nucleus across various regions of the mouse cochlea, including the organ of Corti. Furthermore, its expression underwent developmental regulation during mouse cochlear development. Specifically, HMGB1 was found to be localized in the tympanic border cells at each developmental stage, coinciding with the gradual anatomical in this region during development. In addition, HMGB1 was expressed in the greater epithelial ridge (GER) and supporting cells of the organ of Corti, as validated by the supporting cell marker Sox2 at P1 and P8. However, at P14, the expression of HMGB1 disappeared from the GER, coinciding with the degeneration of the GER into the inner sulcus cells. Moreover, we observed that HMGB1 co-localized with Ki-67-positive proliferating cells in several cochlear regions during late embryonic and early postnatal stages, including the GER, the tympanic border cells, cochlear lateral wall, and cochlear nerves. Furthermore, by dual-staining Ki-67 with neuronal marker TUJ1 or glial marker Sox10, we determined the expression of Ki-67 in the neonatal glial cells. Our spatial-temporal analysis demonstrated that HMGB1 exhibited distinct expression patterns during mouse cochlear development. The co-localization of HMGB1 with Ki-67-positive proliferating cells suggested that HMGB1 may play a role in cochlear development.

High-mobility group (HMG) proteins are non-histone nuclear DNA-binding proteins, classified into three families: HMGA, HMGB, and HMGN.1,2 Among these families, the HMGB protein family is the most abundant, consisting of three members (HMGB1-3), each containing two “box” domains (A and B box) in the N-terminus.3 HMGB1, the most extensively studied and abundant HMG protein, is a 25-kDa protein composed of 215 amino acids. HMGB1 can be actively secreted or passively released from necrotic cells in response to infection, injury and cellular stress. When passively released, HMGB1 acts as a danger-associated molecular pattern (DAMP) molecule protein by binding to receptors for advanced glycation end-products (RAGE) and Toll-like family of receptors (TLRs).4-7 It has also been found that the release of HMGB1 from supporting cells of the organ of Corti is involved in an inflammatory process in the amikacin-poisoned rat cochlea.8 Besides its pathological role, HMGB1 has been proposed to have many potential roles, acting as a transcription factor and growth factor involved in various biological processes, including transcription,9 replication, DNA repair, and recombination.10,11 Studies have suggested that HMGB1 has a critical role in the development of the central nervous system.12 Loss of HMGB1 in vivo impairs neural progenitor cell survival and proliferation.13,14 HMGB1 is also involved in other developmental processes, such as spinal cord regeneration, endothelial cell proliferation, osteogenic differentiation, and myogenesis in other cell types.15-18

HMGB1 displays a complex temporal and spatial distribution pattern during tooth development and in the mouse brain.19,20 Besides HMGB1, another member of the high mobility group family, HMGA2, has been proposed to possibly participate in inner ear development, based on its extended and overlapping expression with the stem cell and cochlear supporting-cell marker Sox2.21 Previous data have indicated changes in HMGB1 expression during the development of the mouse cochlea, suggesting that HMGB1 may participate in the development of the mouse cochlea.22 Two recent studies have shown that abnormal HMGB1 expression in the mouse cochlea is associated with noise-induced hearing loss and contributes to the ototoxicity of cisplatin to the inner ear.23-25 In a recent study, Xiao et al. reported on the cytoplasmic accumulation of HMGB1 in cochlear hair cells, which mediated noise-induced cochlear damage.26

While the crucial role of HMGB1 in the cochlea was confirmed, its exact function of HMGB1 in the mammalian auditory systems remained ambiguous. In the present study, we conducted further investigations into the immunolocalization of HMGB1 in the mouse cochlea from embryonic day (E)18.5 to postnatal day (P) 28, utilizing double immunofluorescence histochemistry. Before the onset of hearing (approximately P14),27 our findings revealed co-localization of HMGB1 with Sox2 in the supporting cells of the greater epithelial ridge (GER), a temporary structure during cochlea development.28 As the hearing commenced, both HMGB1 and Sox2 expression ceased in the GER, aligning spatially and temporally with the degeneration of the GER into the inner sulcus cells by the second postnatal week. Furthermore, we observed specific and stable expression of HMGB1 in the tympanic border cells, located on the lower side of the basilar membrane, gradually transforming from a three- to four-cell layered structure at postnatal day 1 (P1) into a one-cell layer structure in adulthood. 29 Notably, this study first reported that HMGB1 co-localizes with proliferation markers Ki-67 in several regions of the late embryonic and early postnatal mouse cochlea, including the GER, tympanic border cells, cochlear lateral wall, and auditory nerves, where cochlear stem cells were identified. We confirmed the presence of nerve cells expressing Ki-67 within the osseous spiral lamina and spiral ganglion through dual-immunostaining for Ki-67 with the glial marker Sox10.30 Overall, these results suggest that HMGB1 might have a vital role in cochlear development.

The husbandry and management of animals were approved by the Animal Use and Care Committee of Southeast University (approval No. 20200402025). All animal procedures conform to international bioethical guidelines. After mating, gestational age was counted as embryonic day 1 (E1) when a vaginal plug was detected. Individual embryos were staged according to standard mouse development.

BALB/c mice aged from E18.5 P28 were used in the present study. Pregnant (gestational day 18.5) and P1-P28 BALB/c mice were anesthetized using 10% chloral hydrate (0.2 mL/100 g mouse weight). Then, postnatal mice were intracardially perfused using normal saline and ice-cold 4% paraformaldehyde in 0.1M phosphate buffer (PB; pH 7.4). Embryonic mice were immediately decapitated, and the cochleae were dissected. The detailed methods and procedures of immunofluorescence staining were described in previous publications.31,32 Briefly, the cochleae were flushed with the fixative solution through the oval window and then fixed in 4% paraformaldehyde for 35 min at room temperature. The cochlea of mice older than P5 was decalcified in 10% EDTA at 4°C. In our protocol, P14 mouse cochlea was decalcified in 10% EDTA solution at 4°C for 6 h. The cochleae were washed with 0.01M phosphate-buffered saline (PBS; pH 7.4) for 5 min. The cochleae were processed in 15% sucrose for 3 h and then 30% sucrose for 12 h. After air-drying for 3 min, the cochleae were mounted in an optimum cutting temperature compound (OCT) at 4°C 3 h, and frozen at -80°C.

Cochlear tissue was cryosectioned at 6 μm thickness using a Leica (Wetzlar, Germany) cryostat microtome. The sections were incubated with 0.01M PBS containing 10% donkey serum and 0.3% Triton X-100 for 35 min at room temperature. Next, sections were incubated with primary antibodies in 0.01M PBS overnight or longer at 4°C. Primary antibodies used were as follows: rabbit anti-HMGB1 polyclonal antibody (1:200, Abcam, Cambridge, MA, USA), goat anti-Sox2 polyclonal antibody (1:200, Santa Cruz Biochemicals, Dallas, TX, USA), rat anti-Ki-67 monoclonal antibody (1:100; Invitrogen, Waltham, MA, USA), biotinylatedisolectin B4 antibody (1:250, Vector Laboratories, Newark, CA, USA), mouse anti-TUJ1 monoclonal antibody (1:200, Millipore, Burlington, MA, USA), rabbit anti-Sox10 monoclonal antibody (1:50, Abcam). After rinsing with 0.01M PBS three times for 15 min, the cryosections were incubated for 1 h at room temperature with the corresponding secondary antibodies: Alexa fluor 555 donkey anti-rabbit IgG (1:250, Yeasen Biotechnology, Shanghai, China), Alexa fluor 488 donkey anti-rabbit IgG (1:250, Beyotime, Haimen, China), Alexa fluor 488 donkey anti-goat IgG (1:250, Beyotime), Alexa fluor 488 donkey anti-rat IgG (1:250, Beyotime), Streptavidin conjugated with Alexa fluor 488 (1:250, Yeasen), Coralite488-conjugated Phalloidin antibody (1:250, Proteintech, Wuhan, China). Rabbit IgG control polyclonal antibody (Proteintech) and omission of the primary antibodies assessing non-specific secondary antibody binding was used as a negative control. After washing with 0.01M PBS three times for 5 min, sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; 1:600, Biyuntian, Beijing, China) for 5 min to visualize cell nuclei. Cryostat sections were observed and photographed with a LeicaSP8 laser scanning confocal microscope with 63X (NA=1.4) oil-immersion objectives or a Zeiss (LSW900) laser scanning confocal microscope with 20X (NA=0.8), 40X (NA=0.95) and 63X (NA=1.4) oil-objectives at 1024 by 1024 pixels, LAS AF Version 3.2.1.9702 acquisition software and Zen3.0 acquisition software was used. Immunostaining presented in the figures was representative of three individual experiments. Images were cropped and resized using Adobe Photoshop CC 2019.

This study employed double-labeling immunofluorescence to examine the distribution and expression of HMGB1 on cryosections of the mouse cochlea at various developmental stages. In the P1 auditory epithelium, immunofluorescence staining for HMGB1 was observed in the nucleus, and nuclear labeling for HMGB1 in the GER was identified by double-labeling with supporting-cell marker Sox2. Almost a complete co-localization of HMGB1 and Sox2 was observed. HMGB1 also co-expressed with Sox2 in the Deiters’ cells and pillar cells of the organ of Corti. Positive expression of HMGB1 was observed in three-to-four-cell layered tympanic border cells located on the lower side of the basilar membrane (Figure 1 A-C). At P8, co-localization of HMGB1 and Sox2 continued to be detected in the GER and supporting cells of the organ of Corti. HMGB1 immunostaining was maintained in the inner and outer hair cells (IHCs and OHCs), and tympanic border cells (Figure 1 D-F). At P14, the organ of Corti was mature, the expression of HMGB1 and Sox2 disappeared from the GER that degenerated into the inner sulcus cells, and HMGB1 immunolabeling retained its expression in the tympanic border cells and the organ of Corti (Figure 1 G-I). In the adult (P28) mouse cochlea, the general expression pattern of HMGB1 was similar to that seen in P14. HMGB1 continued to be expressed in a one-cell layer of tympanic border cells and the organ of Corti (Figure 1 J-L). The location of HMGB1-positive cells in the IHCs and OHCs was demonstrated by double staining with phalloidin, a specific marker for Factin (Figure 2 A-F). Co-immunostaining with HMGB1 and isolectin B4 (IB4), a specific vascular endothelial marker,33 showed nuclear expression of HMGB1 around and inside IB4-positive blood vessels within the cochlear lateral wall, and HMGB1- labelled nuclei did not colocalize with isolectin B4 (IB4) (Figure 2 G-L).

Ki-67, a nuclear protein marker for proliferating cells, is widely used to identify cells acting as stem cells in developing and adult tissues.34,35 In the mammalian inner ear, Ki-67 is a reliable marker of proliferation.36-38 Several developmental inner ear genes were identified by co-labeling with Ki-67.39,40 In the current study, many Ki-67-positive proliferating cell nuclei were co-expressed with HMGB1-positive nuclei at later embryonic stages (Figure 3 A-C). Consistent with previous reports of expression of Ki-67 in the GER and tympanic border cells at embryonic developmental stages,39,41,42 our results showed that Ki-67 immunostaining was limited to the apical surface of the GER in the apical turn of E18.5; almost all Ki-67-positive GER cells were co-localized with HMGB1-positive GER cells (Figure 3D), whereas no Ki-67 immunostaining was observed in the GER in the middle turns of the E18.5 cochlea (Figure 3G). Ki-67 and HMGB1 co-localized in the Reissner’s membrane, cochlear lateral wall, spiral limbus and cochlear nerve cells within the spiral ganglion region (Figure 3 EF, I-K). We also found that Ki-67-positive cells among the tympanic border cells were co-localized with HMGB1-positive cells. It is worth noting that HMGB1 and Ki-67 were co-localized in the tympanic border cells below the hair cells where the cochlear spiral modiolar artery exists (Figure 3H). No immunofluorescence labeling of HMGB1 and Ki-67 was detected in any parts of the E18.5 cochlea in negative controls omitting the primary antibody (Figure 3L). Also, no antibody binding was observed in negative controls stained with normal rabbit IgG (data not shown). At P1, the distribution of Ki-67 in the GER also differed between the apical, middle, and basal cochlear turns. Only at the apical turn of P1, immunostaining for Ki-67 was detectable in the GER (Figure 4 AC). HMGB1-Ki-67 double-positive cells were abundantly present in the cochlear modiolus (Figure 4 D-F). Several tympanic border cells surrounding the cochlear spiral modiolar artery and located below the outer sulcus cells showed immunostaining for Ki-67 and HMGB1 (Figure 4 G-I), but Ki-67 expression was largely absent from the spiral limbus at P1 (Figure 4J). HMGB1-Ki-67 doublepositive cells were also seen in the stria vascularis, the spiral ligament and the Reissner’s membrane (Figure 4K). In the basal turn of the P1 mouse cochlea, HMGB1-Ki-67 double-positive cells could be detected in the tympanic border cells (Figure 4L). Previous studies suggested that the expression of Ki-67 is only detected in spiral ganglion neurons at E16 and E20 in rat.43 In addition, we have previously shown that most Sox2-positive small and spindle-shaped glial cells were co-labeled with HMGB1 at this stage.22 To investigate this further, dual immunostaining was performed using spiral ganglion neuronal markers for TUJ1 and either HMGB1 or Ki-67. Nuclear expression of HMGB1 in large and spherical spiral ganglion neuron (SGN) was identified by co-staining of TUJ1 with HMGB1, and spindle-shaped glial cells wrapping SGNs were immunolabeled for HMGB1 (Figure 5 A-C). At the same time, Ki-67 immunostaining was seen only in the nuclei of the non-neuronal (TUJ1-negative) cells in the spiral ganglion (Figure 5 D-F). Cochlear nerve cells within the spiral ganglion of the P1 Rosenthal canals were double-labeled with HMGB1 and Ki-67 (Figure 5 G-I). Given that the Sox10 transcription factor is a marker for glial cells in the neonatal and postnatal cochlea and other regions of the nervous system,44-46 which regulates Wnt/β- catenin signaling in diverse developmental processes in normal tissues, dual-immunostaining for Sox10 and Ki-67 was performed to determine further the identification of the nerve cells expressing Ki-67 in the spiral ganglion of the Rosenthal canals. Consistent with previous observations, Sox10-positive nuclei were detectable in the stria vascularis, mainly in the strial marginal cells. Reissner’s membrane, the interdental cells and supporting cells were also positive for Sox1047 (Figure 6 A-C). Interestingly, Ki-67 was co-localized with Sox10 in the GER (Figure 6 D-F). Additionally, to the best of our knowledge, this is the first time that many Sox10-positive neonatal cochlear glial cells within the osseous spiral lamina and the spiral ganglion were found to colabel with Ki-67 (Figure 6 G-L). At P3, no Ki-67-positive cells were seen in the GER throughout the cochlear duct. Ki-67 continued to be expressed in the tympanic border cells, cochlear lateral wall and nerve cells within the spiral ganglion region, and colocalized with HMGB1 (Figure 7 A-F). From P5 onwards, Ki-67-positive cells were scarcely detectable in the Rosenthal canals. The distribution of HMGB1-Ki-67 double-positive cells was constrained to the tympanic border cells and cochlear lateral wall (Figure 7 GI). As previously reported, at P8, the distribution of Ki-67-positive proliferating cells changed with the advancement of development,48 and only a few Ki-67-HMGB1 double-labeled cells were scattered in the cochlear lateral wall and the osseous spiral lamina, but there is no significant colocalization between Ki-67 and HMGB1 in the Rosenthal canals and tympanic border cells at this time point (Figure 7 J-L).

In this study, we investigated the expression and distribution of HMGB1 in the mouse organ of Corti by dual-staining for HMGB1 and Sox2. We observed age-related changes in HMGB1 expression in the GER, a crucial cell population essential for developing the auditory sensory epithelium. This suggests that HMGB1 expression may be associated with cochlear development. As the failure of the GER cell proliferation leads to hypoplasia of the organ of Corti,49 the location of stem cells has been identified in the embryonic and neonatal mouse cochlea using the proliferation marker Ki-67.41-44 Therefore, we performed dual-immunostaining for HMGB1 and Ki-67 on cryosections of the mouse cochlea to further examine the potential roles of HMGB1 in cochlear development. Our results demonstrated that HMGB1 co-express with Ki- 67 in several cochlear regions at later embryonic and early postnatal stages, including the GER, tympanic border cells, cochlear lateral wall and cochlear nerve cells. These findings suggest that HMGB1 might participate in cell proliferation during cochlear development, which aligns with previous studies showing the essential role of HMGB1 in cell proliferation of various cell types.50-52 Knockdown of HMGB1 can significantly reduce the Ki-67 expression and inhibit the proliferative activities.53 Previous studies reported that HMGB1 affects cell proliferation via modulation of multiple signaling pathways such as NF-κB, PI3K/Akt, JNK and RAGE/TLR4.54-56 Given the expression of the RAGE, one of the main receptors for HMGB1 that contribute to cochlear maturation, 57 we hypothesized that HMGB1 could participate in cochlear development via signaling to RAGE. To the best of our knowledge, this is the first study reporting the expression of HMGB1 in the tympanic border cells and its co-localization with Ki-67. Tympanic border cells are Wnt-responsive and act as progenitors for postnatal mouse cochlear cells.42 These cells express Ki-67 and exhibit active proliferation until the second week of postnatal development. Our data showed that HMGB1-Ki-67 double-positive tympanic border cells were located near the cochlear spiral modiolar artery, which is consistent with the specific localization of the slow-cycling cells within the tympanic border zone near the vascular structure. Previous studies reported that acoustic or druginduced injury to the mammalian and chick cochlea induces the proliferation of tympanic border cells,58,59 thus, we assumed that changes in HMGB1 expression in the tympanic border cells following cochlear damage may correlate with cochlear pathology, as demonstrated in the spiral limbus and spiral ligament.

Additionally, we found the presence of HMGB1 in Reissner’s membrane and the co-localization of HMGB1 and Ki-67 in Reissner’s membrane at E18.5 and P1. Several proteins contributing to cochlear development, such as LaminB1 and Pax2,60,61 are expressed similarly in Reissner’s membrane, which acts as a barrier between endolymph and perilymph, maintaining their unique fluid compositions.62,63 The cell nucleus of Reissner’s membrane is resistant to degeneration resulting from acute atoxyl intoxication and is the last cell component to disintegrate.64 The number of cells and cellular proliferation in Reissner’s membrane has also been implicated in endolymphatic hydrops.65,66 Therefore, the expression of HMGB1 in the Reissner’s membrane observed in this study may be essential in cochlear function.

In our previous study, we found that HMGB1 was mainly expressed in the nuclei of the SGNs and Sox2-positive glial cells in the spiral ganglion regions at various developmental stages. Moreover, upregulation of HMGB1 expression in glial cells of the spiral ganglion regions was linked to cochlear pathogenesis after acoustic trauma.24 Similar changes in HMGB1 expression were also observed in the SGNs of amikacin-treated rats.67 Previous studies have shown that HMGB1 can promote neural stem cell proliferation.68,69 In this study, we further extended our previous investigation by demonstrating the co-localization of HMGB1 and Ki-67 in the non-neuronal cells within the spiral ganglion regions during prenatal and early postnatal stages. Additionally, Ki-67 was co-expressed with Sox10 in cochlear neonatal glial cells. Sox10 is known to be essential for the determination, differentiation and maintenance of peripheral glial cells,70 suggesting the potential involvement of HMGB1 in cochlear nerve cell proliferation during cochlear development.

In conclusion, we identified a specific expression pattern of HMGB1 in the tympanic border cells throughout the developmental period. Using double immunofluorescence histochemistry, we revealed that the changes in HMGB1 and Sox2 expression in the GER correlate with the morphological degeneration of the GER during postnatal development. Additionally, we found that Ki-67- positive proliferating cells co-localize with HMGB1 during late embryonic and early postnatal cochlear development. These findings offer some insights into the potential physiological functions of HMGB1 in the cochlea.