Neuritin restores auditory following cochlear spiral ganglion neuron denervation of gerbils

doi:10.21203/rs.3.rs-2630540/v1

Download PDF

Research Article

Neuritin restores auditory following cochlear spiral ganglion neuron denervation of gerbils

https://doi.org/10.21203/rs.3.rs-2630540/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Spiral ganglion neurons (SGNs) transmit sound signals received by hair cells to the auditory center to produce hearing. The quantity and function are important for maintaining normal hearing function. Limited by the regenerative capacity, SGNs are unable to regenerate spontaneously after injury. Various neurotrophic factors play an important role in the regeneration process. Neuritin, a neurite outgrowth factor, is a neurotrophic factor closely related to neural plasticity. In this study, we used bioinformatics analysis to show that neuritin was negatively correlated with cochlear damage. Then, we aimed to establish a cochlear spiral ganglion-specific sensorineural deafness model in gerbils using ouabain and determine the effects of exogenous neuritin protein in protecting damaged cochlear SGNs and repairing damaged auditory nerve function. The provides a new research strategy and scientific basis for the prevention and treatment of sensorineural deafness caused by the loss of SGNs. We found that neuritin is expressed throughout the development of the gerbil cochlea, primarily in the SGNs and Corti regions. The expression of neuritin was negatively correlated with the sensorineural deafness model. In vitro and in vivo revealed that neuritin significantly maintained the number and arrangement of SGNs and nerve fibers in the damaged cochlea and effectively protected the high-frequency listening function of gerbils.

neuritin

sensorineural deafness

hearing loss

spiral ganglion neurons

gerbils

Deafness is one of the most common disabling diseases in clinic[1]. According to the first World Hearing Report released by the World Health Organization in 2021, more than 1.5 billion people worldwide have hearing loss, 430 million of whom have a moderate or high degree of hearing loss. It is estimated that by 2050, one in four people worldwide will have some degree of hearing loss, and at least 700 million will require rehabilitation treatment[2]. Common deafness factors include ototoxic drug use, noise exposure, age, metabolic disorders and genetics[3–5]. The sensory nerve tissue of the cochlea has a very limited capacity for self-repair and regeneration[4, 6]. Mature cochlear cells (including cochlear spiral ganglion and inner and outer hair cells) have no self-differentiate and regenerate ability at all. Therefore, it is very important to find ways to reduce the loss of auditory nerve cells and promote their survival in the treatment of neural deafness.

Current studies have found that drug therapy alleviates some of the symptoms of associated with neural deafness but there is still no clinically effective drug for effective treatment, and the majority of patients still require a hearing aid or cochlear implant[4]. However, cochlear implantation requires the presence of a number of spiral ganglion neurons (SGNs). As the first afferent neuron, SGNs can carry sound information from the cochlea to the central nerve and then produce hearing, which is critical in the sound transmission pathway.

The effect after cochlear implantation largely depends on the number and function of residual SGNs. Therefore, the effective maintenance of the number and function of SGNs has important practical value in the protection and treatment of neural deafness[7]. Current research strategies to maintain and restore SGNs mainly include gene therapy and stem cell transplantation[8, 9]. Gene therapy regulates the expression of deafness in the cochlea through overexpression or knockout of genes related to the occurrence and development of deafness, so as to achieve therapeutic and protective effects, but there are great limitations in clinical treatment. Stem cell transplantation can maintain the survival of cochlear SGNs and promote the recovery of hearing function in the short term, but with the differentiation and apoptosis of stem cells, the therapeutic effect is significantly reduced. Recent studies have found that neurotrophic factors (NTFs) can regulate the cochlear spiral ganglion cell transduction pathway[10], which turn plays a protective role in SGNs. Therefore, it is very important to explore the protective effect of neurotrophic factors on impaired SGNs and the recovery of hearing function, which can provide a theoretical basis for the treatment of neural deafness.

Neuritin is an NTF with neurotrophic plasticity[11], and also a downstream effector other NTFs such as brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3)[12, 13]. The above-mentioned NTFs have been implicated in promoting the recovery of impaired auditory function[14], but there has been no literature that has implicated neuritin in the auditory system. Our research group has been engaged in the study of the function and mechanism of recombinant human neuritin for a long time, and has clarified the role of Neuritin as NTFs. A large number of active recombinant human Neuritin proteins were obtained[15]. Neuritin was found to not only to promote the regeneration and structural reconstruction of nerve fibers after peripheral nerve injury[16], but also to maintain (rescue) the survival and functional repair of neurons after central nervous system injury[17]. Further, neuritin plays a role in inducing the directional differentiation of bone marrow mesenchymal stem cells into nerve cells[18]. In addition, neuritin was found to significantly promote the growth and branching of neural processes, enhance the maturation of synapses, increase the number and length of synaptic connections[19], promote the establishment of functional synaptic connections[20], and contribute to the survival of neurons by inhibiting apoptosis[21, 22]. Neuritin expression in the damaged site was consistent with functional recovery during nerve injury repair, suggesting that its expression was related to structural and functional repair of the damaged site [23].

Ouabain is an ototoxic drug that can selectively inhibit Na⁺/K⁺ ATPase and its transport of Na⁺/K⁺ in SGNs of the cochlea to reduce intracellular Na⁺ excretion. Indeed, ouabain has been shown to inhibit Na⁺/Ca²⁺ exchange and increase intracellular Ca²⁺ concentrations, resulting in intracellular ion exchange disorder and apoptosis that subsequently induce specific loss of cochlear SGNs[24].

Furthermore, Through bioinformatics analysis, we found that neuritin was significantly down-regulated in the cochlea after ouabain-induced injury. It has been suggested that neuritin has a negative correlation with deafness caused by ouabain. Therefore, this study used an ouabain-induced neural deafness model in gerbils to explore the protective effect of neuritin on neural deafness.

2.1 Summary of microarray datasets

Public microarray repositories were curated to search through the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo) using the keywords “ouabain” and “hearing loss”. We found three datasets in the search results and GSE59416 was used for the subsequent bioinformatics analysis. The overall design of GSE59416 was as follows: An ouabain solution was administered in the round window niche of the right ear, while the left ears (i.e., contralateral) were not surgically manipulated and were used as controls. Mice were allowed to recover for 3 days or 7 days following treatments. The cochleas were prepared for RNA extraction and the subsequent global gene expression analysis.

2.2 DEGs enrichment analysis and its visualization of the datasets

R tools was used in this study. Quality control was performed using the ‘simpleaffy’ ‘affyPLM’ packages. After quality control, unqualified sample data were removed. The datasets were then normalized on the base-2 logarithm using the Robust Multi-Array Average and Linear Models for Microarray packages and annotated by converting different probe IDs to gene IDs. Genes significantly dysregulated in acute SGN injury mice as compared to matched data in control samples were defined by an adjusted p-value < 0.01 and log2 fold change of > 2 (upregulated genes) or < − 2 (downregulated genes). Finally, gene ontology enrichment analysis and Kyoto encyclopedia of genes and genomes enrichment analysis were performed using the ‘GOstats’ and ‘GeneAnswers’ packages. We used the ‘Clusterfiler’ and ‘pheatmap’ packagex to visualize the statistical analysis.

2.3 Animals and experimental design

Animal experiments were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Animal Subjects Review Board of the Hangzhou Normal University School of Medicine (permit no. 2019175). Surgeries were carried out under sodium pentobarbital anesthesia.

Mongolian gerbils (2–3 mouths, weighing 60–80 g) were obtained from the Laboratory Animal Centre of Hangzhou Normal University. All animals had free access to both food and water and were kept maintained in cages with a 12 h light: dark cycle at a temperature and humidity of 21–24 ℃ and 40–70%, respectively.

2.4 Neural deafness model and neuritin treatment

Gerbils were assigned to an experimental and control group. First, ouabain (Sigma, O3125, St Louis, MO, USA) was dissolved in normal saline (NS). Next, the cochlear bulla was exposed through a retro-auricular approach followed by creating a small hole in the bulla to expose the round window and the ouabain (20 µL, 1mM) solution was positioned onto the round window membrane of the cochlea. The solution was then incubated for 30 min at 37 ℃, after which small cubes (1 mm³) of gelfoam were soaked in 16/32 µL of a 1 mg/mL solution of neuritin dissolved in NS that positioned onto the round window membrane. The control group received NS instead of neuritin. Hearing was measured weekly after administration using the auditory brainstem response (ABR). Subsequently, the gerbils in each group were euthanized, and the cochlear samples were isolated and taken out for immunofluorescence detection

2.5 Cochlear samples collection

In order to further clarify the relationship between neuritin and ouabain-induced hearing impairment in gerbils, neuritin mRNA and protein expression in the control group and at 6 h and 3, 7, and 56 days after injury were detected by western blot and qPCR respectively. After euthanasia, the gerbil cochlea injured by ouabain was extracted, and the cochlea tissue samples were separated and stored in liquid nitrogen.

The cochlear tissue of the gerbils in the normal group and at 7 days were next assessed by immunofluorescence to detect the expression of neuritin in the cochlea.

2.6 Organotypic culture of the cochleae

Gerbil cochlear organotypic cultures were prepared as previously described [25]. Briefly, a mixture of rat-tail collagen (9:1:1 ratio of Type 1 collagen (BD Biosciences, #4236, Bedford, MA, USA), 10 × basal medium eagle (Sigma, #B9638), and 2% sodium carbonate was prepared, and 10 µL of the mixture was placed in the center of a 35 mm diameter culture dish (Falcon 1008, Becton Dickinson) for 30 min until the mixture solidified. Then, 1.3 mL of serum-free medium [2 g bovine serum albumin (BSA, Sigma A-4919), 2 mL serum-free supplement (Sigma, #I-188), 4.8 mL of 20% glucose (Sigma G-2020), 0.4 mL penicillin G (Sigma P-3414), 2 mL of 200 mM glutamine (Sigma G-6392), and 190.8 mL of 1 × BME (Sigma B-1522)] were added to the culture dish.

On postnatal day 3, gerbil pups were euthanized by carbon dioxide asphyxiation and decapitated. The temporal bones were removed, and the whole basilar membrane containing the organ of Corti and SGNs was carefully dissected out in Hank's balanced salt solution and placed on the surface of the collagen gel in the culture dish. The culture dish was then placed in an incubator for 1 h at 37 ℃ an 5% CO₂ to allow the cochlear explants to attach to the surface of the gel. After, 0.7 mL of serum-free medium was added to the explants, and the culture dish was returned to the incubator overnight. On the second day, the culture medium was removed, and medium containing specific concentrations of ouabain (200 µM) was added to the culture dish. Meanwhile, the explants were treated with or without 16/32 µg/mL neuritin for 3 days and then subjected to immunofluorescence staining and western blotting analysis.

2.7 Electrophysiological recordings

Gerbils were first anesthetized with an intraperitoneal injection of 0.01 g/mL pentobarbital sodium (100 mg/kg body weight). Next, the System 3 digital signal processing package of hardware and software (Tucker Davies Technologies (TDT), FL) was used to present click stimuli ranging from 90 to 10 dB at a rate of 20 s^− 1 in decreasing 10 dB steps. Tone stimuli were presented as pure tone pips lasting 5 ms at 90 dB, at frequencies at 2, 4, 8, 16, and 32 kHz. The sounds were presented to the animal via a ‘closed field’ method a 3 mm diameter, 10 cm tube leading from the speaker was inserted into the auditory meatus until its tip was close to the tympanic membrane. ABR measurements were recorded using 27G subdermal needle electrodes placed at the vertex of the skull (recording electrode) and the ipsilateral mastoid process (reference electrode), with a ground electrode placed dorsally adjacent to the tail. The differential voltage induced by the presentation of the sound stimulus was thus recorded. Each stimulus was presented 1024 times, thus the wave generated represented an average response over this time. All experiments were carried out in a sound-proof, custom-made audiology booth. The ABR data were analyzed using GraphPad Prism 6 software.

2.8 RNA isolation and qPCR

To detect Neuritin mRNA levels, total RNA was isolated from cochlear Corti tissue samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Equivalent amounts of RNA from each sample were used for cDNA synthesis using the FastKing gDNA Dispelling RT SuperMix kit [Tiangen Biotech (Beijing) Co., Ltd.]. Subsequently, the cDNAs were used for qPCR analysis. The primers used were as follows: Neuritin (F) 5’-CGCGGTGCAAATAGCTTACC-3’; Neuritin (R) 5’-TGTTCGTCTTGTCGTCCAGG-3’; β-actin (F) 5’-CTGGCTCCTAGCACCATGAA-3’; β-actin (R) 5’-CGCAGCTCAGTAACAGTCCG-3’. The thermocycling conditions for Nrn1 mRNA were 95°C for 15 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s. The 2^−ΔΔCt method was used to analyze the level of mRNA.

2.9 Western blot analysis

Neuritin protein levels in the cochlear tissues were detected by western blot analysis. Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) with 1% protease inhibitor cocktail (Beyotime Institute of Biotechnology), centrifuged for 10 min at 15,777 g and 4°C, and the supernatant was collected. The amount of total protein was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Total protein was electrophoresed by 12.5% SDS-PAGE and transferred to PVDF membranes at 23 V for 43 min. Membranes were blocked with Tris-buffered saline/5% fat-free skim milk for 2 h at room temperature, and then incubated with rabbit anti-Neuritin monoclonal antibody (1:1,000; ab64186, Abcam) for 16 h at 4°C. The following day, the membranes were incubated with secondary goat anti-rabbit IgG-HRP antibody (1:2,500; ZB-2301, Zsgb-Bio) for 2 h at room temperature. Finally, proteins were detected using Clarity Western ECL substrates (Bio-Rad Laboratories, Inc.). β-actin (Zsgb-Bio) was used as an endogenous control. Adobe photoshop CC 2015 software was used for densitometry.

2.10 Histology

Cochlear tissues were fixed by immersion in 4% paraformaldehyde (PFA; pH 7.4) for 24–48 h at 4 ℃ and decalcified in 0.25 M ethylenediaminetetraacetic acid pH 8.0 for a week at 4 ℃. Samples were then immersed an ascending sucrose/PBS series (15% and 30%, several hours/overnight at each stage). Following embedding in Cryo-M-Bed medium (VWR, Lutterworth, U.K.), tissue was sectioned (12 µm) onto gelatin/ chrome alum-coated glass slides using a Bright cryostat. The tissue sections were defrosted, rehydrated, briefly re-fixed with 4% PFA, rinsed with 0.1% Triton/PBS, and blocked in 2% BSA before incubation overnight at 4 ℃ with the following antibodies: anti-MyoVIIA (1:1000, rabbit monoclonal, Abcam), anti-Neurofilament 200 (NF-200; 1:1000, rabbit monoclonal, Abcam), and anti-Tuj1(1:1000, mouse monoclonal, Abcam). Sections were then incubated with anti-chicken Alexa 633, anti-mouse Alexa 488, and anti-rabbit Alexa 568 (all from Life Technologies, Paisley, U.K.) for 2 h at room temperature and counter-stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) before mounting in Vectashield (Vector Laboratories, Peterborough, U.K.). Images were taken on a Zeiss Axiophot microscope using Axiovision software and figures assembled using Adobe Photoshop 7.0. Five visual fields were randomly selected in each group for quantitative statistics. The SGNs density was calculated as the number of SGNs per 0.01 mm². The HCs and nerve fibers was calculated as the number of cells per 100µm.

2.11 Statistical analyses

For each experimental condition, at least three independent experiments were performed. Data were analyzed using GraphPad Prism 6 software and are presented as the means ± standard errors of the means. Two-tailed, unpaired Student's t tests were used to determine statistical significance when comparing two groups, and two-way ANOVA followed by a Bonferroni post-hoc test was used when comparing more than two groups. A value of P < 0.05 was considered statistically significant.

3.1 Auditory function and neuritin expression and localization in the cochlea of gerbils

The gerbil is sensitive to hearing and has a large auditory bubble organ, and is one of the preferred experimental animals for the study of hearing impairment. The normal hearing threshold of gerbils is 10–20 dB SPL(Fig. 1A). Pure tone detection revealed that gerbils had the most sensitive hearing at 4kHz (Fig. 1B).

In order to clarify the correlation between neuritin and auditory function, we examined its expression changes in the cochlea of gerbils. Specifically, neuritin mRNA and protein expression from cochlear tissue of gerbils at different developmental stages was assessed by western blot and qPCR, respectively. The results showed that neuritin was expressed in varying degrees at all stages of cochlear development in gerbils, and that the expression in the cochlea after birth was significantly higher than that in the embryo. The expression level of neuritin was relatively low at embryonic day 20 (E20); however, with increasing age, the neuritin expression continued to increase from P1 to P56, and the expression level was significantly elevated after P21 (Fig. 1D, E and F). The results indicate that neuritin may play a key role in the development of the cochlea in both juvenile and adult gerbils. Finally, immunofluorescence results indicated that neuritin was highly expressed in the cochlea of gerbils at P56 and, in particular, was found to located in SGNs and Corti organ regions (Fig. 1C).

3.2 Expression changes of different genes after ouabain-induced injury using bioinformatics analysis

Public microarray repositories were curated to search through the GEO database using the keywords “ouabain” and “hearing loss”. We found three datasets in the searching results and GSE59416 was subsequently used for our bioinformatics analysis. The data analysis results showed that the expression levels of a large number of genes in the cochlea of the ouabain injury group changed significantly, and that the expression of neuritin was down-regulated by 2.48 times compared with the normal group. The results suggest that neuritin is inversely associated with ouabain-induced cochlear damage (Fig. 2).

3.3 Establishment of the cochlear SGN injury model

Following ouabain-induced cochlear damage, ABR was used to test the hearing function of gerbils on a weekly basis. The results showed that the hearing threshold of the injured group of gerbils was significantly improved after the 1st week and that this significant improvement continued to the 12th week; the hearing threshold was still significantly higher than the control group, and there was no sign of recovery (Fig. 3C).

Next, immunofluorescent results indicated that, compared with the control group, the number of SGNs was significantly reduced in the injury group, and only a small number were evident (Fig. 3B ,This is shown by the yellow arrow); however, hair cells were still alive (Fig. 3B). It can be seen that ouabain (1 mM) only caused the loss of cochlear SGNs, while the hair cells were not damaged, and which resulted in an upward shift of the auditory threshold of the gerbil cochlea.

3.4 Neuritin expression in the gerbil cochlea after ouabain-induced injury

In order to further clarify the relationship between neuritin and ouabain-induced hearing impairment in gerbils, neuritin mRNA and protein expression in the control group and at 6 h and 3, 7, and 56 days after injury were detected by western blot and qPCR respectively. The results showed that compared with the control group, the expression of neuritin in the cochlea of the injured group was significantly down-regulated (Fig. 4B, C and D), and that the mRNA and protein expression levels were basically the same.

The cochlear tissue of the gerbils in the normal group and at 7 days were next assessed by immunofluorescence to detect the expression of neuritin in the cochlea. Compared with the control group, the fluorescence intensity of the SGNs in the injured group was significantly weakened, but there was no significant difference fluorescence in the Corti region (Fig. 4A).

3.5 Neuritin mitigates impaired auditory function in gerbils

In this experiment, an established animal model of gerbil cochlear SGNs-specific damage was used to conduct experiments in vivo. The experimental results showed that compared with the normal group, the hearing threshold of each frequency of the gerbils cochlea increased significantly 2 weeks after ouabain injury. The hearing thresholds at 16kHz and 32kHz in the Neuritin (16µg) group were 32.5 ± 8.9dB and 61.3 ± 3.5dB, which were significantly lower than those in the injured group (48.3 ± 16.0dB and 85 ± 5.5dB). The hearing thresholds at 8kHz, 16kHz and 32kHz in the Neuritin (32µg) treatment group were 25 ± 10.7dB, 31.3 ± 9.9dB and 55 ± 5.3dB, which were also significantly lower than those in the injured group. With the increase of Neuritin concentration, the protection of hearing function tended to the low and middle frequency regions. The Neuritin (32µg) group recovered about 30dB at 32 kHz compared with the injured group, but only 20dB in the 16µg group. It was shown that Neuritin dose-dependent enhanced auditory recovery in gerbils (Fig. 5A).

After the ABR auditory function test was completed, the tissue level was observed. The cochlear tissues of gerbils in each group were collected, fixed, decalcified, dehydrated, embedded, frozen and sectioned, and then immunofluorescence detection was performed. The results showed that a large number of SGNs in each gyrus of cochlea was lost and only a small amount remained after injury. After Neuritin treatment, the number of residual SGNs in the cochlear gyrus was significantly higher than that in the injured group. The Neuritin (32µg) group was more significant than the Neuritin (16µg) group (Fig. 5B). The result of histomorphology was consistent with that of ABR.

At the same time, the basal membrane of the cochlea was taken from each group and the changes of hair cells were detected by immunofluorescence assay. It was found that there was almost no loss of hair cells in each group, and there was no significant difference from the normal group (Fig. 6).

3.6 Neuritin protects damaged SGNs in gerbils

In this study, the tissue of Corti's organ of newly-born gerbils was cultured in vitro and the ouabain injury model was established in vitro. The protective effects of Neuritin on damaged gerbils cochlea cells were investigated by adding exogenous Neuritin proteins at different concentrations. The results showed that the SGNs (25.7 ± 0.6/100µm²) and the nerve fiber (13.4 ± 1.1/100µm) were arranged in order under normal conditions. After ouabain injury, the number of SGNs (7.3 ± 2.2/100µm²) and nerve fibers (4.6 ± 1.1/100µm) was significantly reduced. Interestingly, the number of spiral ganglion cells (13.2 ± 1.2/100µm²) and nerve fibers (8.2 ± 1.1/100µm) were higher after Neuritin (16µg/mL) treatment than in the injured group, and the nerve fibers were arranged in a better order. The Neuritin (32µg/mL) treatment group had a more significant effect, with 16 ± 2.6/100µm² and 11.6 ± 1.5/100µm SGNs and nerve fibers, respectively (Fig. 7).

Meanwhile, the morphological changes of hair cells in each group were detected, and the results showed that there was no significant loss of hair cells in each group, and no significant difference from the control group (Fig. 8). In conclusion, exogenous Neuritin intervention can significantly protect the number of SGNs and nerve fibers, maintain the structural connection, and maintain the integrity of auditory nerve pathway.

4.1 Expression of NTFs in the auditory system

NTFs are proteins necessary for the growth, differentiation, and survival of nerve cells[26]. They can affect the development of the nervous system in multiple ways, regulate the distribution of neurons, and promote the repair and regeneration of neurons and synapses. Studies have shown that a variety of factors, including BDNF, NT3, CNTF, GDNF, NT4/5, neurturin, artemin, TGF, and FGF[8, 27–29], are involved in the development of inner ear tissue and survival of hair cells and SGNs.

However, the expression of neuritin in the gerbil is still unclear. We found that neuritin has different degrees of expression in all stages of gerbil development. At E20, the expression level was low; however, from birth to P56, the expression of neuritin in the cochlea continued to increase. The results suggest that neuritin may play an important role in the development of the cochlea in both the juvenile and adult gerbil.

In the study of rat cochlear development, it was found that the expression of BDNF mRNA in the cochlea began to decrease from E17, and reached a minimum level on the 4th day after birth. However, the expression of NT-3 mRNA was still high at E17, however the expression decreased after E21[30]. In addition, studies have shown that NT-3, CNTF, and GDNF have lower expression levels before birth and increased expression after birth; similar to the expression of neuritin in the gerbil cochlea. The expression levels of neurturin and artemin did not change significantly before and after birth, and were in a relatively constant state. There is still a certain spatial and temporal difference in the expression of CNTF and BDNF in the cochlea. BDNF expression is higher in the cochlear gyrus during the development of the cochlea, but lower in the low gyrus, whereas CNTF expression is the opposite [10]. The results indicate that the expression of different NFTs in the development of the cochlea is very different.

4.2 Correlation between neuritin and sensorineural deafness

According to the above experimental results, neuritin plays an important role in the development of gerbils from birth to adulthood. However, there is no report on the correlation between neuritin and ouabain-induced sensorineural deafness in gerbils. The results of this study showed that the expression of neuritin was down-regulated in the gerbil cochlea after ouabain-induced injury. The result indicated that neuritin was associated with ouabain-induced sensorineural deafness and was negatively correlated. The results are consistent with the bioinformatics analysis. It is suggested that exogenous neuritin may have a certain effect on the protection and treatment of deafness. A recent study indicated that the expression of several NTFs including NT-3, CNTF, GDNF, and BDNF in the cochlear tissue of rats after kanamycin-induced injury was also down-regulated. However, neurturin and artemin did not show significant changes before or after injury[10]. The results indicate that the expression of different NTFs in the development of the cochlea and the development of auditory function are complex, and not all NTFs are related to the development of deafness.

4.3 Protective effect of NTFs on cochlear SGNs

Here, the results of in vitro and in vivo experiments indicated that neuritin not only had significant protective effects on damaged cochlear spiral ganglion neurons, moreover, it effectively protected nerve fibers and maintained their ordered arrangement. Further, neuritin promoted the structural connection between hair cells and SGNs, maintained the integrity of the entire auditory signal transmission chain, and thus achieved the purpose of protecting hearing. In our previous research neuritin was also found to effectively protect the number and arrangement of cochlear nerve fibers and maintain the effective connection of the auditory nerve chain in mice. In addition, recombinant human neuritin has been shown to effectively maintain the arrangement of nerve fibers after rat sciatic nerve injury, thereby promoting the recovery of damaged nerve structures[16].

In previous studies, various NTFs have been shown to promote protective effects against neuronal damage. NTF-mediated maintenance of synaptic connections is also another in preventing neuronal degeneration[31]. BDNF, NT-3, and GDNF can effectively promote SGN survival after noise exposure or ototoxic drug damage[32, 33]. In addition, others have found that cochlear electrical stimulation (ES) can effectively prevent SGN degeneration caused by hair cell loss. A combined cochlear ES and GDNF overexpression therapy approach provided robust anti-SGN degeneration effects[34]. Taken together, the data indicate that NTFs play a role in the maintenance of SGNs in varying degrees, which is in agreement with the results of the current study and further demonstrates that neuritin has the potential to treat sensorineural deafness. The treatment paradigm presented here provides a theoretical basis and offers novel therapy options.

4.4 Discussion on the protective effect of recombinant human neuritin on hearing function

Research on hearing, especially the study of hearing function recovery, has always been a clinical problem, and researchers have tried to achieve the effect of recovery of damaged hearing function through different strategies. In this experiment, an SGN-specific injury model was established by ouabain, after which the round window was treated with exogenous neuritin and sustainedly released with gelatin sponge. It was found that neuritin effectively recovered the high-frequency hearing function of the damaged cochlea. Further, neuritin (16 µg) treatment recovered about 20 dB at a frequency of 32 kHz compared to the injured group. At the same frequency, neuritin (32 µg) treatment recovered 30 dB. Tissue level assays showed that neuritin dose-dependently protected the number and arrangement of SGNs and nerve fibers in the damaged cochlea of gerbils. Interestingly, a variety of NTFs have protective effects on damaged neurons. Specifically, the sustained release of gelatin sponge and the instillation of BDNF through the round window effectively promoted the survival of SGNs but that ABR detection showed the I wave amplitude was not significantly different from the injury group[32].

The results of the current study indicate that under such damaged conditions, neuritin has a good protective effect on the high frequency region of the cochlea in gerbils, suggesting that it has the potential to treat sensorineural deafness. However, the specific protective mechanism is still unclear.

In summary, we demonstrate that neuritin is expressed during the development of the cochlea of gerbils, especially during adolescence and adulthood. Further, neuritin is primarily expressed in the SGNs and Corti regions of the cochlea. In vitro and in vivo experiment revealed that neuritin significantly maintained the number and arrangement of SGNs and nerve fibers in the damaged cochlea and effectively protected the high-frequency hearing function of gerbils.

Acknowledgements

The authors would like to thank the Animal center of Hangzhou Normal University for breeding the mice. Dr Xiaofeng Chu (Animal Management Office, Hangzhou Medical College, Hangzhou, china) kindly provided the gerbils.

Funding

This research was supported by The Key R&D program of Zhejiang Province (grant No. 2019C03060) and Zhejiang Provincial Natural Science Foundation under grant No. LY18H260002.

Availability of data and materials

The data used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

Yang Lei, Song Xiaoming, and Huang Jin contributed to the conception or design of the study. Gui Fei, Song Dandan and Wang Haiyan contributed to the acquisition, analysis, or interpretation of the gerbils data. Sun Xiaopin contributed to the acquisition, analysis, or interpretation of all cell-level data. Zhang Shuai and Song Dandan contributed to the acquisition, analysis and interpretation of the bioinformatics data. Gui Fei, Hong Yu, Yang Yi and Chen Rong contributed to the drafting of the the article and revising it critically for all content. All authors contributed to revising the work critically for important intellectual content.

Ethics approval and consent to participate

All experimental procedures were approved by the Animal Ethics committee of Hangzhou Normal University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Pogson JM, Halmagyi GM. Hearing but not understanding: word deafness from a brainstem lesion. Lancet, 399(10326), 756 (2022).
McMahon CM, Nieman CL, Thorne PR, Emmett SD, Bhutta MF. The inaugural World Report on Hearing: From barriers to a platform for change. Clin Otolaryngol, 46(3), 459-463 (2021).
Kil J, Harruff EE, Longenecker RJ. Development of ebselen for the treatment of sensorineural hearing loss and tinnitus. Hear Res, 413, 108209 (2022).
Wang J, Puel JL. Toward Cochlear Therapies. Physiol Rev, 98(4), 2477-2522 (2018).
Carol Liu YC, Ibekwe T, Kelso JM et al. Sensorineural hearing loss (SNHL) as an adverse event following immunization (AEFI): Case definition & guidelines for data collection, analysis, and presentation of immunization safety data. Vaccine, 38(30), 4717-4731 (2020).
Hickman TT, Hashimoto K, Liberman LD, Liberman MC. Cochlear Synaptic Degeneration and Regeneration After Noise: Effects of Age and Neuronal Subgroup. Front Cell Neurosci, 15, 684706 (2021).
Yu Y, Yang J, Luan F et al. Sensorineural Hearing Loss and Mitochondrial Apoptosis of Cochlear Spiral Ganglion Neurons in Fibroblast Growth Factor 13 Knockout Mice. Front Cell Neurosci, 15, 658586 (2021).
Mukherjee S, Kuroiwa M, Oakden W et al. Local magnetic delivery of adeno-associated virus AAV2(quad Y-F)-mediated BDNF gene therapy restores hearing after noise injury. Mol Ther, 30(2), 519-533 (2022).
Kanzaki S, Toyoda M, Umezawa A, Ogawa K. Application of Mesenchymal Stem Cell Therapy and Inner Ear Regeneration for Hearing Loss: A Review. Int J Mol Sci, 21(16) (2020).
Bailey EM, Green SH. Postnatal expression of neurotrophic factors accessible to spiral ganglion neurons in the auditory system of adult hearing and deafened rats. J Neurosci, 34(39), 13110-13126 (2014).
Nedivi E, Hevroni D, Naot D, Israeli D, Citri Y. Numerous candidate plasticity-related genes revealed by differential cDNA cloning. Nature, 363(6431), 718-722 (1993).
Karamoysoyli E, Burnand RC, Tomlinson DR, Gardiner NJ. Neuritin mediates nerve growth factor-induced axonal regeneration and is deficient in experimental diabetic neuropathy. Diabetes, 57(1), 181-189 (2008).
Vink HA, Versnel H, Kroon S, Klis SFL, Ramekers D. BDNF-mediated preservation of spiral ganglion cell peripheral processes and axons in comparison to that of their cell bodies. Hear Res, 400, 108114 (2021).
Hashimoto K, Hickman TT, Suzuki J et al. Protection from noise-induced cochlear synaptopathy by virally mediated overexpression of NT3. Sci Rep, 9(1), 15362 (2019).
Zhang Y, Zhang S, Xian L et al. Expression and purification of recombinant human neuritin from Pichia pastoris and a partial analysis of its neurobiological activity in vitro. Appl Microbiol Biotechnol, 99(19), 8035-8043 (2015).
Wang H, Li X, Shan L et al. Corrigendum: Recombinant hNeuritin Promotes Structural and Functional Recovery of Sciatic Nerve Injury in Rats. Front Neurosci, 11, 65 (2017).
Gao R, Li X, Xi S et al. Exogenous Neuritin Promotes Nerve Regeneration After Acute Spinal Cord Injury in Rats. Hum Gene Ther, 27(7), 544-554 (2016).
Zhu J, Meng P, Wang Q et al. Effects of neuritin on the differentiation of bone marrowderived mesenchymal stem cells into neuronlike cells. Mol Med Rep, 16(3), 3201-3207 (2017).
Cantallops I, Haas K, Cline HT. Postsynaptic CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in vivo. Nat Neurosci, 3(10), 1004-1011 (2000).
Fujino T, Leslie JH, Eavri R et al. CPG15 regulates synapse stability in the developing and adult brain. Genes Dev, 25(24), 2674-2685 (2011).
Putz U, Harwell C, Nedivi E. Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis. Nat Neurosci, 8(3), 322-331 (2005).
Sharma TP, Liu Y, Wordinger RJ, Pang IH, Clark AF. Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush. Cell Death Dis, 6, e1661 (2015).
He Y, Yang G, Wang Y et al. Expression of candidate plasticity-related gene 15 is increased following traumatic brain injury. Neurol Res, 35(2), 174-180 (2013).
Liu Y, Chu HQ, Sun YB et al. Expression of alpha2-Na/K-ATPase in C57BL/6J Mice Inner Ear and Its Relationship with Age-related Hearing Loss. Curr Med Sci, 41(1), 153-157 (2021).
Ding D, Prolla T, Someya S, Manohar S, Salvi R. Roles of Bak and Sirt3 in Paraquat-Induced Cochlear Hair Cell Damage. Neurotox Res, 39(4), 1227-1237 (2021).
Schulze J, Staecker H, Wedekind D, Lenarz T, Warnecke A. Expression pattern of brain-derived neurotrophic factor and its associated receptors: Implications for exogenous neurotrophin application. Hear Res, 413, 108098 (2022).
Rak K, Volker J, Frenz S et al. Effects of the neurotrophic factors BDNF, NT-3, and FGF2 on dissociated neurons of the cochlear nucleus. Neuroreport, 25(12), 960-964 (2014).
Stover T, Scheper V, Diensthuber M, Lenarz T, Wefstaedt P. [In vitro neurite outgrowth induced by BDNF and GDNF in combination with dexamethasone on cultured spiral ganglion cells]. Laryngorhinootologie, 86(5), 352-357 (2007).
Feng J, Bendiske J, Morest DK. Postnatal development of NT3 and TrkC in mouse ventral cochlear nucleus. J Neurosci Res, 88(1), 86-94 (2010).
Wheeler EF, Bothwell M, Schecterson LC, von Bartheld CS. Expression of BDNF and NT-3 mRNA in hair cells of the organ of Corti: quantitative analysis in developing rats. Hear Res, 73(1), 46-56 (1994).
Glazner GW, Mu X, Springer JE. Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system. J Comp Neurol, 391(1), 42-49 (1998).
Havenith S, Versnel H, Agterberg MJ et al. Spiral ganglion cell survival after round window membrane application of brain-derived neurotrophic factor using gelfoam as carrier. Hear Res, 272(1-2), 168-177 (2011).
Budenz CL, Pfingst BE, Raphael Y. The use of neurotrophin therapy in the inner ear to augment cochlear implantation outcomes. Anat Rec (Hoboken), 295(11), 1896-1908 (2012).
Scheper V, Paasche G, Miller JM et al. Effects of delayed treatment with combined GDNF and continuous electrical stimulation on spiral ganglion cell survival in deafened guinea pigs. J Neurosci Res, 87(6), 1389-1399 (2009).

Download PDF

Version 1

posted

You are reading this latest preprint version

Neuritin restores auditory following cochlear spiral ganglion neuron denervation of gerbils

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Summary of microarray datasets

2.2 DEGs enrichment analysis and its visualization of the datasets

2.3 Animals and experimental design

2.4 Neural deafness model and neuritin treatment

2.5 Cochlear samples collection

2.6 Organotypic culture of the cochleae

2.7 Electrophysiological recordings

2.8 RNA isolation and qPCR

2.9 Western blot analysis

2.10 Histology

3. Results

3.1 Auditory function and neuritin expression and localization in the cochlea of gerbils

3.2 Expression changes of different genes after ouabain-induced injury using bioinformatics analysis

3.3 Establishment of the cochlear SGN injury model

3.4 Neuritin expression in the gerbil cochlea after ouabain-induced injury

3.5 Neuritin mitigates impaired auditory function in gerbils

3.6 Neuritin protects damaged SGNs in gerbils

4. Discussion

4.1 Expression of NTFs in the auditory system

4.2 Correlation between neuritin and sensorineural deafness

4.3 Protective effect of NTFs on cochlear SGNs

4.4 Discussion on the protective effect of recombinant human neuritin on hearing function

Declarations

References

Status:

Version 1