A Novel Central-Peripheral Interface: The Auditory Nerve Glial Transition Zone Exhibits Enhanced Age-Related Immune and Glial Cell Dysfunction

auditory nerve degeneration, glial transition zone, age-related hearing loss, 15 demyelination, cochlear macrophage, microglia, aging 16 *Correspondence: 17 Shelby Payne 18 [email protected] 19 Hainan Lang 20 [email protected] 21

22 Age-related hearing loss (ARHL) is a rapidly growing public health concern, affecting two-thirds 23 of adults over 65 years old, with no effective therapeutics available. As the aging population 24 grows at an unprecedented rate, the burden of ARHL will only increase. The causes of ARHL are 25 multifactorial, but an understudied major contributor is glial dysfunction. The auditory nerve 26 (AN) conducts sound from the cochlea to the brainstem and holds a diverse population of 27 immune cells and myelinating glia. As the AN fibers bundle together within the cochlea to 28 project to the brainstem, they are first myelinated by Schwann cells in the peripheral AN, then 29 myelinated by oligodendrocytes in the central AN. The region where myelination shifts from 30 Schwann cells to oligodendrocytes is the glial transition zone (GTZ), located in the cochlear 31 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 2 modiolus, creating a unique biological niche. While central-peripheral interfaces are recognized 32 in other cranial nerves, the AN GTZ is understudied. This region integrates the peripheral and 33 central microenvironments within the confined bony cochlea, positioning it as a niche for glial 34 dysfunction in pathological conditions, such as aging. We hypothesize that the GTZ is a site of 35 enhanced glial dysfunction contributing to age-related AN demyelination, an important 36 contributor to ARHL. We evaluated this in an ARHL mouse model combining RNA-sequencing, 37 quantitative immunohistochemistry, and 3D high-resolution imaging. We examined the AN GTZ 38 from human temporal bone donors. RNA-sequencing of the AN revealed age-associated 39 increases in abnormal myelination/glial function and inflammation. There was a significant age-40 dependent increase in Iba1+ macrophages/microglia, with accumulation at the AN GTZ, and an 41 increase in cellular volume and surface area, suggesting greater age-related activation. 42 Macrophages/microglia contained significantly more internalized myelin debris in the AN 43 (peripheral, central, and GTZ) with aging. More importantly, we found structurally intact myelin 44 within macrophages/microglia only at the GTZ, suggesting a unique microenvironment at the 45 GTZ altering phagocytic activity in aging. Together, our data suggest that the GTZ, a previously 46 unrecognized central-peripheral interface, is a critical site of immune-glial interactions and 47 especially vulnerable to age-related demyelination and neuroinflammation. This study highlights 48 the GTZ as a potential target for preserving AN myelination and mitigating ARHL. 49 1. Introduction 50 Two-thirds of adults over the age of 65 years old are affected by age-related hearing loss 51 (ARHL) (National Center for Health Statistics (U.S.) et al., 2021). Although there are numerous 52 contributors to the pathology of ARHL, glial dysfunction remains understudied. The auditory 53 nerve (AN), which is one branch of cranial nerve VIII, conducts sound from the cochlea to the 54 brainstem (Bordoni, Mankowski and Daly, 2025). This nerve also contains a diverse population 55 of immune cells and myelinating glia (Hu, Zhang and Frye, 2018). The peripheral AN is 56 myelinated by Schwann cells, while the central AN is myelinated by oligodendrocytes (Bercury 57 and Macklin, 2015; Long et al., 2018). The region where myelination shifts from Schwann cells 58 to oligodendrocytes is called the glial transition zone (GTZ) (i.e. Obersteiner-Redlich zone) and 59 creates a unique biological niche due to the close proximity of both peripheral and central glia 60 (Fontenas, 2023). While such central-peripheral interfaces have been defined in other cranial 61 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 3 nerves and the spinal cord, the GTZ is understudied in the auditory system (Fraher, 1992; 62 Knipper et al., 1998; Osen, Furness and Hackney, 2011; Bojrab et al., 2017; Li et al., 2022). The 63 GTZ of the AN is a one-of-a-kind region of glial diversity because it integrates the peripheral and 64 central microenvironments within the bony confines of the cochlea, which is housed in the 65 petrous portion of the temporal bone, the hardest bone in the human body. This makes the GTZ 66 of the AN a niche for concentrated glial dysfunction in pathological conditions, such as aging, 67 which is the focus of this study. 68 Previous studies in the spinal cord suggest that such central-peripheral transition zones serve as 69 important immune interfaces that regulate myelin turnover during development and after injury 70 (Borjini et al., 2019; Wu et al., 2022; Fontenas, 2023; Xin et al., 2025). Within the brain, both 71 resident microglia and peripheral macrophages play important roles in myelination during 72 development and myelin maintenance, consistent with the dynamic nature of myelination 73 throughout an individual’s lifetime (Chapman and Hill, 2020; Santos and Fields, 2021; Kent and 74 Miron, 2023; Gao et al., 2024). However, in the aging nervous system, these processes become 75 dysregulated, leading to myelin breakdown, chronic inflammation, and axonal degeneration 76 (Beirowski et al., 2014; Raj et al., 2017; Watson et al., 2017; Borucki et al., 2020; Beirowski, 77 2022; Seicol, Lin and Xie, 2022; Huang et al., 2025). Myelin is critical to AN function because it 78 preserves temporal synchrony required for speech perception (Long et al., 2018; Harris et al., 79 2021, 2022; Kister and Kister, 2023). However, the effects of aging on the immune and glial cell 80 populations at the AN GTZ housed within the cochlea have not been defined. 81 Understanding the dynamics of the AN immune cell compartment and associated changes to 82 myelination within the aging AN is critical for determining the mechanisms of ARHL and other 83 cochlear pathologies. Although prior studies have described macrophages in the cochlea during 84 development, aging, and acute noise injury (Fischer et al., 2020; Noble et al., 2022; Seicol, Lin 85 and Xie, 2022; Song et al., 2022; Lang et al., 2023), the effects of aging on the interactions of 86 AN immune cells and myelinating glia remain largely unexplored. Furthermore, the extent to 87 which these age-related changes differ between the central AN, the peripheral AN, and the GTZ 88 has not been addressed in mouse models or in human temporal bones. 89 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 4 The goal of this study is to better understand aging effects on AN myelination and immune-glial 90 interactions, specifically focusing on age-related differences within the peripheral AN, central 91 AN, and GTZ, a unique niche of glial-immune interactions in the context of age-related AN 92 degeneration. Utilizing RNA-sequencing, quantitative immunohistochemistry, and 3D high-93 resolution imaging in an established ARHL mouse model, we examined aging effects on the AN 94 GTZ, macrophage/microglia populations, myelination, glia-immune interactions, and phagocytic 95 activity within the central AN, the peripheral AN, and the AN GTZ. We also assessed the AN 96 GTZ from human temporal bone donors. 97 2. Materials and Methods 98 2.1 Animals 99 All studies were performed in accordance with the guidelines of the Institutional Animal Care 100 and Use Committee of the Medical University of South Carolina (MUSC) and the Ralph H. 101 Johnson V A Health Care System (V AHCS) in Charleston, South Carolina. Breeding pairs for 102 CBA/CaJ mice were originally purchased from The Jackson Laboratory (JAX#000654) and bred 103 at the MUSC Animal Research Facility. Mice were bred and housed in a low-noise vivarium with 104 a 12/12-hour light/dark cycle and given standard lab chow and water ad libitum. Young adult (3-105 4 months) and aged (>2.5 years) CBA/CaJ mice (of both sexes) were used in this study. The 106 number of mice per group is reported in the figure legends. Mice with signs of external ear canal 107 obstruction, middle ear obstruction, or infection were excluded. 108 2.2 Assessment of auditory function 109 All mice used in this study underwent auditory brainstem recordings (ABRs) as previously 110 described (Brown et al., 2017; Panganiban et al., 2022; Fabrizio-Stover et al., 2025). Mice were 111 anesthetized with an intraperitoneal injection (10 mg/kg xylazine and 100 mg/kg ketamine). 112 ABR recordings were performed in an acoustically isolated booth (IAC Acoustics), and mice 113 were placed on a 37°C heating pad, with artificial tear ointment on both eyes. Subdermal needle 114 electrodes (F-EZ-24, Genuine Grass Reusable Subdermal Needle Electrodes) were placed on the 115 vertex (recording), ipsilateral mastoid (reference), and hind limb (ground). ABRs were digitized 116 at 15 kHz using a low-impedance head stage connected to a pre-amplifier (RA4LI/RA4PA, 117 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 5 Tucker Davis Technologies). Electrode impedance was checked at the start of each session to 118 ensure it did not surpass 3 kΩ. The pre-amplifier was connected to an RZ6 input/output device 119 (Tucker Davis Technologies) and responses were recorded with BioSigRZ software (Tucker 120 Davis Technologies). Audiometric thresholds were evaluated with 1.1 ms tone pips with 0.55 ms 121 cosine rise-fall time presented from 90 dB SPL to 5 dB SPL using 5 dB steps. Stimuli were 122 presented in a closed-field through an MF1 speaker (Tucker Davis Technologies) coupled to a 3 123 mm diameter plastic tube and earpiece inserted into the ear canal. Presentation rate of stimuli 124 was 21 Hz with an average of 512 repetitions. ABR Wave I thresholds at 11.3 kHz were 125 determined via the lowest stimulus level with an identifiable and reproducible Wave I 126 (Supplementary Figure 1). 127 128 2.3 Mouse cochlear tissue collection and preparation 129 Mice were euthanized and transcardial perfusion was performed with 4% paraformaldehyde 130 (Electron Microscopy Sciences) solution in 1x phosphate buffered saline, pH 7.4 (1xPBS). 131 Temporal bones were then collected and immediately bath-fixed at 4 °C with 4% 132 paraformaldehyde solution in 1xPBS and perfused via the round and oval windows. Temporal 133 bones were kept in fixative for 24 hours at 4 °C. Fixed cochleae were then decalcified in 10% 134 Ethylenediamine tetraacetic acid (EDTA) for 1-3 days depending on age of the mouse. Each 135 cochlea was then cryoprotected in a 15%/20%/30% sucrose gradient over 2 days. After 136 cryoprotection, cochleae were embedded in Tissue-Tek OCT compound and sectioned at a 137 thickness of 30 μm, then stored at -20°C until needed for staining. The sectioning plane of mouse 138 temporal bones was oriented such that the cutting face was parallel to the AN within the cochlear 139 modiolus to ensure that sections were mid-modiolar to allow optimal viewing of the AN GTZ. 140 2.4 Human cochlear tissue collection and preparation 141 Procedures for the collection and preparation of human temporal bones have been previously 142 reported (Cunningham et al., 2001; Xing et al., 2012; Noble et al., 2019). All specimens were 143 obtained from the MUSC Hearing Research Program’s temporal bone archive and the MUSC 144 Carroll A. Campbell, Jr. Neuropathology Laboratory Brain Bank. In all cases of human temporal 145 bone collection, written informed consent was obtained from the next-of-kin in accordance with 146 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 6 South Carolina laws and regulations. Temporal bone research was approved by the MUSC 147 Institutional Review Board as not human subject research (Pro0030845). No hearing history is 148 available for the human temporal bone (89+ year old female donor) used in this study. After 149 removal of the temporal bone from the skull, scalar perfusion was performed with a 4% solution 150 of paraformaldehyde. Fixation was continued by immersion for at least 48 hours at 4°C. Each 151 temporal bone was then rinsed with 1x PBS and decalcified in 0.35 M EDTA, pH 8.0 for a period 152 of 4-6 weeks as described previously (Cunningham et al., 2000). During this decalcification 153 period, each specimen was trimmed to remove the hard bone encasing the cochlea and vestibule 154 of the inner ear. Once fully decalcified and trimmed, the inner ear portions of the temporal bone 155 were processed for frozen sectioning at a thickness of 12 μm. 156 2.5 Immunohistochemistry on mouse and human cochlear sections 157 Cochlear sections were air dried with a fan for 30 minutes, then submerged in -20°C acetone for 158 5 minutes followed by -20°C methanol for 10 minutes. Sections were permeabilized and blocked 159 in donkey serum buffer with Triton X-100 (16% donkey serum, 0.3% Triton X-100, 450 mM 160 NaCl, 20 mM Phosphate Buffer) at room temperature for 3 hours. Primary antibodies were 161 prepared in donkey serum buffer with Triton X-100 and added to the sections, then incubated 162 overnight at 4°C. The next day, sections were washed with wash buffer with Triton X-100 (0.3% 163 Triton X-100, 450 mM NaCl, 20 mM Phosphate Buffer) and the appropriate biotinylated 164 secondary antibodies conjugated with fluorescent avidin (Vector Labs) were added as previously 165 described (Lang et al., 2011). For myelin staining with FluoroMyelinTM (ThermoFisher 166 Scientific), slides were washed and blocked, then stained overnight at 4°C, based on the protocol 167 recommended by the manufacturer. The following day slides were washed using wash buffer 168 with Triton X-100, stained for nuclei (Hoechst), then washed again before being mounted with 169 Vectashield (Vector Laboratories). The primary antibodies, secondary antibodies, and stains used 170 for immunohistochemistry are listed in Table 1. 171 2.6 Confocal imaging with Airyscan and post-acquisition analysis 172 Slice and confocal image stacks were collected using a Zeiss LSM 880 NLO with Airyscan using 173 ZEN acquisition software (Zeiss United States). For human cochlear section preparations, 174 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 7 images were taken at a resolution of 0.52 µm x 0.52 µm to evaluate the AN and AN GTZ. 175 Imaging was performed at a resolution of 0.16 µm x 0.16 µm to analyze macrophage/microglia 176 morphology and myelination in human temporal bone sections. For mouse cochlear section 177 preparations, images were taken at a resolution of 0.83 µm x 0.83 µm for evaluating the AN and 178 AN GTZ. For analysis of macrophage/microglia morphology and myelination (including 179 assessment of internalized myelin), image stacks were taken at a resolution of 0.13 µm x 0.13 180 µm with a z-step of 0.60 µm. Images were processed using ZEN 3.9 Blue Edition (Carl Zeiss 181 Microscopy GmbH). 182 The Imaris volume rendering function (Imaris 10.2, Oxford Instruments) was used for 3D 183 reconstructions and analysis of macrophage/microglia morphology, myelination, and 184 identification and quantification of internalized myelin within AN macrophages/microglia. 185 Immunofluorescence image stacks from 30 µm sections in CBA/CaJ mice of both age groups 186 were imported into Imaris 10.2. For the 3D reconstruction of Iba1+ cells, surfaces were created 187 using the Machine Learning Segmentation Tool (Imaris 10.2, Oxford Instruments). Training was 188 performed on each image using 3-5 repetitions as needed, based on complexity of 189 macrophage/microglia morphology. Any touching objects were split. Surfaces were hand-filtered 190 to remove any objects that were not part of the Iba1+ macrophage/microglia mask. All surface 191 parameters were exported into a separate .csv file for each image stack. 192 Myelination was quantified in the same manner, using the Machine Learning Segmentation Tool 193 (Imaris 10.2, Oxford Instruments), which generated surfaces of each myelinated fiber in the 194 section and output the surface area and volume of the myelinated fibers contained within each 195 region of interest (ROI). For the 3D reconstruction of myelinated fibers in the AN to evaluate 196 surface area and volume, the same procedure was used as described above for generating 197 surfaces of Iba1+ macrophages/microglia. After the surfaces of the myelinated fibers were 198 created for each image, the statistics were exported, including the total volume and surface area 199 of each surface. All surface parameters were exported into a separate .csv file for each image 200 stack. 201 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 8 The quantity of internalized myelin was determined based on the volume of the myelin surface 202 contained within the Iba1+ surface to ensure only fully enclosed myelin components were 203 included in the analysis. For quantification of total internalized myelin within Iba1+ 204 macrophages/microglia, Object-Object Statistics were performed in Imaris (Imaris 10.2, Oxford 205 Instruments). For each individual image, the Iba1+ macrophage/microglia surface was used to 206 create a mask against the myelinated fiber surface generated for each stack. Then, a surface was 207 created that contained only myelin-stained objects within the Iba1+ macrophage/microglia 208 surface. The statistics for the internalized myelin surface were then exported to determine total 209 volume of internalized myelin within the Iba1+ macrophages/microglia for each image stack. 210 2.7 Epoxy resin embedding preparation of the auditory nerve 211 CBA/CaJ mice were processed and imaged via electron microscopy as previously described 212 (Lang et al., 2011). Briefly, mice (CBA/CaJ 1 year old) were perfused via cardiac perfusion with 213 10 mL normal saline with 0.1% sodium nitrate, then 15 mL of 4% paraformaldehyde and 2% 214 glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Cochleae were decalcified in 50 mL of 120 215 mM solution of ethylenediaminetetraacetic acid (EDTA), pH 7.2, with gentle stirring at room 216 temperature for 2-3 days with daily changes of EDTA solution. Tissues were post-fixed in 1% 217 osmium tetroxide for 1 hour, dehydrated, and embedded in Epon LX 112 resin. Semi-thin 218 sections were cut at approximately 1 µm thick and stained with Toluidine blue. 219 2.8 Light sheet microscopy and three-dimensional high-resolution imaging of the auditory 220 nerve glial transition zone 221 Young and aged mouse temporal bones were tissue-cleared and imaged with large-field light 222 sheet microscopy to visualize the AN GTZ. Tissue clearing was performed using the MACS 223 Clearing Kit (Miltenyi Biotec) and their standard protocol with the addition of a decalcification 224 step (10% EDTA for 1 day) before permeabilization. Mouse temporal bones were stained with 225 CD68 (Miltenyi Biotec) and Myelin Basic Protein (Miltenyi Biotec). Detailed descriptions of 226 antibodies used are listed in Table 1. Cleared whole mouse temporal bones were mounted and 227 imaged with an Ultramicroscopy II Scope (Miltenyi Biotec) with a Super Plan configuration, 228 equipped with a sC-MOS camera (4.2 Megapixel, Andor Technology), and objective lenses with 229 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 9 dipping caps specifically designed and optimized for large-field light sheet imaging as previously 230 described (Chen et al., 2025). All 3D image stacks were acquired using a 561 nm laser with a 231 595/40 nm emission filter to capture CD68 fluorescence, and a 639 nm laser with a 680/30 nm 232 emission filter to capture Myelin Basic Protein fluorescence. Each mouse temporal bone was 233 mounted on the sample holder and oriented such that volume was acquired with the z-steps 234 moving from apex to base through the AN to ensure the capture of the full AN GTZ. The entire 235 AN GTZ was imaged using a 12x objective at 0.6x magnification with a resolution of 0.903 x 236 0.903 (X,Y) with a 1 µm z-step interval. The number of CD68+ cells was quantified every 25 µm 237 within the AN for each temporal bone in young and aged CBA/CaJ mice. 238 2.9 RNA-sequencing analysis of the auditory nerves in young and aged mice 239 To examine expression of genes relating to myelination, glial cell function, and abnormal 240 inflammation in young and aged mouse AN, analysis was performed on bulk RNA-sequencing 241 data obtained from a prior study involving young adult (3 months) and aged (>2.5 years) ANs 242 from CBA/CaJ mice (accession GSE141865) (Panganiban et al., 2022). AN tissues in the 243 previous study were isolated by microdissection of the modiolus from the other structures of the 244 cochlear bulla (such as the cochlear lateral wall and sensory epithelium), with the tissue collected 245 from the two cochleae of each mouse pooled to make each biological replicate; three biological 246 replicates of each age group were used. Raw sequencing data (fastq files) were analyzed using 247 Partek Flow software (Illumina Inc). Reads were aligned to the mouse genome assembly mm39 248 by STAR (version 2.7.8a) (Dobin et al., 2013) and quantified to annotation model (Partek E/M) 249 built from mm39 Ensembl Transcripts release 104 using default parameter settings (Strict paired-250 end compatibility=true; Require junction reads to match introns=true; Minimum read overlap 251 with feature=100%). Normalization and comparative analysis was done with DESeq2 (Love, 252 Huber and Anders, 2014). Normalized count data is archived in NCBI Gene Expression Omibus 253 (accession GSE320402). Differential gene expression was defined as adjusted p-value (FDR step 254 up) 2, yielding 1240 genes (Supplementary Table 1). 255 Biological process enrichment analysis was conducted with ToppGene (Chen et al., 2009). 256 2.10 Statistical analysis 257 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 10 Sample sizes for experiments and morphological observations are listed in the appropriate results 258 section and figure legends. Images presented here are representative of each experimental group 259 for fluorescence staining. All statistical values are presented as a mean ± standard deviation 260 unless stated otherwise. All data sets were tested for normality using a Kolmogorov-Smirnov test 261 with reference to a normal distribution. Mann-Whitney U-test was used for pairwise comparisons 262 between groups. Exact p-values are reported for all comparisons. For differential expression 263 analyses of RNA-sequencing data, a p-adjusted value (FDR step up) of 2 was considered significant. Graphs were plotted and statistics were performed in Igor 265 Pro 9 (Wavemetrics) and GraphPad Prism 8 (GraphPad Software). 266 3. Results 267 Previous studies on aging effects on neural degeneration of the auditory system have focused on 268 the peripheral AN fibers in the region where they project to the sensory epithelia, specifically on 269 the spiral ganglion and ribbon synapses within the inner hair cells (Sergeyenko et al., 2013; Wan 270 and Corfas, 2017; Long et al., 2018; Heeringa et al., 2020, 2020; Budak et al., 2021; Panganiban 271 et al., 2022). Other studies have focused on the central auditory system, including the cochlear 272 nucleus and auditory cortex (Cramer and Rubel, 2016; Milinkeviciute et al., 2019; Eggink et al., 273 2022; Seicol, Lin and Xie, 2022). Here, this study investigated aging effects on AN myelination 274 and inflammation, focusing on the GTZ which houses both peripheral and central immune cells 275 and myelinating glia, in an established ARHL mouse model and human temporal bone sections 276 from an older donor. 277 3.1 The glial transition zone of the mouse auditory nerve 278 To better evaluate the interactions between macrophages/microglia and the myelinating glia of 279 the AN GTZ, we used FluoroMyelinTM stain on 30 µm frozen sections of young and aged 280 CBA/CaJ mice (Figure 1A, D; see Table 1 for detailed description). Auditory function was 281 assessed for mice in this study, and aged mice had elevated hearing thresholds compared to 282 young mice (Supplementary Figure 1). Sections were also stained with the primary antibody Iba1 283 as well as a nuclear marker (Hoechst) to aid in the identification of the cell bodies of Iba1+ 284 macrophages/microglia. Three labeling approaches utilized in this study reveal the GTZ, identify 285 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 11 a gap in myelin-related structures between central and peripheral portions of the mouse AN 286 (indicated by the dashed white lines), and distinguish the peripheral AN from central AN (Figure 287 1; Supplementary Figure 2). These labeling approaches include 1) frozen sections stained with 288 FluoroMyelinTM stain (Figure 1A,D), 2) Epon LX 112 resin embedded sections stained with 289 Toluidine blue (Figure 1G, G’), and 3) frozen sections stained with NG2 antibody for Schwann 290 cells (peripheral AN) and CC1 and NG2 antibodies for oligodendrocytes (central AN) (Figure 291 1C, F; Supplementary Figure 2). FluoroMyelinTM staining revealed region-specific structural 292 differences between the central and peripheral AN. Central AN myelin has a different depth of 293 staining compared to peripheral AN myelin (Figure 1A, D) and appears to have a lower nuclei 294 density than that of the peripheral AN (Figure 1C, F). This observation of increased central 295 myelin staining is consistent with known compositional differences between central and 296 peripheral myelin. Previous studies report that myelin basic protein makes up approximately 297 30% of the protein mass of central myelin, while it only accounts for 5-18% of the protein mass 298 of peripheral myelin (Garbay et al., 2000; Kister and Kister, 2023). Like other cranial nerve 299 transition zones, the higher nuclear density seen in the peripheral AN compared to the central AN 300 reflects the differences in myelinating cell types across the GTZ. This is because the central AN 301 is myelinated by oligodendrocytes, which surround numerous AN fibers with one cell body, 302 while the peripheral AN is myelinated by Schwann cells, which surround only one individual FR. 303 Iba1 staining showed macrophages and microglia dispersed throughout the AN in young and 304 aged sections (Figure 1B, E). In these low-magnification mid-modiolar sections, observable 305 differences in the Iba1+ cell populations throughout the AN and at the GTZ are apparent, with the 306 aged sections having a greater number of Iba1+ cells throughout the AN and Iba1+ cells 307 appearing to accumulate at the GTZ (Figure 1B, E). 308 To examine the entire AN and the GTZ, a temporal bone from a young mouse and an aged 309 mouse were cleared and imaged via large-field light sheet microscopy (Power and Huisken, 310 2017; Chen et al., 2025). Temporal bones were immunostained with a fluorescently tagged 311 Myelin Basic Protein antibody, a reliable marker of AN myelin sheaths (Xing et al., 2012). 312 Examining young and aged mouse temporal bones shows that the GTZ spans the entirety of the 313 AN and resides within the cochlear modiolus in a cone-shaped morphology (Figure 2E’, P’). A 314 visible transition from peripheral AN to central AN is again evident based on the differences in 315 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 12 Myelin Basic Protein staining intensity throughout the AN in both the young and aged samples. 316 To identify phagocytic and active immune cell subpopulations, temporal bones were also stained 317 with a fluorescently tagged CD68 antibody (Hendrickx et al., 2017; Choudhary and Malek, 318 2023; Swanson et al., 2023). CD68+ cells were observed more frequently within the AN in the 319 aged temporal bones compared to the young temporal bones, mostly within the central AN as 320 well as accumulating near the AN GTZ (Figure 2T’, V’). 321 3.2 Aging auditory nerves have more immune cells, with focal accumulation at the glial 322 transition zone 323 Previous studies have reported age-related changes to cochlear macrophages/microglia within the 324 spiral ganglia and cochlea (Noble et al., 2019; Fischer et al., 2020). To assess the effects of aging 325 on AN macrophages/microglia at the AN GTZ, we performed quantitative 326 immunohistochemistry to evaluate Iba1+ cells by region of the AN. Immunostained sections of 327 the peripheral AN, GTZ, and central AN are shown in young (Figure 3A-F) and aged (Figure 3G-328 L) mice. 329 To evaluate the activation state of Iba1+ cells within the AN, we assessed their morphology, 330 including surface area and volume (Kopper et al., 2021; You et al., 2023). Generally, Iba1+ cells 331 in aged mice seem to have greater surface areas and volumes compared to young, however, there 332 is no significant difference between groups (Surface Area: A VGYoung = 6.90 ± 2.35 x 103 µm2, 333 A VGOld = 10.29 ± 2.82 x 103 µm2, p = 0.111; V olume: A VGYoung = 4.86 ± 1.71 x 103 µm3, A VGOld 334 = 9.71 ± 5.67 x 103 µm3, p =0.064) (Figure 3M, N). 335 To assess phagocytic activity of the Iba1+ cells that populate the AN, we stained for CD68, an 336 established marker of phagocytic activation, in the AN with aging (Hendrickx et al., 2017; 337 Choudhary and Malek, 2023; Swanson et al., 2023). Iba1 acts as a marker of cells of myeloid 338 lineage, such as macrophages and microglia, while CD68 is marker of phagocytic activity by 339 macrophages and microglia(Schwabenland et al., 2021). Using serial sections extracted from the 340 cleared mouse temporal bones, we quantified the number of CD68+ cells within the entire AN in 341 young mice (3 months) and aged mice (2.5 years) (Figure 2). The aged mice had a significantly 342 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 13 greater quantity of CD68+ cells compared to the young mice (A VGYoung = 3.00 ± 2.16, A VGold = 343 67.00 ± 11.05, p = 0.025) (Figure 3O). 344 Aged mice contained significantly more Iba1+ cells compared to young mice across all regions of 345 the AN (A VGYoung = 3.44 ± 0.18, A VGOld = 10.72 ± 1.91, p = 0.032) (Figure 3P). Of all three 346 regions of the AN, the GTZ had the greatest density of Iba1+ cells in aged mice compared to 347 young mice (Peripheral AN: A VGYoung = 3.50 ± 0.41, A VGOld = 7.93 ± 0.32, p = 3.67E-04; GTZ: 348 A VGYoung = 3.89 ± 0.16, A VGOld = 12.52 ± 2.90, p = 0.050; Central AN: A VGYoung = 1.67 ± 0.94, 349 A VGOld = 12.57 ± 2.90, p = 0.019) (Figure 3Q). Iba1+ cells are still present in young mice but 350 sparsely populate the AN in both the peripheral and central regions. Interestingly, Iba1+ cells in 351 the AN GTZ were also found with projections in the central and peripheral regions of the AN 352 (Figure 3B, E, H, K). 353 3.3 Aging is associated with disruption of auditory nerve myelin 354 Myelin staining revealed a difference in morphology between the peripheral AN and central AN 355 in young and aged mice (Figure 4). The peripheral AN had a homogenous myelin stain, while the 356 central AN had a more heterogeneous appearance in both young and aged mice. We observed an 357 increase in disrupted myelin sheaths throughout the peripheral and central AN regions with age 358 (Figure 4A, B, D, E), which confirms previous electron microscopy studies in the peripheral AN 359 (Xing et al., 2012; Panganiban et al., 2022). To quantify age-related changes in myelination in 360 the AN, we measured the volume and surface area of myelinated fibers in young (3 - 4 months) 361 and aged (≥ 2.5 years) CBA/CaJ mice using Imaris. Aged mice had no significant difference in 362 surface area of myelinated fibers compared to young mice (Figure 4C). Aged mice also had no 363 significant difference in volume of myelinated fibers compared to young mice (Figure 4F). 364 Although not significant, young mice have a larger average surface area and volume of 365 myelinated fibers in the AN compared to aged mice (Surface Area: A VGYoung = 183.75 ± 61.00 x 366 102 µm2, A VGOld = 133.74 ± 38.30 x 102 µm2, p = 0.342; V olume: A VGYoung = 290.74 ± 52.63 x 367 103 µm2, A VGOld = 226.50 ± 60.45 x 103 µm2, p = 0.200). 368 3.4 Aging effects on the auditory nerve transcriptome – abnormal immune cell and 369 myelinating glial function 370 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 14 To further validate our observations of age-related myelin degeneration and increased immune 371 cell activity within the AN and its enhanced effects at the AN GTZ, bulk RNA-sequencing data 372 obtained from a prior study of AN from young adult mice (3 months) and aged mice (2.5 years) 373 was analyzed anew, using updated reference databases. Samples in the study contained central 374 AN and peripheral AN within the cochlear modiolus and internal auditory meatus. Analysis of 375 the RNA-sequencing data revealed significant age-dependent transcriptional changes in 1240 376 genes (FDR 2) (Figure 5A). Functional enrichment analysis of 377 these genes detected significant effects on pathways relating to myelination and immune 378 activation, including abnormal myelination (q = 2.30 x 10-2) and abnormal inflammatory 379 response (q = 1.76 x 10-9) (Figure 5B, C). Significantly upregulated genes mapping to these 380 pathways included Clec7a, Trem2, and Itgb2 (abnormal inflammatory response). A complete list 381 of differentially expressed genes, and their association(s) with these pathways, is given in 382 Supplementary Table 1. Detection of an effect on abnormal myelination aligns with our observed 383 immunohistochemistry results in old AN. Together, these findings demonstrate that aging is 384 associated with dysregulation of myelin-associated processes and inflammatory signaling. 385 3.5 Accumulation of myelin debris found in aging auditory nerve macrophages/microglia 386 Given the proven function of macrophages/microglia in phagocytic processes associated with 387 myelin maintenance, we evaluated this process in aging Iba1+ cells (Benmamar-Badel, Owens 388 and Wlodarczyk, 2020; Borucki et al., 2020; Hughes and Appel, 2020; Goddery et al., 2021; 389 Santos and Fields, 2021; Kent and Miron, 2023, 2023; Swanson et al., 2023; Beiter, Sheehan and 390 Schafer, 2024; Berglund et al., 2024). Upon close examination of the Iba1+ cells within the AN, 391 many contained internalized myelin debris, especially in the aged ANs (Figure 6). The increased 392 presence of Iba1+ cells burdened with internalized myelin suggests possible immune dysfunction 393 and dysregulation of the myelination process (Moreno-García et al., 2018; Burns et al., 2020; 394 Swanson et al., 2023; Beiter, Sheehan and Schafer, 2024). Young mice had very little to no 395 internalized myelin debris within Iba1+ cells (Figure 6A-C), while aged mice had large amounts 396 of internalized myelin debris within their cell bodies (Figure 6D-F). This is evident in the 397 immunofluorescence images, as well as in the Imaris reconstructions (Figure 6D’, B’). These 398 age-related differences were consistent across the peripheral, central, and GTZ regions of the 399 AN. Quantifying the volume of internalized myelin contained within Iba1+ cells, aged mice had 400 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 15 significantly more internalized myelin compared to young mice (Internalized Myelin V olume: 401 A VGYoung = 9.80 ± 10.69 µm3, A VGOld = 686.71 ± 414.09 µm3, p = 0.004) (Figure 6H). To 402 further explore the effects of aging on phagocytic functions, our bulk RNA-sequencing results 403 were re-examined with the finding that phagocytic categories were significantly enriched in the 404 differentially expressed gene set, including “phagocytosis, engulfment” (GO:0006911; q = 3.67 x 405 10-4). Review of expression patterns for phagocytic engulfment genes illustrated that they were 406 predominantly upregulated in aged AN (Figure 6G). 407 3.6 Iba1+ cells at the auditory nerve glial transition zone contain healthy-like myelin 408 Examining morphological differences in the internalized myelin within Iba1+ 409 macrophages/microglia in the peripheral AN, central AN, and the AN GTZ reveals a unique 410 morphology of internalized myelin found only in the aging AN GTZ. Young mice had very few 411 Iba1+ cells with any internalized myelin across regions of the AN, including the AN GTZ (Figure 412 7A-G). By contrast, aged mice had numerous Iba1+ cells with internalized myelin debris 413 throughout the AN, but the AN GTZ macrophages/microglia contained myelin that appeared 414 healthy and structurally intact (Figure 7H-N). Upon close inspection of the higher magnification 415 images, there are pockets within the Iba1+ cell bodies containing intensely FluoroMyelinTM-416 stained myelin structures, which have a similar morphology to the healthy myelinated fibers in 417 these sections (Figure 7I-N). Additionally, the morphology of this healthy-like myelin has the 418 appearance of an open cylinder, which is different from the disorganized appearance of myelin 419 debris shown in Figure 6. These striking observations suggest that the phagocytic processes 420 performed by immune cells at the GTZ involves both myelin debris and healthy-like, structurally 421 intact myelin sheaths of aged AN fibers. 422 3.7 Human temporal bones have Iba1+ cells with internalized myelin at the auditory nerve 423 glial transition zone 424 To further validate the age-related changes in the immune-glial interactions determined in mice, 425 we analyzed human temporal bone sections at the AN GTZ. We used sections of the temporal 426 bones from an aged donor (89+ years old) to look at aging effects on this region of the AN. 427 Locating mid-modiolar sections from human temporal bones is challenging, and identifying the 428 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 16 specific sections that contain the GTZ is difficult due to the large number of sections (>200 per 429 specimen) and the exact angle required to capture the AN GTZ. Once we located the appropriate 430 human temporal bone sections, sections were immunostained and then imaged to evaluate the 431 Iba1+ macrophages/microglia present at the AN GTZ (Figure 8). We identified Iba1+ cells 432 throughout the human AN. The GTZ was again identified in human sections based on the 433 difference in density of nuclear staining (more nuclei peripherally due to myelination by 434 Schwann cells and fewer nuclei centrally due to myelination by oligodendrocytes) (Figure 8A). 435 Additionally, we often found Iba1+ cells around the AN GTZ, similar to the mouse AN GTZ. 436 High-resolution imaging revealed Iba1+ cells in direct contact with myelin sheaths of the AN 437 both centrally and peripherally (Figure 8B). Iba1+ cells containing internalized myelin debris 438 were also observed (Figure 8B, C), perhaps suggesting phagocytic processes occur in the aging 439 human AN and are amplified at the AN GTZ. 440 4. Discussion 441 The present study identifies the AN GTZ as a dynamic neuroimmune interface with age-related 442 structural changes that may play a role in AN myelin degeneration and functional declines 443 associated with ARHL. By integrating quantitative immunohistochemistry, morphological 444 analyses, 3D high-resolution imaging, and transcriptomic profiling, we demonstrate that this 445 central-peripheral interface at the AN GTZ is not just a boundary between Schwann cells and 446 oligodendrocytes, but a dynamic region characterized by enhanced immune activation and 447 immune-glial interactions in the aging AN. Aging was associated with reduced myelination and 448 disrupted peripheral and central myelin sheaths as well as a substantial increase in Iba1+ 449 macrophages/microglia throughout the AN, with significant enrichment of abnormal immune 450 activity at the GTZ. This heightened immune presence at the AN GTZ suggests enhanced focal 451 vulnerability to immune dysregulation contributing to impaired myelin homeostasis and AN 452 degeneration. Validation of mouse AN observations in human temporal bones from older donors 453 further supports the abnormal phagocytic activities by immune cells within the AN and its GTZ, 454 highlighting demyelination and enhanced glial dysfunction as key contributors to AN 455 degeneration and ARHL. Together, these findings position the GTZ as a unique nexus of 456 neuroimmune activity with broad implications, not just for understanding and treating AN 457 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 17 degeneration, but also for better understanding the specialized biology of GTZs throughout the 458 central and peripheral nervous systems. 459 4.1 A unique central-peripheral immune interface at the auditory nerve glial transition 460 zone 461 The AN connects the peripheral sensory epithelia housed within the cochlea to the central 462 auditory pathways, yet the glial architecture and immune microenvironment at its central-463 peripheral transition remains largely unexplored. Previous studies noted the existence of GTZ 464 regions and their development across spinal and cranial nerves (Moll and Meier, 1983; Fraher, 465 1992; Fraher and Cheong, 1995; Toma, McPhail and Ramer, 2006; Ziyal and Ozgen, 2007). In 466 the auditory system, coordinated migration of oligodendrocytes at the AN GTZ during 467 development creates a border between peripheral and central in cat, rat, and mouse models 468 (Knipper et al., 1998; Osen, Furness and Hackney, 2011; Bojrab et al., 2017). 469 Because this region represents a structural and immunological boundary, we examined how 470 immune cells and myelinating glia interact at this unique central-peripheral interface at the AN 471 GTZ. It is well established that macrophages are required for normal auditory function, but can 472 also contribute to the pathogenesis of injury and disease (Sung et al., 2019, 2024; Chokr et al., 473 2022; Seicol, Lin and Xie, 2022; Shimada et al., 2023). Traditionally, the AN studies focused on 474 either the peripheral AN, maintained by macrophages and Schwann cells, or the central auditory 475 system and cochlear nucleus, maintained by microglia and oligodendrocytes (Wan and Corfas, 476 2017; Long et al., 2018; Xin et al., 2025). Numerous studies report cochlear macrophage activity 477 during development and their roles in spiral ganglion neuron health after hair cell injury and 478 noise exposure (Hirose et al., 2005; Shi, 2010; Kaur et al., 2015; Hirose, Rutherford and 479 Warchol, 2017; Dong et al., 2018; Kishimoto et al., 2019; Miwa et al., 2024; Murali et al., 480 2025). The role of microglia has been investigated in the central auditory system, including the 481 cochlear nucleus and auditory brainstem during development and after injury (Baizer et al., 482 2015; Cramer and Rubel, 2016; Fuentes-Santamaría et al., 2017; Milinkeviciute and Cramer, 483 2018; Milinkeviciute et al., 2019; Wang et al., 2020). Here, our study offers a new approach for 484 studying the cochlear macrophage/microglia populations and their immune-glial interactions 485 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 18 across both the central and peripheral nervous systems, both of which are contained within the 486 cochlear modiolus. 487 Our data revealed that the AN GTZ contains a sharply demarcated shift in myelination between 488 the peripheral and central AN regions accompanied by a myeloid compartment 489 (macrophages/microglia) that is far less segregated in the GTZ than assumed, based on the 490 canonical understanding of the central and peripheral nervous systems (Figure 1). At the AN 491 GTZ the processes of some Iba1+ cells extend to both the peripheral and central AN (Figure 3), 492 suggesting that macrophages/microglia move freely across this boundary, and exhibit 493 modification in their phagocytic activity specifically within the GTZ, indicated by the numerous 494 Iba1+ cells with internalized healthy-like myelin found only at the GTZ with aging (Figure 7). 495 This central-peripheral interface located within the cochlear modiolus also represents a potential 496 immunological niche where macrophages and microglia coexist and may interact to coordinate 497 their responses to pathologic conditions, such as aging. Notably, there is no physical barrier 498 separating the peripheral and central immune microenvironments; rather, the GTZ is defined by a 499 discrete gap in myelination where the shift between Schwann cells and oligodendrocytes occurs 500 (Fraher, 1992; Bojrab et al., 2017). One study reported that glia can adapt and change their 501 phenotype in response to dysfunctional neighboring glia (Beachum et al., 2025). It has also been 502 reported that microglia mount a specialized response at root entry zones in both the peripheral 503 and central portions of the sciatic nerve after injury (Gai, Zhou and Rush, 1996; Liu, Rudin and 504 Kozlova, 2000). Additionally, studies have reported that the various glial cells, including 505 oligodendrocytes, astrocytes, Schwann cells, and macrophages/microglia, have plasticity in 506 phenotype in response to injury (Luo et al., 2019; Chokr, Bui-Tran and Cramer, 2024). In our 507 study, the AN GTZ displays region-specific pathological changes with aging, including a marked 508 increase in macrophage/microglia density, suggesting that the GTZ may serve as a specialized 509 site of immune surveillance and signaling distinct from the surveillance processes that occur in 510 the central or peripheral compartments. 511 4.2 Abnormal phagocytic activity may be a key contributor to age-associated demyelination 512 of the auditory nerve 513 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 19 A defining feature of the aging AN in our study was the coordinated emergence of demyelination 514 and impaired phagocytic activity in the AN, with evidence of immune system dysregulation at 515 the AN GTZ. It is well established that aging is associated with demyelination in the brain (Réu 516 et al., 2017; Hill, Li and Grutzendler, 2018; Hou et al., 2023). Additionally, aging has been 517 linked with phagocytic dysfunction in the brain, especially in the context of neurodegenerative 518 disease, some of which can cause hearing loss (Leszek et al., 2016; Raj et al., 2017; Zheng et al., 519 2017; Colonna and Brioschi, 2020; Butler et al., 2021; Beirowski, 2022, 2022; Silvin et al., 520 2022; Huang et al., 2025). Our data showed that aging is associated with a reduction in myelin 521 integrity throughout the peripheral and central AN, reflected by a disruption in myelin sheaths 522 and an accumulation of myelin debris within macrophages/microglia (Figures 4, 6, and 7). This 523 is particularly interesting because previous studies in the brain have reported that myelin-loaded 524 microglia is evidence of phagocytic dysfunction, characteristic of a proinflammatory phenotype, 525 and can exacerbate disease progression (O’Neil et al., 2018; Thomas et al., 2022; Quick et al., 526 2023; Gao et al., 2024). However, future studies should focus on identifying specific 527 macrophage and microglia phenotypes and their phagocytic function that may be contributing to 528 AN degeneration and should use advanced omics and direct assay of phagocytic activity in the 529 AN. This convergence of reduced myelination throughout the AN and evidence of 530 proinflammatory phagocytic dysfunction in the Iba1+ cell population suggests that the GTZ may 531 be a focal point for age-related myelin degeneration. 532 Although the presence of Iba1+ cells in the cochlea and AN has been established, their function 533 in AN myelination and aging remains unclear (Defourny, Lallemend and Malgrange, 2011; 534 Ginhoux and Guilliams, 2016; Bojrab et al., 2017; Brown et al., 2017; Driver and Kelley, 2020; 535 Yu, Gao and Wan, 2021; Hough et al., 2022). Myelination is an ongoing process that occurs 536 throughout a person’s lifetime (Bacmeister et al., 2020; Langley, Triplet and Scarisbrick, 2020; 537 Franklin, Frisén and Lyons, 2021; Sen et al., 2022; Kent and Miron, 2023). The roles of 538 macrophages/microglia in myelin maintenance have been reported in both the peripheral and 539 central nervous systems (Koike and Katsuno, 2021; Kopper et al., 2021; Ryan et al., 2022; Sen 540 et al., 2022). In the aging brain, deficits in phagocytic activity associated with myelin turnover 541 and maintenance are often found in neurodegenerative and demyelinating diseases such as 542 Alzheimer’s Disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis (Bitsch et al., 2000; 543 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 20 Cunha et al., 2020; Gaitsch et al., 2024). Our morphological analyses revealed that Iba1+ 544 macrophages/microglia in aged mice often contained myelin debris, suggesting active but 545 incomplete phagocytic engagement (Figures 6 and 7). Together with transcriptomic analysis of 546 aged ANs, our data suggests a decline in effective phagocytosis, with aged AN Iba1+ cells 547 exhibiting signs of stalled or dysfunctional clearance rather than efficient debris removal. This 548 dysfunctional clearance may be linked to the upregulation of Clec7a, Trem2, and Itgb2 (Figure 549 6). These genes have been linked to dysregulation of phagocytic processes and are expressed by 550 disease-associated microglia in the brain (Jay, V on Saucken and Landreth, 2017; Deczkowska et 551 al., 2018; Deerhake and Shinohara, 2021; Silvin et al., 2022; Gao et al., 2023; Martins-Ferreira 552 et al., 2025). However, our study utilized bulk RNA sequencing of the ANs which does not 553 allow regional resolution between the central AN, peripheral AN, and GTZ. Future studies 554 should focus on heterogeneity of the Iba1+ cell populations in the AN and cochlea using 555 advanced omics approaches such as single-cell RNA sequencing and spatial omics assays to 556 better define the spatial organization of age-related changes of AN immune cells. 557 In addition, lipid-burdened macrophages/microglia can take on a proinflammatory state and 558 exacerbate disease progression (Burns et al., 2020; Franklin and Simons, 2022). Additionally, 559 when myelination is disrupted, this can lead to axonal damage and neuronal cell death 560 (Beirowski, 2022). This impaired phagocytic function likely contributes to the persistence of 561 myelin debris observed throughout the aged AN and may exacerbate degeneration by failing to 562 restore a supportive microenvironment for AN axon health and conduction (Sepp, Schulte and 563 Auld, 2001; Beirowski et al., 2014, 2014; Leszek et al., 2016; Arancibia-Cárcamo et al., 2017; 564 Beirowski, 2022, 2022; Grüter et al., 2022; Freire et al., 2023). 565 More importantly, this decrease in myelination was not restricted to either canonical peripheral 566 or central AN compartment. Both the Schwann cell-associated and oligodendrocyte-associated 567 regions exhibited signs of compromised myelin turnover or repair (Figure 4), indicating that 568 aging induces a similar maladaptive immune-glial response across the entire AN, with enhanced 569 effects at the GTZ. Together, our findings support a model in which aging leads to widespread 570 demyelination coupled with a dysfunctional immune cell compartment with insufficient AN self-571 repair mechanisms, culminating in progressive AN degeneration and ARHL (Figure 9). 572 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 21 4.3 A dynamic glial transition zone is conserved in the human auditory nerve 573 To extend our studies from the mouse AN to humans, we examined the structure and immune 574 composition of the GTZ in the human AN from an 89+ year old donor, revealing a similarly 575 dynamic region with enriched immune-glial interactions. The GTZ of the AN is a unique 576 neuroanatomical boundary where central and peripheral myelination converge, which we observe 577 in our study in human temporal bones (Skinner, 1931; Guclu et al., 2009). Our study confirmed 578 that an active myeloid population is present in the human AN GTZ. Another important 579 observation was that Iba1+ cells were identified containing internalized myelin debris (Figure 8), 580 suggesting that phagocytic processes also occur in humans to support myelin homeostasis and 581 normal function of the AN. It has been shown that microglia turnover in the human brain is a 582 slow continuous process, making this slowly renewing population particularly vulnerable to 583 pathologic disturbances like aging and demyelination (Réu et al., 2017). Additionally, the unique 584 biology of the AN GTZ region may be a contributor to the increased rates of schwannoma in the 585 CNVIII compared to other cranial nerves, as well as a driver of dysfunction in these glia leading 586 to the development of schwannomas across the nervous system (Nickele et al., 2012; Lan et al., 587 2020; Eggink et al., 2022). Some studies have linked variation in central myelin morphology in 588 the trigeminal nerve to the development of trigeminal neuralgia (Nomura et al., 2019). 589 Additionally, studies are focusing on this region due to its regenerative potential in the context of 590 spinal cord injury (Carlstedt, 1997; Monje, Deng and Xu, 2021; Ghosh and Pearse, 2023). The 591 localization of Iba1+ cells to this region and the evidence of phagocytic activity point to potential 592 roles in myelin maintenance and remodeling. These processes are critical to AN function and any 593 dysregulation could contribute to age-related demyelination, AN degeneration, and ARHL. 594 Our findings raise the possibility that targeting immune-glial interactions at the GTZ could be a 595 novel strategy to mitigate age-related demyelination in the AN. Preclinical models have shown 596 that modulating glial subtype can have a protective role, particularly in neurodegenerative 597 disease (Rahimian et al., 2022; Silvin et al., 2022; Barclay et al., 2023; Gao et al., 2023; Ball et 598 al., 2024; Martins-Ferreira et al., 2025). Modulating macrophage/microglia activation state to 599 restore phagocytic function may help maintain myelin integrity and preserve axonal conduction 600 in aging. Interventions aimed at supporting this dynamic immune niche at the GTZ could be 601 potential strategies to ultimately slow or prevent the progression of AN degeneration and ARHL. 602 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 22 Data Availability Statement 603 The original contributions presented in this study are included in the article; further inquiries can 604 be sent to the corresponding authors. 605 Ethics Statement 606 All animal studies for this project were reviewed and approved by the Institutional Animal Care 607 and Use Committee of the Medical University of South Carolina (MUSC) and the Ralph H. 608 Johnson V A Health Care System (V AHCS) in Charleston, South Carolina. 609 Author Contributions 610 SP and HL designed the study. HL, SP, PC, and HY acquired the funding. SP, HA, and HL 611 prepared samples. SP performed confocal imaging. PC, JC, and HY developed and performed 612 light sheet imaging. JB analyzed the RNA-seq dataset. SP developed unpublished analysis 613 routines. SP analyzed the data. SP wrote the manuscript. SP, HL, and HA revised the manuscript. 614 All authors contributed to the article and approved the submitted version. 615 Funding 616 This work was partially funded by NIH/NIDCD R01 DC021436 (HL), V A RR&D Merit Award 617 BX006478 (HL), NIH/NIDCD F30 DC023098 (SP), SC INBRE P20GM103499 (HL, SP), 618 Interdisciplinary Research Training in Otolaryngology and Communication Sciences 619 5T32DC014435 (SP), NIH/NIGMS P20 GM121342 (HY and PC), NIH/NIDCR R01 DE021134 620 (HY), Musculoskeletal Transplant Foundation grant (PC), and the Department of Pathology and 621 Laboratory Medicine. Confocal imaging core facilities were supported in part by the Cell & 622 Molecular Imaging Shared Resource, MUSC Cancer Center Support Grant (P30 CA138313), the 623 SC COBRE in Digestive and Liver Diseases (P20 GM130457), the MUSC Digestive Disease 624 Research Cores Center (P30 DK123704), and the Shared Instrument Grants S10 OD018113 and 625 S10 OD028663. 626

627 We thank Jiaying Wu for her technical assistance and Tyreek Jenkins and Emily Fabrizio-Stover 628 for their comments and suggestions. 629 Conflict of Interest 630 The authors declare that the research was conducted in the absence of any commercial or 631 financial relationships that could be construed as a potential conflict of interest. 632 633 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 23

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(2007) “Microanatomy of the central myelin-peripheral myelin 1060 transition zone of the trigeminal nerve,” Neurosurgery, 60(3), pp. E582; author reply E582. 1061 Available at: https://doi.org/10.1227/01.NEU.0000255367.19228.C4. 1062 1063 1064 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 35 1065 Table 1. Antibodies and stains used for this study. Antibody Source Vendor Catalog Number Dilution Iba1 Rabbit Abcam AB178847 1:150 CC-1 Mouse Millipore MABC200 1:100 NG2 Rabbit Millipore AB5320 1:100 Biotinylated anti-rabbit IgG Goat Vector Laboratories X1104 1:100 Biotinylated anti-mouse IgG Horse Vector Laboratories BA2000 1:100 Biotinylated anti-rat IgG Rabbit Vector Laboratories BA4000 1:100 Streptavidin Texas Red™ N/A Vector Laboratories SA5006 1:100 Streptavidin Fluorescein N/A Vector Laboratories SA5001 1:100 Anti-mouse IgG Alexa Fluor™ 488 Rabbit ThermoFisher Scientific A11029 1:200 Myelin Basic Protein, REAfinity Vio R667 N/A Miltenyi Biotec 130-131-152 1:50 CD68, REAfinity Vio G570 N/A Miltenyi Biotec 130-133-803 1:50 Toluidine Blue N/A Electron Microscopy Sciences 22050 1% FluoroMyelinTM Green N/A Invitrogen F34651 1:700 Hoechst 33342 N/A ThermoFisher Scientific H3570 1:500 1066 1067 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 36 Figure 1 1068 1069 Figure 1. Mouse mid-modiolar sections demonstrating age-related changes to the AN GTZ. 1070 (A-F) Thirty µm mid-modiolar sections of the auditory nerve (AN) glial transition zone (GTZ) in 1071 a young (3 months) (A-C) and an aged (2.5 years) (D-F) CBA/CaJ mouse. Sections are stained 1072 with FluoroMyelinTM (green), macrophage/microglia cell marker Iba1 (red), and Hoechst nuclear 1073 stain (blue). The dashed white line indicates where the AN GTZ is located in a mid-modiolar 1074 section. Scale bar = 100 µm. (G) Representative image from Epon LX 112 resin-embedded 1075 sections (stained with Toluidine blue) of the AN GTZ in a 1 year-old mouse. (G’) Enlarged 1076 images of the white outlined region of the AN GTZ are shown to illustrate the visible gap 1077 between central and peripheral myelination in the AN. 1078 1079 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 37 Figure 2 1080 1081 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 38 Figure 2. Stacked serial sections of the mouse AN GTZ identifying more CD68+ cells in 1082 aged mice. (A – X) Serial sections taken 25 μm apart from tissue cleared mouse temporal bones 1083 showing the entire AN within the cochlea and the AN GTZ in a young (3 months) (A-L) and an 1084 aged (2.5 years) (M-X) CBA/CaJ mouse. (D’, E’, I’) Enlarged images of the white outlined 1085 regions of the AN GTZ in a young mouse are shown. (P’, T’, V’) Enlarged images of the white 1086 outlined regions of the AN GTZ in an aged mouse are shown with a white arrow indicating 1087 CD68+ cells within the AN, while no CD68+ cells were seen in the AN of the young mouse (D’, 1088 E’, I’). Dashed white lines in D’, E’, I’, and P’ show the cone-shaped morphology of the AN 1089 GTZ. Dashed white lines in T’ and V’ show the GTZ in the basal turn/hook of the cochlea. Each 1090 mouse temporal bone was stained with Myelin Basic Protein (green) and CD68 (red). Scale bars 1091 = 200 µm for all images. 1092 1093 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 39 Figure 3 1094 1095 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 40 Figure 3. Increased density and morphological alterations of immune cells at the AN GTZ 1096 in aged mice. (A-L) Immunostained sections of the AN GTZ in young (A-F) and aged (G-L) 1097 mice showing Iba1+ cells in the peripheral AN (A, D, G, J), GTZ (B, E, H, K), and the central 1098 AN (C, F, I, L) with aging. White arrows indicated Iba1+ cells that cross both the peripheral and 1099 central AN. (M) Average surface area of Iba1+ cells per region of interest (ROI) in young (filled 1100 red circles) and aged (open red circles) mice. N = 4 mice per group. 4 ROIs per mouse (1 ROI 1101 for peripheral AN, 1 ROI for central AN, 2 ROIs for AN GTZ). (N) Average volume of Iba1+ 1102 cells per ROI per age group. N = 4 per group. 4 ROIs per animal (1 ROI for peripheral AN, 1 1103 ROI for central AN, 2 ROIs for AN GTZ). (O) Quantity of CD68+ cells within the entire AN in 1104 every section, 25 µm apart in young mice (n=3) and aged mice (n=3). (P) Average number of 1105 Iba1+ cells per ROI in young and aged mice. N = 3 per group, 9 ROIs (3 ROIs for peripheral AN, 1106 3 ROIs for central AN, 3 ROIs for AN GTZ) per mouse. The difference between young and old 1107 was significant (p = 0.03). (Q) Average number of Iba1+ cells in the peripheral AN, GTZ, and 1108 central AN in young and aged mice. N = 3 mice per group, 3 ROIs per region per age group. All 1109 distributions were evaluated for normality using a Kolmogorov-Smirnoff test. Significance was 1110 determined using a Mann-Whitney U-test, α = 0.05. Diamonds show the mean, and error bars 1111 represent the standard deviation. *p<0.05. ****p<0.0001. 1112 1113 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 41 Figure 4 1114 1115 Figure 4. No significant difference in myelin surface area and volume, but more disrupted 1116 myelin sheaths in the peripheral AN and central AN in aged mice. (A-B) FluoroMyelinTM-1117 stained sections in the peripheral AN (A) and central AN (B) in a young (3 months) and aged 1118 (2.5 years) CBA/CaJ mouse. Boxes on the top-right are enlarged images from the dashed boxes 1119 in A and B. (C) Quantification of myelinated AN fiber surface area in young (filled green circles) 1120 and aged (open green circles). N= 4 mice per age group. 4 ROIs per mouse (2 ROIs for 1121 peripheral AN, 2 ROIs for central AN). The difference between the young and the aged was not 1122 significant. (D-E) FluoroMyelinTM-stained sections in the peripheral AN (D) and central AN (E) 1123 in an aged CBA/CaJ mice. Magenta dashed boxes indicate the region of the pop-out image in the 1124 magenta outlined box. Boxes on the top-right are enlarged images from the dashed boxes in D 1125 and E. White arrows indicate the disrupted myelin sheaths in the peripheral AN (D) and central 1126 AN (E). (F) Quantification of myelinated AN fiber volume by age group. N = 4 mice per age 1127 group. 4 ROIs per animal. The difference between the young and the aged was not significant. 1128 Diamonds represent the mean and error bars represent the standard deviation for each group. All 1129 distributions were tested for normality using a Kolmogorov-Smirnoff test. Significance was 1130 determined using a Mann-Whitney U-test, α = 0.05. Scale bar = 10 µm for all images. 1131 1132 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 42 Figure 5 1133 1134 Figure 5. Gene expression profiles of the AN revealed age-dependent effects on 1135 inflammation and myelination. (A) Analysis of AN RNA-sequencing data from young (3 1136 months) and aged (2.5 years) CBA/CaJ mice detects 1240 differentially expressed genes 1137 (Supplementary Table 1). (B) Enrichment analysis of differentially expressed genes detects 1138 significant effects on abnormal myelination and abnormal inflammatory response. (C) 1139 Expression patterns of differentially expressed genes linked to abnormal myelination, abnormal 1140 inflammatory response or both categories. For abnormal inflammatory response, the 15 genes 1141 showing highest upregulation are listed on the right. 1142 1143 1144 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 43 Figure 6 1145 1146 Figure 6. Increased internalized myelin debris and upregulated expression of phagocytosis-1147 related genes appeared in the aged AN. (A-F) Sections of the peripheral AN (A, D), GTZ (B, 1148 E), central AN (C, F) and Imaris 3D reconstructions (D’, B’) showing myelin debris inside Iba1+ 1149 cells. (G) Differentially expressed genes linked to phagocytic engulfment are predominantly 1150 upregulated in aged (2.5 years) versus young (3 months). (H) V olume of internalized myelin in 1151 young mice and aged mice. 4 ROIs per mouse, N = 3 mice per age group. All distributions were 1152 assessed for normality using a Kolmogorov-Smirnoff test. Comparisons between young and old 1153 for significance were determined using a Mann-Whitney U-test, α = 0.05. **p < 0.01. 1154 1155 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 44 Figure 7 1156 1157 Figure 7. Increased healthy-like myelin found in Iba1+ cells in the aging AN GTZ. (A-N) 1158 Sections of the AN GTZ in young (A-G) and old (H-N) mice showing Iba1+ cells with 1159 internalized myelin. Dotted boxes in A and H are regions that were enlarged to show the 1160 structurally intact myelin within Iba1+ macrophages/microglia at the AN GTZ (B-G, I-N). 1161 Images are individual slices from the z-stack to show the structurally intact myelin within Iba1+ 1162 cells. 1163 1164 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 45 Figure 8 1165 1166 Figure 8. Internalized myelin found in human AN Iba1+ cells. (A) Mid-modiolar cochlear 1167 section containing the AN GTZ in a temporal bone from an 89+ year old donor. Large white 1168 dashed line is where the AN GTZ is located. (B, C) Enlarged images from A showing Iba1+ cells 1169 in close association with AN myelin and containing internalized myelin throughout the AN. 1170 Dashed box in A is shown in B and C. Sections are stained with FluoroMyelinTM (green), Iba1 1171 (red), and Hoechst (blue). Scale bar = 100 µm. 1172 1173 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint Payne et al. Auditory Nerve Glial Transition Zone 46 Figure 9 1174 1175 Figure 9. Summary of Aging Effects on the AN and GTZ. Schematic demonstrating age-1176 related decreased myelination, increased numbers of Iba1+ macrophages/microglia, and enhanced 1177 dysfunction at the AN GTZ. The model highlights regional differences in age-related immune 1178 and glial changes across the peripheral AN, central AN, and GTZ compared to the young AN. In 1179 the aging AN, Iba1+ cells throughout all regions of the AN contain myelin debris, while the GTZ 1180 exhibits a marked increase in Iba1+ cells that also contain structurally intact myelin, suggesting 1181 region-specific functional declines that may contribute to age-related AN demyelination. 1182 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted March 31, 2026. ; https://doi.org/10.64898/2026.03.27.714751doi: bioRxiv preprint