Regenerative treatment of middle ear mucosa with topical retinoid administration

Regenerative treatment of middle ear mucosa with topical retinoid administration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Regenerative treatment of middle ear mucosa with topical retinoid administration Sayuri Terada, Chikako Shinkawa, Yu Koizumi, Motoyasu Sugiyama, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6273919/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Middle ear dysfunction can lead to hearing loss and diseases, most commonly cholesteatoma. Although radical treatment of cholesteatoma involves complete removal of the lesion, incomplete regeneration of the middle ear mucosa can lead to hearing loss and recurrence of cholesteatoma. Recently, several middle ear mucosal regeneration therapies have been reported; however, none have been widely used clinically. Therefore, we focused on retinoids that have been reported to have regenerative effects on the nasal mucosal epithelium. We investigated the efficacy of retinoids in an animal model of middle ear mucosal damage using guinea pigs. Our results suggest that the regenerated ciliated epithelium has histological and functional characteristics similar to those of the normal epithelium. We additionally investigated the ototoxicity of retinoids and found no evidence of such. Retinoids may be useful as a novel therapy for middle ear mucosal regeneration. Health sciences/Diseases Health sciences/Medical research Cholesteatoma cilia middle ear mucosa mucosal regeneration retinoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The middle ear has an air-containing cavity called the tympanic cavity, which consists of bony tissue covered with a thin layer of mucosal epithelium. The middle ear cavity is important for efficient transmission of sound to the inner ear; poor sound transmission results in hearing loss if pneumatization of the middle ear cavity is compromised. Maintaining middle ear ventilation and middle ear cavity clearance is important for preserving normal middle ear function. Middle ear ventilation is controlled by the Eustachian tube and passive gas exchange in the middle ear mucosa 1 . In addition, the ciliated epithelium around the Eustachian tube plays an important role in maintaining middle ear clearance 2 3 4 . Impaired middle ear ventilation and clearance can lead to hearing loss and a variety of middle ear disorders, including cholesteatoma, which destroys the temporal bone. In addition, cholesteatoma can cause hearing loss, dizziness, and facial paralysis because the areas around the middle ear cavity contain important structures such as the cochlea, semicircular canal, and facial nerve. Curative treatment for cholesteatoma is complete surgical removal, which requires combined removal of the pathological mucosa surrounding the cholesteatoma. However, postoperative regeneration of the middle ear mucosa can be challenging, resulting in hearing loss and frequent recurrence of cholesteatoma. Prevention of recurrence is clinically important as the postoperative recurrence rate of cholesteatoma is relatively high at 17–37% 5 6 7 . To achieve sufficient postoperative pneumatization and reduce the recurrence rate of cholesteatoma, several methods of middle ear mucosal regeneration have been reported in vivo 8 9 . Recently, autologous nasal mucosal epithelial cell sheets have been developed for postoperative middle ear mucosal regeneration. Yamamoto et al. reported that transplantation of such cultured cell sheets promoted middle ear mucosal regeneration in rabbits 10 and this cell sheet transplantation method has been approved in humans 11 . However, this treatment has not been widely adopted due to several problems, such as the preparation time before transplantation, the need for high-quality culture facilities, and the high cost. A more clinically applicable regeneration treatment of the middle ear mucosa is needed to reduce the recurrence rate of cholesteatoma. In this study, we focused on retinoids, a generic term for vitamin A and its related compounds, which are widely used clinically for systemic treatment of hematological and skin diseases. More recently, retinoids have been reported to promote differentiation and proliferation of keratinocytes 12 and have been used for topical treatment of skin conditions such as psoriasis and acne vulgaris 13 . Although there are few studies on retinoids in the field of otorhinolaryngology, a regenerative effect of retinoids on ciliated nasal epithelial cells has been reported 14 15 . Based on those reports, we created a model of middle ear mucosal damage in guinea pigs and investigated the regenerative effect of retinoids on the middle ear mucosa in vivo. This could lead to reconstruction of a normal ventilated middle ear cavity and development of treatments to prevent cholesteatoma recurrence with improved postoperative hearing. Results Histological evaluation of mucosal regeneration due to retinoid administration Twelve guinea pigs were divided into two groups: the retinoid treatment group and control group, which was treated with ointment base only. A 0.02% retinoid solution was created by mixing tretinoin (NANOEGG Research Laboratories, Inc. Tokyo, Japan) 16 with ointment base (Squalane, hydrogenated lecithin, cholesterol; NANOEGG Research Laboratories, Inc.). The surgical procedure was performed via a transcanal approach using a 1.9-mm endoscope (Karl Storz SE & Co. KG, Tuttlingen, Germany). The middle ear mucosa around the Eustachian tube was removed with a pick and the drug was injected into the middle ear cavity. Mucosal regeneration was evaluated histologically at 2 weeks postoperatively. The frozen sections were evaluated by hematoxylin and eosin (HE) staining. The sections were observed at three fields of view: the center of the healing area, outer 200 µm, and inner 200 µm. We defined "healing" as regeneration of cilia similar to that of the normal mucosa in all three fields, "improvement" as regeneration of cilia in any of the three fields, and "unchanged" as the absence of cilia in all three fields. According to HE staining, five of the six animals in the retinoid group demonstrated regeneration of the ciliated epithelium (Fig. 1 a, c). Four of the six animals exhibited healing, and one showed improvement. One unchanged animal showed no epithelial regeneration. All six animals in the control group were unchanged, without any regeneration of the ciliated epithelium. The control animals did not show obvious epithelial regeneration but showed denser submucosal tissue growth compared with spontaneously healing tissue after mucosal stripping with no drugs (Fig. 1 b, d). Similar results were seen in the high-power field (Fig. 2 a–d). The retinoid group showed significantly more ciliary regeneration than the control group (P = 0.0183; Table 1 ). Table 1 Regeneration effect on ciliated epithelium Healing Improvement Unchanged Retinoid 4 1 1 Control 0 0 6 Of the six animals, four showed healing, one showed improvement, and one showed no change in the retinoid group, whereas all 6 animals in the control group showed no change. (p = 0.0183) In the immunohistological study, all five animals in the retinoid group with regenerated ciliated epithelium showed positive staining of the epithelial cell markers E-cadherin and pan-cytokeratin and the ciliary marker tubulin (Fig. 3 ). Evaluation of ciliary motility Three guinea pigs were treated with retinoids after stripping the middle ear mucosa. Specimens of the middle ear mucosa were harvested 2 weeks after treatment. Three guinea pigs that did not undergo surgery were used as the normal middle ear mucosa group, in which the middle ear mucosa was harvested following the same method. The harvested middle ear mucosa was observed, and videos of ciliary mobility were recorded. Videos were taken in three arbitrary fields of view per sample and analyzed using Cilia FA 17 , a plug-in software for ImageJ ( https://imagej.nih.gov/ij/ ), to measure the mean ciliary beat frequencies (CBF). CBF was measured as the number of times the cilia rippled in 1 s, an indicator of ciliary motility. The mean values from the three fields of view were compared between two groups. CBF in the retinoid and normal groups were 8.02 ± 0.60 and 8.24 ± 0.34 Hz, respectively. There was no significant difference between two groups (Supplementary Fig. 1). Evaluation of retinoid ototoxicity Hearing changes evaluated with auditory brainstem response (ABR) and inner ear histology were evaluated to assess the ototoxic potential of retinoids. Six guinea pigs were divided into two groups: the retinoid treatment group and control group not treated with any drugs. A 0.05% retinoid solution was created by mixing tretinoin (NANOEGG Research Laboratories, Inc.) with ointment base (NANOEGG Research Laboratories, Inc.). The three guinea pigs in the retinoid treatment group were injected with retinoids without stripping the middle ear mucosa after opening the tympanic membrane using a pick. The three guinea pigs in the control group were not injected with any drug after opening the tympanic membranes using a pick. The postoperative hearing changes in the retinoid and control groups are shown in Fig. 4 . Hearing changes were calculated by subtracting the preoperative hearing threshold from the hearing threshold at each week. In the retinoid group, hearing loss of approximately 15 dB was observed at 4, 8, and 32 kHz at 2 weeks after surgery. However, the hearing loss gradually recovered to the preoperative hearing level at 8 weeks (Fig. 4 a). In the control group, hearing loss of approximately 10 dB was observed at 4 and 8 kHz at 2 weeks after surgery, and it also recovered to the preoperative hearing level at 8 weeks (Fig. 4 b). Latency-intensity (L-I) curves, which show the relationship between ABR latency and sound pressure, in the retinoid group at 2 weeks postoperatively showed prolonged latency compared with preoperative values at all frequencies, regardless of sound pressure. In contrast, the L–I curves in the control group at 2 weeks postoperatively showed prolonged latency at 4 and 8 kHz, but not at 16 or 32 kHz. (Supplementary Fig. 2). Hair cells of the organ of Corti at 2 weeks after retinoid treatment are shown in Fig. 5 a to d. All the outer hair cells and inner hair cells were preserved after retinoid treatment. In addition, the spiral ganglia after retinoid administration were equivalent to the normal ganglia (Fig. 5 e, f). Evaluation of retinoid receptor expression in the middle ear mucosa The mRNA expression of retinoid receptors was investigated in the middle ear mucosa using reverse-transcription PCR to confirm the presence of retinoid receptors. Retinoid receptors belong to two families, RARs and RXRs, and each family comprises three isotypes, α, β, and γ, all of which were examined. Expression of mRNA for all retinoid receptors was observed in the middle ear mucosa of guinea pigs (Fig. 6 ). Discussion Maintaining middle ear ventilation and middle ear cavity clearance is essential for normal pneumatization in the middle ear cavity, with the middle ear mucosa playing a particularly important role. The histology of the mucosa in the human middle ear cavity varies depending on anatomical location. Specifically, the area around the Eustachian tube in the anterior part of the middle ear cavity has a single layer of ciliated columnar epithelium, whereas the area near the mastoids in the posterior part has a single layer of cuboidal or squamous epithelium 18 . The function of each of these mucosal epithelia is not completely clear. However, the histological features suggest that the anterior ciliated columnar epithelium maintains clearance of the middle ear cavity via secretions directed toward the Eustachian tube 2 3 4 , whereas the posterior cuboidal or squamous epithelium plays a role in middle ear ventilation by passive gas exchange 19 . According to Lim et al., the middle ear cavity of guinea pigs has the same variations in mucosal histology as that of humans. The mucosal epithelium around the Eustachian tube was evaluated in this study because there is abundant ciliated epithelium around the Eustachian tube 20 . Cholesteatoma is a serious complication of otitis media caused by middle ear cavity dysfunction. Although various theories have been proposed as to the cause of cholesteatoma 21 , the most common theory is that middle ear function is impaired after inflammation of the middle ear, resulting in negative pressure in the middle ear cavity, which in turn induces a depression in part of the tympanic membrane and deposition of debris in that area 22 . Pauna et al. performed a histological study of human temporal bone from a case of cholesteatoma and reported that the ciliated epithelium was reduced in the anterior region of the middle ear cavity compared with normal temporal bone 23 . Thus, disorders of the middle ear mucosa are important causes of cholesteatoma. In addition, surgery for cholesteatoma requires comprehensive removal of the diseased mucosa surrounding the cholesteatoma to remove it completely. However, the postoperative middle ear cavity is covered by scar tissue, not normal middle ear mucosal epithelium. Postoperative dysfunction of the middle ear cavity can lead to hearing loss due to negative pressure and adhesions within the tympanic cavity, as well as cholesteatoma recurrence with retraction of the tympanic membrane. Staged surgery is used to create a ventilated cavity to improve postoperative middle ear function. However, the postoperative middle ear cavity is covered by scar tissue rather than normal mucosal epithelium. Staged surgery does not completely prevent cholesteatoma recurrence because scar tissue has neither gas exchange nor excretory function. Practical middle ear mucosal regeneration therapy is needed to solve this clinical problem. Therefore, in this study, we used retinoids, which have been used clinically to treat other diseases. There are several reports on the regenerative effects of retinoids on the epithelium 24 25 . For example, in the field of otorhinolaryngology, Hwang et al. reported that topical administration of retinoids to damaged rabbit nasal mucosal epithelium resulted in regeneration of nasal ciliated epithelium 15 . Normal human middle ear epithelial cells and human cholesteatoma epithelial cells morphologically differentiate into ciliated epithelium after retinoid exposure in culture 26 27 . Therefore, retinoids may be important for differentiation of the middle ear mucosa into normal ciliated epithelium. To the best of our knowledge, this is the first report on middle ear mucosal regeneration by topical retinoid administration; neither histological nor functional studies have been reported. According to our HE stained specimens, five of six animals in the retinoid group showed regeneration of their ciliated epithelium similar to normal middle ear mucosa, indicating a substantial regenerative effect on ciliated epithelium compared with the control group. In addition, our immunohistological analysis revealed characteristics of ciliated epithelium in the regenerated mucosa, similar to normal mucosal epithelium, in the retinoid group. In previous reports on topical administration of retinoids, the concentration of retinoids ranged between 0.01% and 0.25% 14 15 . In a pilot study, we examined retinoid concentrations of 0.01%, 0.02%, 0.05%, and 0.25%. The highest rate of regeneration of the ciliated epithelium was 0.02% and harmful granulation tissue was observed in the middle ear cavity with poor pneumatization in all cases at the highest concentration (0.25%). It has been reported that retinoids also have a granulation effect during the wound healing process 28 , and this effect may have led to the appearance of granulation tissue at the 0.25% retinoid concentration. Based on the results of these pilot studies, we used a retinoid concentration of 0.02% in this study, which gave good results. We examined the expression of retinoid receptors because there have been no previous reports on the expression of retinoid receptors in the middle ear mucosa. The active form of retinoids is all-trans-retinoic acid (retinoic acid), which specifically binds to the nuclear retinoic acid receptor (RAR). RAR functions as a transcription factor by forming a dimer with retinoid X receptor (RXR), which is a homologous nuclear receptor, and each of these receptors has three isotypes termed as α, β, and γ. Retinoid receptors are widely expressed throughout the body. However, their expression patterns differ among tissues. For example, most of the retinoid receptors expressed in the skin are RARγ and RXRα, whereas RXRγ is not expressed 29 . Furthermore, although all isotypes are expressed in the intestinal mucosa, their expression levels differ 30 . This suggests that each retinoid receptor isotype has unique effects and functions, which may vary among tissues depending on the expression pattern 31 . In this study, all retinoid receptors were expressed in normal guinea pig middle ear mucosa. In the future, it will be necessary to investigate the differences in the expression levels of each receptor. We also analyzed ciliary motility, as it is important to assess the function of the ciliated epithelium in addition to histology. We calculated the CBF using image analysis software, as reported by Ueda et al. 32 . CBF has been widely used as an index of the speed of ciliary motility 33 34 . The CBF of the normal human middle ear mucosa was reported to be 8.7 ± 2.4 Hz 34 . In this study, the CBF was almost the same between the retinoid and control groups, with no significant difference found. These results suggest that the regenerated ciliated epithelium induced by retinoid administration may have functional motility similar to that of normal cells. Our results confirm that retinoids are effective for regeneration of the middle ear mucosa. We also evaluated hearing and inner ear histology after retinoid administration to assess the ototoxicity of retinoids for clinical applications. There was a hearing loss of approximately 15 dB at 4, 8, and 32 kHz at 2 weeks after retinoid administration, but by 8 weeks, hearing had recovered to almost the same level as before surgery. Temporary hearing loss also occurred in the control group. However, the degree of hearing loss was greater in the retinoid group; this is thought to be due to granulation caused by the "retinoid reaction" 35 , which temporarily occurs after retinoid administration. The retinoid reaction is an inflammatory response that occurs within 2–5 days after topical administration of retinoids and is associated with various cytokines; however, the response is temporary 36 . In this study, when the tympanic membrane was observed 1–2 weeks after retinoid administration, granulation-like white lesions behind the tympanic membrane were observed by endoscopy. These white lesions decreased in size and disappeared over time. This was thought to be due to a retinoid reaction at the edge of the tympanic membrane perforation during drug injection. These granulation-like lesions may have restricted the mobility of the tympanic membrane and caused a greater degree of temporary hearing loss in the retinoid than control group. We also evaluated the L–I curves to determine whether the increased ABR threshold in the retinoid group was caused by conductive or sensorineural hearing loss due to inner ear damage. The L–I curve is a graph of sound pressure (intensity) on the horizontal axis and latency on the vertical axis, based on wave V of the ABR in humans. We used wave II to create L–I curves because wave II reactivity is stable in guinea pigs. The L–I curve is one of the most used clinical tests for differentiating sensorineural from conductive hearing loss. Specifically, the latency at each sound pressure was prolonged in parallel with conductive hearing loss compared with normal hearing loss. In sensorineural hearing loss, however, the latency is longer than normal at sound pressures around the threshold. However, as the sound pressure increases, the latency approaches normal range. Additionally, at sufficiently high sound pressures, the latency is within normal range. The phenomenon of sensorineural hearing loss describes the recruitment phenomenon, which is when the subjective sense of change in sound loudness in the hearing-impaired ear is much greater than the objective change in sound intensity in the normal ear 37 . This recruitment phenomenon is thought to be due mainly to damage to the outer hair cells 38 . In contrast, in conductive hearing loss, the latency is prolonged beyond normal, regardless of the sound pressure, because the inner ear function, including hair cells, is not damaged. The L–I curves in our retinoid group showed a parallel prolongation of latency at each sound pressure compared with the preoperative normal ABR, which is characteristic of conductive hearing loss rather than sensorineural hearing loss. We believe that the primary cause of the transiently elevated ABR threshold in the retinoid group was conductive hearing loss due to reduced mobility of the tympanic membrane. There was no evidence of retinoid ototoxicity because the histological evaluation showed that both hair cells and the spiral ganglion had the same normal morphology after retinoid administration. This study has three limitations. First, we did not investigate the effect of retinoids on mucosal regeneration other than on the ciliated epithelium. There is not only ciliated epithelium but also trabecular and squamous epithelium in the middle ear cavity of guinea pigs. Previous reports suggest that mucosal epithelium play a role in passive gas exchange during middle ear ventilation. Therefore, further studies on the regeneration of trabecular and squamous epithelium are warranted. Second, the functional assessment of cilia was performed using CBF only. Functional assessment of cilia requires assessment of directionality and synchrony of ciliary movement as well as CBF 32 . Ciliary transport functions should be assessed using fluorescent microsphere imaging. Third, we have not been able to identify a detailed mechanism of action at the molecular level of the effect of retinoids on ciliary epithelial regeneration. In the future, we would like to investigate the pathways downstream of the retinoid receptors. Despite these limitations, this study is the first to demonstrate the presence of retinoid receptors in the middle ear mucosa and to show that retinoids are effective for regenerating the ciliated epithelium in the middle ear mucosa. In practice, patients with cholesteatoma are treated with transcanal endoscopic ear surgery 39 40 41 42 . We propose that this experimental treatment with retinoids using the same endoscopic approach is the first step in the development of a new therapy for cholesteatoma. Methods Animals Four-week-old male Hartley guinea pigs (Kumagai-Shigeyasu Co., Ltd. Sendai, Japan), ranging in weight from 250 to 350 g, were used. All guinea pigs had free access to chow diet and water until the end of the study period. This study was approved by the Animal Research Committee of Yamagata University Faculty of Medicine (Project Identification No. R4080). Mucosal regeneration by topical administration of retinoids All procedures were performed under general anesthesia. Anesthesia was achieved by intraperitoneal systemic injection of midazolam (4 mg/kg; Astellas Pharma Inc., Tokyo, Japan), medetomidine hydrochloride (0.3 mg/kg; Kyoritsu Pharma, Co., Ltd., Tokyo, Japan), and butorphanol tartrate (5 mg/kg; Meiji Seika Pharma, Co., Ltd., Tokyo, Japan) 43 . The surgical procedure was performed via a transcanal approach using a 1.9-mm endoscope (Karl Storz SE & Co. KG, Tuttlingen, Germany) connected to a built-in video camera using a charge-coupled device (Olympus, Tokyo, Japan) on the left side, as shown in Supplementary Fig. 3. The anterior half of the tympanic membrane was opened using a pick (Supplementary Fig. 3b). The middle ear mucosa around the Eustachian tube was stripped using the pick (Supplementary Fig. 3c), and the drug was injected into the middle ear (Supplementary Fig. 3d). Surgery was terminated by placing a 0.1-mm-thick, 4-mm-diameter silicone sheet over the site of the tympanic membrane opening (Supplementary Fig. 3e). Histological evaluation was performed at 2 weeks postoperatively. Ciliary motility study The harvested middle ear mucosa was immediately immersed in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. Ciliary motility was observed at room temperature using the IX-71 inverted microscope (Olympus). The images were captured using the VW-6000 high-speed camera (Keyence, Tokyo, Japan) with a frame rate of 125 fps and shutter speed of 1/250 s (Supplementary Video). Hearing assessment to evaluate inner ear ototoxicity after retinoid administration A hearing assessment was performed to investigate the possibility of inner ear toxicity caused by the retinoids. The auditory brainstem response (ABR) thresholds were evaluated in a soundproof room before and at 2, 4, 6, and 8 weeks after the surgery in each group. The Tucker–Davis Technologies system (Tucker–Davis Technologies, Inc., Alachua, FL, USA) was used to generate tone burst stimuli of 10 ms duration at frequencies of 4 and 8 kHz. Electrical responses were sampled via needle electrodes subcutaneously inserted on both sides of the auricle, with a ground electrode placed on the back. Responses (1280) to stimuli were averaged at each sound pressure level using PowerLab® software (AD Instruments, Sydney, Australia). ABR waveforms were recorded in 5 dB steps. The lowest stimulus level at which wave II could be discriminated reproducibly was set as the hearing threshold. Retinoid receptor expression Total RNA was extracted from the harvested normal middle ear mucosa using the TRIzol® Plus RNA Purification Kit (Thermo Fisher Scientific). cDNA was synthesized from the total RNA using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). The retinoid receptor isotypes were detected by PCR using Tks Gflex DNA polymerase (Takara Bio, Inc.) and specific primers (Table 2 ). The thermal cycling conditions for PCR were as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles of denaturation at 98°C for 10 s and annealing and extension at 60°C (except for RXRγ: 68°C) for 15 s. The PCR products were separated by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. This study was performed on three individual animals to confirm the reproducibility of the results. Table 2 Primers used for PCR to detect guinea pig retinoid receptor isotypes. Target Primer sequence Product length (bp) RARα forward 5’-TCCACCCACCTTCTGACTGT-3’ 674 reverse 5’-CCGAGCTGTTGTTCGTAGTG-3’ RARβ forward 5’-AAGACCAACAGCCTACGTGC-3’ 444 reverse 5’-TGCGGAAAAAGCCCTTACATC-3’ RARγ forward 5’-TACAGTCACCTCCCATGCTG-3’ 548 reverse 5’-TACGTCGGAAGAAGCCCTTG-3’ RXRα forward 5’-GTGAACTCCTCGTCCCTCAA-3’ 765 reverse 5’-GGGGTCATTTGGCGAGCTA-3’ RXRβ forward 5’-CCACCCACGAGAAAGATTCC-3’ 882 reverse 5’-CCTGGTCATCCAGAGGCAAA-3’ RXRγ forward 5’-TAGCCAGGGTGATTTGGGCT-3’ 959 reverse 5’-AGACCCTTGGCATCTGGGTTA-3’ β-actin forward 5’-CCCATGCCATCCTGCGTCTG-3’ 426 reverse 5’-GCATCCTGTCGGCAATGCCT-3’ RAR, retinoic acid receptor; RXR, retinoid X receptor Histological evaluation of mucosal regeneration and inner ear ototoxicity The guinea pigs were euthanized under anesthesia and perfused transcardially with 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.4). The left temporal bone was collected and immersed in 4% paraformaldehyde overnight, after which it was incubated in 0.25 M ethylenediaminetetraacetic acid (pH 7.4) for 7 days using a microwave-based rapid decalcification device (Azumaya, Tokyo, Japan). The decalcified temporal bone was embedded in optimum cutting temperature compound (Funakoshi, Tokyo, Japan). Frozen specimens of the temporal bone were cut into 8 µm sections using a cryostat (HM525 NX, Thermo Fisher Scientific) and stored at − 20°C. The frozen sections were evaluated by hematoxylin and eosin (HE) staining and immunohistochemistry. The sections were rinsed with PBS and incubated in blocking solution (10% donkey serum albumin with 0.3% Triton X-100) for 30 min at room temperature. For the immunohistochemical analysis, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-E-cadherin (1:500; BD Biosciences, Franklin Lakes, NJ, USA) 8 and mouse anti-pan cytokeratin (1:500; Novus Biologicals, Minneapolis, MN, USA) as epithelial cell markers and rat anti-tubulin (1:1000; Abcam, Cambridge, UK) 44 as a ciliary marker. After washing with PBS, the sections were incubated with the following secondary antibodies, diluted 500-fold, for 2 h at room temperature: Alexa568 donkey anti-mouse IgG (Thermo Fisher Scientific), Alexa488 donkey anti-rat IgG (Thermo Fisher Scientific), Alexa568 donkey anti-rabbit IgG (Thermo Fisher Scientific), and Alexa488 donkey anti-rabbit IgG (Thermo Fisher Scientific). In addition, 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. After washing with PBS, the samples were mounted with glycerol and observed. The LSM-700 confocal microscope (Carl Zeiss, Jena, Germany) was used to observe the immunohistochemical results. Hematoxylin and eosin (HE) staining was performed by conventional methods. The BX-53 upright microscope (Olympus) was used to observe HE staining. For evaluation of inner ear ototoxicity, cochlear specimens were harvested from the decalcified temporal bone at 2 weeks postoperatively and prepared for the whole-mount method to evaluate ototoxicity due to retinoids. Whole-mount cochlear tissue in which the organ of Corti was well preserved was observed using ImageJ software and the response regions at 4, 8, 16 and 32 kHz were analysed. Immunohistochemical analysis of the cochlear specimens was performed exactly as described above for the temporal bone sections, with the exception of the antibodies used. The primary antibodies used were rabbit anti-myosin7a (1:500; Proteus Biosciences, Waltham, MA, USA) 45 , as a hair cell marker, and rabbit anti-NF200 (1:1000; Sigma-Aldrich, St. Louis, MO, USA) 45 , as a spiral ganglion marker. The secondary antibodies used were Alexa568 donkey anti-rabbit IgG (Thermo Fisher Scientific) and Alexa488 donkey anti-rabbit IgG (Thermo Fisher Scientific). Statistical analysis For evaluation of lineage epithelial regeneration, the HE staining results were scored as follows: 2, healing; 1, improvement; 0, unchanged. The differences in the HE scores between the retinoid and control groups were statistically analyzed using the two-tailed Mann–Whitney U test. Similarly, in the ciliary motility study, CBF was compared between the retinoid and control groups using the two-tailed Mann-Whitney U test. The results are presented as means ± standard error of the mean. All statistical analyses were performed using Prism 5 (Graph Pad Software, Inc., La Jolla, CA, USA). Statistical significance was set at P < 0.05. Declarations Competing interests All authors declare no financial or non-financial competing interests. Funding: This study was supported by the Ministry of Education, Science, Sports, and Culture (Grant in Aid for Basic Research, C). Author Contribution All authors contributed to the study conception and design. Material preparation, data collection, and data analysis were performed by S.T. and T.I. The first draft of the manuscript was written by S.T., and all authors commented on the previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement This work was supported by JSPS KAKENHI Grant Number 23K08955. Data availability All data associated with this study are present in the paper or the Supplementary Information. All relevant data are available from the corresponding author upon reasonable request. References Gaihede, M., et al. 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Topical pretreatment of diabetic rats with all-trans retinoic acid improves healing of subsequently induced abrasion wounds. Diabetes 54, 855–861 (2005). Choi, J. Y., et al. Retinoic acid depletion induces keratinizing squamous differentiation in human middle ear epithelial cell cultures. Acta Otolaryngol 123, 466–470 (2003). Choi, J. Y., Cho, K. N. & Yoon, J. H. All-trans retinoic acid induces mucociliary differentiation in a human cholesteatoma epithelial cell culture. Acta Otolaryngol 124, 30–35 (2004). Paquette, D., Badiavas, E. & Falanga, V. Short-contact topical tretinoin therapy to stimulate granulation tissue in chronic wounds. J Am Acad Dermatol 45, 382–386 (2001). Fisher, G. J. & Voorhees, J. J. Molecular mechanisms of retinoid actions in skin. Faseb j 10, 1002–1013 (1996). Dong, P., Tao, Y., Yang, Y. & Wang, W. Expression of retinoic acid receptors in intestinal mucosa and the effect of vitamin a on mucosal immunity. Nutrition 26, 740–745 (2010). Pohl, E. & Tomlinson, C. W. E. Classical pathways of gene regulation by retinoids. Methods Enzymol 637, 151–173 (2020). Ueda, Y., et al. Evaluation of regenerated tracheal cilia function on a collagen-conjugated scaffold in a canine model. Interact Cardiovasc Thorac Surg 31, 644–649 (2020). Nakamura, R., Katsuno, T., Tateya, I. & Omori, K. Evaluation of cilia function in rat trachea reconstructed using collagen sponge scaffold seeded with adipose tissue-derived stem cells. Anat Rec (Hoboken) 303, 471–477 (2020). Agius, A. M., Wake, M., Pahor, A. L. & Smallman, L. A. Nasal and middle ear ciliary beat frequency in chronic suppurative otitis media. Clin Otolaryngol Allied Sci 20, 470–474 (1995). Fisher, G. J., et al. Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin. J Invest Dermatol 96, 699–707 (1991). Kim, B. H., Lee, Y. S. & Kang, K. S. The mechanism of retinol-induced irritation and its application to anti-irritant development. Toxicol Lett 146, 65–73 (2003). Coats, A. C. Human auditory nerve action potentials and brain stem evoked responses. Arch Otolaryngol 104, 709–717 (1978). Kiang, N. Y., Liberman, M. C., Sewell, W. F. & Guinan, J. J. Single unit clues to cochlear mechanisms. Hear Res 22, 171–182 (1986). Ito, T., et al. Transcanal endoscopic ear surgery for pediatric population with a narrow external auditory canal. Int J Pediatr Otorhinolaryngol 79, 2265–2269 (2015). Kakehata, S., et al. Extension of indications for transcanal endoscopic ear surgery using an ultrasonic bone curette for cholesteatomas. Otol Neurotol 35, 101–107 (2014). Marchioni, D., Mattioli, F., Alicandri-Ciufelli, M. & Presutti, L. Endoscopic approach to tensor fold in patients with attic cholesteatoma. Acta Otolaryngol 129, 946–954 (2009). Tarabichi, M. Endoscopic management of limited attic cholesteatoma. Laryngoscope 114, 1157–1162 (2004). Kawai, S., Takagi, Y., Kaneko, S. & Kurosawa, T. Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp Anim 60, 481–487 (2011). Hara, K., et al. Clarithromycin suppresses il-13-induced goblet cell metaplasia via the tmem16a-dependent pathway in guinea pig airway epithelial cells. Respir Investig 57, 79–88 (2019). Koizumi, Y., Ito, T., Mizutari, K. & Kakehata, S. Regenerative effect of a rock inhibitor, y-27632, on excitotoxic trauma in an organotypic culture of the cochlea. Front Cell Neurosci 14, 572434 (2020). Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6273919","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432316259,"identity":"c169d9ab-37ed-4f58-b202-9754a990d750","order_by":0,"name":"Sayuri Terada","email":"","orcid":"","institution":"Yamagata University","correspondingAuthor":false,"prefix":"","firstName":"Sayuri","middleName":"","lastName":"Terada","suffix":""},{"id":432316260,"identity":"667c58a5-6e66-4772-aa95-10291415546f","order_by":1,"name":"Chikako Shinkawa","email":"","orcid":"","institution":"Yamagata University","correspondingAuthor":false,"prefix":"","firstName":"Chikako","middleName":"","lastName":"Shinkawa","suffix":""},{"id":432316261,"identity":"851e7664-7b97-4e3c-a9bd-3c6c973a1b37","order_by":2,"name":"Yu Koizumi","email":"","orcid":"","institution":"Yamagata University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Koizumi","suffix":""},{"id":432316262,"identity":"93df8ae3-b410-429a-bae6-81f84ceb299d","order_by":3,"name":"Motoyasu Sugiyama","email":"","orcid":"","institution":"Yamagata University","correspondingAuthor":false,"prefix":"","firstName":"Motoyasu","middleName":"","lastName":"Sugiyama","suffix":""},{"id":432316263,"identity":"8eaaa16a-68d2-4cc2-8969-a4461b3023be","order_by":4,"name":"Akiko Amano","email":"","orcid":"","institution":"Yamagata University","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Amano","suffix":""},{"id":432316264,"identity":"6eed3c15-6fcf-403b-b417-7bcbe86f2f09","order_by":5,"name":"Seiji Kakehata","email":"","orcid":"","institution":"Ota General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Seiji","middleName":"","lastName":"Kakehata","suffix":""},{"id":432316265,"identity":"7a68bf1e-66f5-480a-9700-802c213e3525","order_by":6,"name":"Tsukasa Ito","email":"data:image/png;base64,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","orcid":"","institution":"Yamagata University","correspondingAuthor":true,"prefix":"","firstName":"Tsukasa","middleName":"","lastName":"Ito","suffix":""}],"badges":[],"createdAt":"2025-03-21 04:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6273919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6273919/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79734481,"identity":"5c522834-f5a2-40f5-b814-a51ba310aa71","added_by":"auto","created_at":"2025-04-02 06:41:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":832230,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin staining in the retinoid and control groups (low-power field).\u003c/p\u003e\n\u003cp\u003ea Normal middle ear cavity.\u003c/p\u003e\n\u003cp\u003eb The middle ear cavity in which the middle ear mucosa was stripped, and no drugs were administered.\u003c/p\u003e\n\u003cp\u003ec The middle ear cavity in the retinoid group. Arrowheads indicate damaged mucosal area.\u003c/p\u003e\n\u003cp\u003ed The middle ear cavity in the control group. Arrowheads indicate damaged mucosal area.\u003c/p\u003e\n\u003cp\u003ee Illustration of the temporal bone slice surface.\u003c/p\u003e\n\u003cp\u003eThe retinoid group showed regeneration of the single layer of epithelium similar to that of the normal mucosa (a, c), whereas the middle ear cavity of the control group (stripped only) showed no obvious epithelial regeneration (b, d).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/97c1534e67b7ad2fc796bd77.png"},{"id":79734737,"identity":"45da0519-9c53-4cb6-88e2-4099a5fcf041","added_by":"auto","created_at":"2025-04-02 06:49:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1291543,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin staining in the retinoid and control groups (high-power field)\u003c/p\u003e\n\u003cp\u003ea Normal middle ear mucosa.\u003c/p\u003e\n\u003cp\u003eb Damaged mucosal area in which the middle ear mucosa was stripped, and no drugs were administered.\u003c/p\u003e\n\u003cp\u003ec Damaged mucosal area in the retinoid group.\u003c/p\u003e\n\u003cp\u003ed Damaged mucosal area in the control group.\u003c/p\u003e\n\u003cp\u003eUnder the high-power field, the retinoid group showed regeneration of the ciliated columnar epithelium similar to that of the normal mucosa (a, c). The control group (stripped only) showed no obvious epithelial structures but relatively dense submucosal tissue growth compared with the middle ear mucosa (b, d).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/85a234f96749255d9f91b767.png"},{"id":79734487,"identity":"d16fa80d-9c2b-4c78-83d3-6c34edd346f1","added_by":"auto","created_at":"2025-04-02 06:41:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":598445,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of E-cadherin, pan cytokeratin (PCK-26), and tubulin in the retinoid and control groups.\u003c/p\u003e\n\u003cp\u003ea, b. Normal middle ear mucosa.\u003c/p\u003e\n\u003cp\u003ec, d. Damaged mucosal area in which the middle ear mucosa was stripped, and no drugs were administered.\u003c/p\u003e\n\u003cp\u003ee, f. Damaged mucosal area in the retinoid group.\u003c/p\u003e\n\u003cp\u003eg, h. Damaged mucosal area in the control group.\u003c/p\u003e\n\u003cp\u003eIn the retinoid group, staining of the epithelial cell markers E-cadherin and pan cytokeratin (PCK-26) and the ciliary marker tubulin were positive in the normal mucosa, whereas none of these markers showed staining in the control group.\u003c/p\u003e\n\u003cp\u003eDAPI: 4',6-diamidino-2-phenylindole (nuclear staining)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/9a711f5514570f3e053e38a7.png"},{"id":79734482,"identity":"d679c2d5-f007-4245-9b55-080c2d48e272","added_by":"auto","created_at":"2025-04-02 06:41:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46259,"visible":true,"origin":"","legend":"\u003cp\u003ePostoperative hearing changes in the retinoid and control groups.\u003c/p\u003e\n\u003cp\u003ea. In the retinoid group, hearing loss of approximately 15 dB was observed at 4, 8, and 32 kHz at 2 weeks after surgery. However, the hearing loss recovered gradually to almost the preoperative hearing level at 8 weeks.\u003c/p\u003e\n\u003cp\u003eb. In the control group, hearing loss of approximately 10 dB was observed at 4 and 8 kHz at 2 weeks after surgery. However, it recovered to almost the preoperative hearing level at 8 weeks (Fig. 4b).\u003c/p\u003e\n\u003cp\u003eHearing changes were calculated by subtracting the preoperative hearing threshold from the hearing threshold at each week.\u003c/p\u003e\n\u003cp\u003eerror bar: ± standard error of the mean\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/c0ad9f9ea5b03dee5987ab36.png"},{"id":79734735,"identity":"04a8c6bf-2e0d-4fb1-88ea-6052e2434b6d","added_by":"auto","created_at":"2025-04-02 06:49:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1772351,"visible":true,"origin":"","legend":"\u003cp\u003eHair cells of the organ of Corti at 2 weeks after retinoid treatment and comparison of the spiral ganglion at 2 weeks after retinoid treatment versus without treatment.\u003c/p\u003e\n\u003cp\u003ea Hair cells at 4 kHz.\u003c/p\u003e\n\u003cp\u003eb Hair cells at 8 kHz.\u003c/p\u003e\n\u003cp\u003ec Hair cells at 16 kHz.\u003c/p\u003e\n\u003cp\u003ed Hair cells at 32 kHz.\u003c/p\u003e\n\u003cp\u003ee Normal spiral ganglion.\u003c/p\u003e\n\u003cp\u003ef Retinoid-treated spiral ganglion.\u003c/p\u003e\n\u003cp\u003eNeither the outer nor inner hair cells were disappeared after retinoid treatment at any of the frequencies (a–d). The spiral ganglion showed no dropout of the neurons after retinoid treatment compared with no treatment (e, f).\u003c/p\u003e\n\u003cp\u003eMyosin 7A: hair cell marker\u003c/p\u003e\n\u003cp\u003eDAPI: 4',6-diamidino-2-phenylindole (nuclear staining)\u003c/p\u003e\n\u003cp\u003eRhodamine phalloidin: actin staining\u003c/p\u003e\n\u003cp\u003eNF-200: spiral ganglion marker\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/539065e9cb620ac64710c07d.png"},{"id":79734486,"identity":"8514edeb-129a-48cd-b7b3-9c3b4b944476","added_by":"auto","created_at":"2025-04-02 06:41:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":149086,"visible":true,"origin":"","legend":"\u003cp\u003eRT-PCR analysis of retinoid receptor isotypes in normal guinea pig mucosa\u003c/p\u003e\n\u003cp\u003eRT-PCR analysis of retinoid receptors in the normal middle ear mucosa of guinea pigs detected mRNA expression of all retinoid receptors evaluated.\u003c/p\u003e\n\u003cp\u003eRT-PCR, reverse-transcription PCR\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/61c5c5977862f81f1fff2976.png"},{"id":81209149,"identity":"6d7eae45-53cb-4e18-8d38-0c7b4d6f8b4d","added_by":"auto","created_at":"2025-04-23 12:53:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4755568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/73e5f867-7d88-4b5c-813b-5e59f9cfaab3.pdf"},{"id":79734741,"identity":"a469f201-67bf-4d29-8fcc-52389f5340b5","added_by":"auto","created_at":"2025-04-02 06:49:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":77309866,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6273919/v1/bbc108dbf76f163801b4e24f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regenerative treatment of middle ear mucosa with topical retinoid administration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe middle ear has an air-containing cavity called the tympanic cavity, which consists of bony tissue covered with a thin layer of mucosal epithelium. The middle ear cavity is important for efficient transmission of sound to the inner ear; poor sound transmission results in hearing loss if pneumatization of the middle ear cavity is compromised. Maintaining middle ear ventilation and middle ear cavity clearance is important for preserving normal middle ear function. Middle ear ventilation is controlled by the Eustachian tube and passive gas exchange in the middle ear mucosa \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In addition, the ciliated epithelium around the Eustachian tube plays an important role in maintaining middle ear clearance \u003csup\u003e2 3 4\u003c/sup\u003e. Impaired middle ear ventilation and clearance can lead to hearing loss and a variety of middle ear disorders, including cholesteatoma, which destroys the temporal bone. In addition, cholesteatoma can cause hearing loss, dizziness, and facial paralysis because the areas around the middle ear cavity contain important structures such as the cochlea, semicircular canal, and facial nerve. Curative treatment for cholesteatoma is complete surgical removal, which requires combined removal of the pathological mucosa surrounding the cholesteatoma. However, postoperative regeneration of the middle ear mucosa can be challenging, resulting in hearing loss and frequent recurrence of cholesteatoma. Prevention of recurrence is clinically important as the postoperative recurrence rate of cholesteatoma is relatively high at 17\u0026ndash;37% \u003csup\u003e5 6 7\u003c/sup\u003e. To achieve sufficient postoperative pneumatization and reduce the recurrence rate of cholesteatoma, several methods of middle ear mucosal regeneration have been reported in vivo \u003csup\u003e8 9\u003c/sup\u003e. Recently, autologous nasal mucosal epithelial cell sheets have been developed for postoperative middle ear mucosal regeneration. Yamamoto et al. reported that transplantation of such cultured cell sheets promoted middle ear mucosal regeneration in rabbits \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and this cell sheet transplantation method has been approved in humans \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, this treatment has not been widely adopted due to several problems, such as the preparation time before transplantation, the need for high-quality culture facilities, and the high cost. A more clinically applicable regeneration treatment of the middle ear mucosa is needed to reduce the recurrence rate of cholesteatoma.\u003c/p\u003e \u003cp\u003eIn this study, we focused on retinoids, a generic term for vitamin A and its related compounds, which are widely used clinically for systemic treatment of hematological and skin diseases. More recently, retinoids have been reported to promote differentiation and proliferation of keratinocytes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and have been used for topical treatment of skin conditions such as psoriasis and acne vulgaris \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Although there are few studies on retinoids in the field of otorhinolaryngology, a regenerative effect of retinoids on ciliated nasal epithelial cells has been reported \u003csup\u003e14 15\u003c/sup\u003e. Based on those reports, we created a model of middle ear mucosal damage in guinea pigs and investigated the regenerative effect of retinoids on the middle ear mucosa in vivo. This could lead to reconstruction of a normal ventilated middle ear cavity and development of treatments to prevent cholesteatoma recurrence with improved postoperative hearing.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHistological evaluation of mucosal regeneration due to retinoid administration\u003c/h2\u003e \u003cp\u003eTwelve guinea pigs were divided into two groups: the retinoid treatment group and control group, which was treated with ointment base only. A 0.02% retinoid solution was created by mixing tretinoin (NANOEGG Research Laboratories, Inc. Tokyo, Japan) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e with ointment base (Squalane, hydrogenated lecithin, cholesterol; NANOEGG Research Laboratories, Inc.). The surgical procedure was performed via a transcanal approach using a 1.9-mm endoscope (Karl Storz SE \u0026amp; Co. KG, Tuttlingen, Germany). The middle ear mucosa around the Eustachian tube was removed with a pick and the drug was injected into the middle ear cavity. Mucosal regeneration was evaluated histologically at 2 weeks postoperatively.\u003c/p\u003e \u003cp\u003eThe frozen sections were evaluated by hematoxylin and eosin (HE) staining. The sections were observed at three fields of view: the center of the healing area, outer 200 \u0026micro;m, and inner 200 \u0026micro;m. We defined \"healing\" as regeneration of cilia similar to that of the normal mucosa in all three fields, \"improvement\" as regeneration of cilia in any of the three fields, and \"unchanged\" as the absence of cilia in all three fields.\u003c/p\u003e \u003cp\u003eAccording to HE staining, five of the six animals in the retinoid group demonstrated regeneration of the ciliated epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c). Four of the six animals exhibited healing, and one showed improvement. One unchanged animal showed no epithelial regeneration. All six animals in the control group were unchanged, without any regeneration of the ciliated epithelium. The control animals did not show obvious epithelial regeneration but showed denser submucosal tissue growth compared with spontaneously healing tissue after mucosal stripping with no drugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d). Similar results were seen in the high-power field (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d). The retinoid group showed significantly more ciliary regeneration than the control group (P\u0026thinsp;=\u0026thinsp;0.0183; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRegeneration effect on ciliated epithelium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealing\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImprovement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnchanged\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRetinoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eOf the six animals, four showed healing, one showed improvement, and one showed no change in the retinoid group, whereas all 6 animals in the control group showed no change. (p\u0026thinsp;=\u0026thinsp;0.0183)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the immunohistological study, all five animals in the retinoid group with regenerated ciliated epithelium showed positive staining of the epithelial cell markers E-cadherin and pan-cytokeratin and the ciliary marker tubulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of ciliary motility\u003c/h3\u003e\n\u003cp\u003eThree guinea pigs were treated with retinoids after stripping the middle ear mucosa. Specimens of the middle ear mucosa were harvested 2 weeks after treatment. Three guinea pigs that did not undergo surgery were used as the normal middle ear mucosa group, in which the middle ear mucosa was harvested following the same method.\u003c/p\u003e \u003cp\u003eThe harvested middle ear mucosa was observed, and videos of ciliary mobility were recorded. Videos were taken in three arbitrary fields of view per sample and analyzed using Cilia FA \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, a plug-in software for ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), to measure the mean ciliary beat frequencies (CBF). CBF was measured as the number of times the cilia rippled in 1 s, an indicator of ciliary motility. The mean values from the three fields of view were compared between two groups.\u003c/p\u003e \u003cp\u003eCBF in the retinoid and normal groups were 8.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 and 8.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 Hz, respectively. There was no significant difference between two groups (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003ch3\u003eEvaluation of retinoid ototoxicity\u003c/h3\u003e\n\u003cp\u003eHearing changes evaluated with auditory brainstem response (ABR) and inner ear histology were evaluated to assess the ototoxic potential of retinoids. Six guinea pigs were divided into two groups: the retinoid treatment group and control group not treated with any drugs. A 0.05% retinoid solution was created by mixing tretinoin (NANOEGG Research Laboratories, Inc.) with ointment base (NANOEGG Research Laboratories, Inc.). The three guinea pigs in the retinoid treatment group were injected with retinoids without stripping the middle ear mucosa after opening the tympanic membrane using a pick. The three guinea pigs in the control group were not injected with any drug after opening the tympanic membranes using a pick. The postoperative hearing changes in the retinoid and control groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Hearing changes were calculated by subtracting the preoperative hearing threshold from the hearing threshold at each week. In the retinoid group, hearing loss of approximately 15 dB was observed at 4, 8, and 32 kHz at 2 weeks after surgery. However, the hearing loss gradually recovered to the preoperative hearing level at 8 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the control group, hearing loss of approximately 10 dB was observed at 4 and 8 kHz at 2 weeks after surgery, and it also recovered to the preoperative hearing level at 8 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLatency-intensity (L-I) curves, which show the relationship between ABR latency and sound pressure, in the retinoid group at 2 weeks postoperatively showed prolonged latency compared with preoperative values at all frequencies, regardless of sound pressure. In contrast, the L\u0026ndash;I curves in the control group at 2 weeks postoperatively showed prolonged latency at 4 and 8 kHz, but not at 16 or 32 kHz. (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eHair cells of the organ of Corti at 2 weeks after retinoid treatment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea to d. All the outer hair cells and inner hair cells were preserved after retinoid treatment. In addition, the spiral ganglia after retinoid administration were equivalent to the normal ganglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEvaluation of retinoid receptor expression in the middle ear mucosa\u003c/h3\u003e\n\u003cp\u003eThe mRNA expression of retinoid receptors was investigated in the middle ear mucosa using reverse-transcription PCR to confirm the presence of retinoid receptors. Retinoid receptors belong to two families, RARs and RXRs, and each family comprises three isotypes, α, β, and γ, all of which were examined.\u003c/p\u003e \u003cp\u003eExpression of mRNA for all retinoid receptors was observed in the middle ear mucosa of guinea pigs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMaintaining middle ear ventilation and middle ear cavity clearance is essential for normal pneumatization in the middle ear cavity, with the middle ear mucosa playing a particularly important role. The histology of the mucosa in the human middle ear cavity varies depending on anatomical location. Specifically, the area around the Eustachian tube in the anterior part of the middle ear cavity has a single layer of ciliated columnar epithelium, whereas the area near the mastoids in the posterior part has a single layer of cuboidal or squamous epithelium \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The function of each of these mucosal epithelia is not completely clear. However, the histological features suggest that the anterior ciliated columnar epithelium maintains clearance of the middle ear cavity via secretions directed toward the Eustachian tube \u003csup\u003e2 3 4\u003c/sup\u003e, whereas the posterior cuboidal or squamous epithelium plays a role in middle ear ventilation by passive gas exchange \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. According to Lim et al., the middle ear cavity of guinea pigs has the same variations in mucosal histology as that of humans. The mucosal epithelium around the Eustachian tube was evaluated in this study because there is abundant ciliated epithelium around the Eustachian tube \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCholesteatoma is a serious complication of otitis media caused by middle ear cavity dysfunction. Although various theories have been proposed as to the cause of cholesteatoma \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, the most common theory is that middle ear function is impaired after inflammation of the middle ear, resulting in negative pressure in the middle ear cavity, which in turn induces a depression in part of the tympanic membrane and deposition of debris in that area \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Pauna et al. performed a histological study of human temporal bone from a case of cholesteatoma and reported that the ciliated epithelium was reduced in the anterior region of the middle ear cavity compared with normal temporal bone \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, disorders of the middle ear mucosa are important causes of cholesteatoma. In addition, surgery for cholesteatoma requires comprehensive removal of the diseased mucosa surrounding the cholesteatoma to remove it completely. However, the postoperative middle ear cavity is covered by scar tissue, not normal middle ear mucosal epithelium. Postoperative dysfunction of the middle ear cavity can lead to hearing loss due to negative pressure and adhesions within the tympanic cavity, as well as cholesteatoma recurrence with retraction of the tympanic membrane. Staged surgery is used to create a ventilated cavity to improve postoperative middle ear function. However, the postoperative middle ear cavity is covered by scar tissue rather than normal mucosal epithelium. Staged surgery does not completely prevent cholesteatoma recurrence because scar tissue has neither gas exchange nor excretory function. Practical middle ear mucosal regeneration therapy is needed to solve this clinical problem. Therefore, in this study, we used retinoids, which have been used clinically to treat other diseases.\u003c/p\u003e \u003cp\u003eThere are several reports on the regenerative effects of retinoids on the epithelium \u003csup\u003e24 25\u003c/sup\u003e. For example, in the field of otorhinolaryngology, Hwang et al. reported that topical administration of retinoids to damaged rabbit nasal mucosal epithelium resulted in regeneration of nasal ciliated epithelium \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Normal human middle ear epithelial cells and human cholesteatoma epithelial cells morphologically differentiate into ciliated epithelium after retinoid exposure in culture \u003csup\u003e26 27\u003c/sup\u003e. Therefore, retinoids may be important for differentiation of the middle ear mucosa into normal ciliated epithelium. To the best of our knowledge, this is the first report on middle ear mucosal regeneration by topical retinoid administration; neither histological nor functional studies have been reported.\u003c/p\u003e \u003cp\u003eAccording to our HE stained specimens, five of six animals in the retinoid group showed regeneration of their ciliated epithelium similar to normal middle ear mucosa, indicating a substantial regenerative effect on ciliated epithelium compared with the control group. In addition, our immunohistological analysis revealed characteristics of ciliated epithelium in the regenerated mucosa, similar to normal mucosal epithelium, in the retinoid group. In previous reports on topical administration of retinoids, the concentration of retinoids ranged between 0.01% and 0.25% \u003csup\u003e14 15\u003c/sup\u003e. In a pilot study, we examined retinoid concentrations of 0.01%, 0.02%, 0.05%, and 0.25%. The highest rate of regeneration of the ciliated epithelium was 0.02% and harmful granulation tissue was observed in the middle ear cavity with poor pneumatization in all cases at the highest concentration (0.25%). It has been reported that retinoids also have a granulation effect during the wound healing process \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and this effect may have led to the appearance of granulation tissue at the 0.25% retinoid concentration. Based on the results of these pilot studies, we used a retinoid concentration of 0.02% in this study, which gave good results.\u003c/p\u003e \u003cp\u003eWe examined the expression of retinoid receptors because there have been no previous reports on the expression of retinoid receptors in the middle ear mucosa. The active form of retinoids is all-trans-retinoic acid (retinoic acid), which specifically binds to the nuclear retinoic acid receptor (RAR). RAR functions as a transcription factor by forming a dimer with retinoid X receptor (RXR), which is a homologous nuclear receptor, and each of these receptors has three isotypes termed as α, β, and γ. Retinoid receptors are widely expressed throughout the body. However, their expression patterns differ among tissues. For example, most of the retinoid receptors expressed in the skin are RARγ and RXRα, whereas RXRγ is not expressed \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Furthermore, although all isotypes are expressed in the intestinal mucosa, their expression levels differ \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This suggests that each retinoid receptor isotype has unique effects and functions, which may vary among tissues depending on the expression pattern \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, all retinoid receptors were expressed in normal guinea pig middle ear mucosa. In the future, it will be necessary to investigate the differences in the expression levels of each receptor.\u003c/p\u003e \u003cp\u003eWe also analyzed ciliary motility, as it is important to assess the function of the ciliated epithelium in addition to histology. We calculated the CBF using image analysis software, as reported by Ueda et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. CBF has been widely used as an index of the speed of ciliary motility \u003csup\u003e33 34\u003c/sup\u003e. The CBF of the normal human middle ear mucosa was reported to be 8.7 ± 2.4 Hz \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In this study, the CBF was almost the same between the retinoid and control groups, with no significant difference found. These results suggest that the regenerated ciliated epithelium induced by retinoid administration may have functional motility similar to that of normal cells.\u003c/p\u003e \u003cp\u003eOur results confirm that retinoids are effective for regeneration of the middle ear mucosa. We also evaluated hearing and inner ear histology after retinoid administration to assess the ototoxicity of retinoids for clinical applications. There was a hearing loss of approximately 15 dB at 4, 8, and 32 kHz at 2 weeks after retinoid administration, but by 8 weeks, hearing had recovered to almost the same level as before surgery. Temporary hearing loss also occurred in the control group. However, the degree of hearing loss was greater in the retinoid group; this is thought to be due to granulation caused by the \"retinoid reaction\" \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, which temporarily occurs after retinoid administration. The retinoid reaction is an inflammatory response that occurs within 2–5 days after topical administration of retinoids and is associated with various cytokines; however, the response is temporary \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, when the tympanic membrane was observed 1–2 weeks after retinoid administration, granulation-like white lesions behind the tympanic membrane were observed by endoscopy. These white lesions decreased in size and disappeared over time. This was thought to be due to a retinoid reaction at the edge of the tympanic membrane perforation during drug injection. These granulation-like lesions may have restricted the mobility of the tympanic membrane and caused a greater degree of temporary hearing loss in the retinoid than control group.\u003c/p\u003e \u003cp\u003eWe also evaluated the L–I curves to determine whether the increased ABR threshold in the retinoid group was caused by conductive or sensorineural hearing loss due to inner ear damage. The L–I curve is a graph of sound pressure (intensity) on the horizontal axis and latency on the vertical axis, based on wave V of the ABR in humans. We used wave II to create L–I curves because wave II reactivity is stable in guinea pigs. The L–I curve is one of the most used clinical tests for differentiating sensorineural from conductive hearing loss. Specifically, the latency at each sound pressure was prolonged in parallel with conductive hearing loss compared with normal hearing loss. In sensorineural hearing loss, however, the latency is longer than normal at sound pressures around the threshold. However, as the sound pressure increases, the latency approaches normal range. Additionally, at sufficiently high sound pressures, the latency is within normal range.\u003c/p\u003e \u003cp\u003eThe phenomenon of sensorineural hearing loss describes the recruitment phenomenon, which is when the subjective sense of change in sound loudness in the hearing-impaired ear is much greater than the objective change in sound intensity in the normal ear \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This recruitment phenomenon is thought to be due mainly to damage to the outer hair cells \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In contrast, in conductive hearing loss, the latency is prolonged beyond normal, regardless of the sound pressure, because the inner ear function, including hair cells, is not damaged. The L–I curves in our retinoid group showed a parallel prolongation of latency at each sound pressure compared with the preoperative normal ABR, which is characteristic of conductive hearing loss rather than sensorineural hearing loss. We believe that the primary cause of the transiently elevated ABR threshold in the retinoid group was conductive hearing loss due to reduced mobility of the tympanic membrane.\u003c/p\u003e \u003cp\u003eThere was no evidence of retinoid ototoxicity because the histological evaluation showed that both hair cells and the spiral ganglion had the same normal morphology after retinoid administration.\u003c/p\u003e \u003cp\u003eThis study has three limitations. First, we did not investigate the effect of retinoids on mucosal regeneration other than on the ciliated epithelium. There is not only ciliated epithelium but also trabecular and squamous epithelium in the middle ear cavity of guinea pigs. Previous reports suggest that mucosal epithelium play a role in passive gas exchange during middle ear ventilation. Therefore, further studies on the regeneration of trabecular and squamous epithelium are warranted. Second, the functional assessment of cilia was performed using CBF only. Functional assessment of cilia requires assessment of directionality and synchrony of ciliary movement as well as CBF \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Ciliary transport functions should be assessed using fluorescent microsphere imaging. Third, we have not been able to identify a detailed mechanism of action at the molecular level of the effect of retinoids on ciliary epithelial regeneration. In the future, we would like to investigate the pathways downstream of the retinoid receptors.\u003c/p\u003e \u003cp\u003eDespite these limitations, this study is the first to demonstrate the presence of retinoid receptors in the middle ear mucosa and to show that retinoids are effective for regenerating the ciliated epithelium in the middle ear mucosa. In practice, patients with cholesteatoma are treated with transcanal endoscopic ear surgery \u003csup\u003e39 40 41 42\u003c/sup\u003e. We propose that this experimental treatment with retinoids using the same endoscopic approach is the first step in the development of a new therapy for cholesteatoma.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eFour-week-old male Hartley guinea pigs (Kumagai-Shigeyasu Co., Ltd. Sendai, Japan), ranging in weight from 250 to 350 g, were used. All guinea pigs had free access to chow diet and water until the end of the study period. This study was approved by the Animal Research Committee of Yamagata University Faculty of Medicine (Project Identification No. R4080).\u003c/p\u003e\u003ch3\u003eMucosal regeneration by topical administration of retinoids\u003c/h3\u003e\u003cp\u003eAll procedures were performed under general anesthesia. Anesthesia was achieved by intraperitoneal systemic injection of midazolam (4 mg/kg; Astellas Pharma Inc., Tokyo, Japan), medetomidine hydrochloride (0.3 mg/kg; Kyoritsu Pharma, Co., Ltd., Tokyo, Japan), and butorphanol tartrate (5 mg/kg; Meiji Seika Pharma, Co., Ltd., Tokyo, Japan) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The surgical procedure was performed via a transcanal approach using a 1.9-mm endoscope (Karl Storz SE \u0026amp; Co. KG, Tuttlingen, Germany) connected to a built-in video camera using a charge-coupled device (Olympus, Tokyo, Japan) on the left side, as shown in Supplementary Fig.\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eThe anterior half of the tympanic membrane was opened using a pick (Supplementary Fig.\u0026nbsp;3b). The middle ear mucosa around the Eustachian tube was stripped using the pick (Supplementary Fig.\u0026nbsp;3c), and the drug was injected into the middle ear (Supplementary Fig.\u0026nbsp;3d). Surgery was terminated by placing a 0.1-mm-thick, 4-mm-diameter silicone sheet over the site of the tympanic membrane opening (Supplementary Fig.\u0026nbsp;3e). Histological evaluation was performed at 2 weeks postoperatively.\u003c/p\u003e\u003ch2\u003eCiliary motility study\u003c/h2\u003e\u003cp\u003eThe harvested middle ear mucosa was immediately immersed in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. Ciliary motility was observed at room temperature using the IX-71 inverted microscope (Olympus). The images were captured using the VW-6000 high-speed camera (Keyence, Tokyo, Japan) with a frame rate of 125 fps and shutter speed of 1/250 s (Supplementary Video).\u003c/p\u003e\u003ch2\u003eHearing assessment to evaluate inner ear ototoxicity after retinoid administration\u003c/h2\u003e\u003cp\u003eA hearing assessment was performed to investigate the possibility of inner ear toxicity caused by the retinoids. The auditory brainstem response (ABR) thresholds were evaluated in a soundproof room before and at 2, 4, 6, and 8 weeks after the surgery in each group. The Tucker–Davis Technologies system (Tucker–Davis Technologies, Inc., Alachua, FL, USA) was used to generate tone burst stimuli of 10 ms duration at frequencies of 4 and 8 kHz. Electrical responses were sampled via needle electrodes subcutaneously inserted on both sides of the auricle, with a ground electrode placed on the back. Responses (1280) to stimuli were averaged at each sound pressure level using PowerLab® software (AD Instruments, Sydney, Australia). ABR waveforms were recorded in 5 dB steps. The lowest stimulus level at which wave II could be discriminated reproducibly was set as the hearing threshold.\u003c/p\u003e\u003ch2\u003eRetinoid receptor expression\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from the harvested normal middle ear mucosa using the TRIzol® Plus RNA Purification Kit (Thermo Fisher Scientific). cDNA was synthesized from the total RNA using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio Inc., Shiga, Japan). The retinoid receptor isotypes were detected by PCR using Tks Gflex DNA polymerase (Takara Bio, Inc.) and specific primers (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The thermal cycling conditions for PCR were as follows: initial denaturation at 94°C for 1 min, followed by 35 cycles of denaturation at 98°C for 10 s and annealing and extension at 60°C (except for RXRγ: 68°C) for 15 s. The PCR products were separated by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. This study was performed on three individual animals to confirm the reproducibility of the results.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used for PCR to detect guinea pig retinoid receptor isotypes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProduct length (bp)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRARα\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-TCCACCCACCTTCTGACTGT-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e674\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-CCGAGCTGTTGTTCGTAGTG-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRARβ\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-AAGACCAACAGCCTACGTGC-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-TGCGGAAAAAGCCCTTACATC-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRARγ\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-TACAGTCACCTCCCATGCTG-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e548\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-TACGTCGGAAGAAGCCCTTG-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRXRα\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-GTGAACTCCTCGTCCCTCAA-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e765\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-GGGGTCATTTGGCGAGCTA-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRXRβ\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-CCACCCACGAGAAAGATTCC-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e882\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-CCTGGTCATCCAGAGGCAAA-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRXRγ\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-TAGCCAGGGTGATTTGGGCT-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e959\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-AGACCCTTGGCATCTGGGTTA-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-CCCATGCCATCCTGCGTCTG-3’\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e426\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereverse\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5’-GCATCCTGTCGGCAATGCCT-3’\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eRAR, retinoic acid receptor; RXR, retinoid X receptor\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003ch2\u003eHistological evaluation of mucosal regeneration and inner ear ototoxicity\u003c/h2\u003e\u003cp\u003eThe guinea pigs were euthanized under anesthesia and perfused transcardially with 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.4). The left temporal bone was collected and immersed in 4% paraformaldehyde overnight, after which it was incubated in 0.25 M ethylenediaminetetraacetic acid (pH 7.4) for 7 days using a microwave-based rapid decalcification device (Azumaya, Tokyo, Japan). The decalcified temporal bone was embedded in optimum cutting temperature compound (Funakoshi, Tokyo, Japan). Frozen specimens of the temporal bone were cut into 8 µm sections using a cryostat (HM525 NX, Thermo Fisher Scientific) and stored at − 20°C.\u003c/p\u003e\u003cp\u003eThe frozen sections were evaluated by hematoxylin and eosin (HE) staining and immunohistochemistry. The sections were rinsed with PBS and incubated in blocking solution (10% donkey serum albumin with 0.3% Triton X-100) for 30 min at room temperature. For the immunohistochemical analysis, the sections were incubated with the following primary antibodies overnight at 4°C: mouse anti-E-cadherin (1:500; BD Biosciences, Franklin Lakes, NJ, USA) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and mouse anti-pan cytokeratin (1:500; Novus Biologicals, Minneapolis, MN, USA) as epithelial cell markers and rat anti-tubulin (1:1000; Abcam, Cambridge, UK) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e as a ciliary marker. After washing with PBS, the sections were incubated with the following secondary antibodies, diluted 500-fold, for 2 h at room temperature: Alexa568 donkey anti-mouse IgG (Thermo Fisher Scientific), Alexa488 donkey anti-rat IgG (Thermo Fisher Scientific), Alexa568 donkey anti-rabbit IgG (Thermo Fisher Scientific), and Alexa488 donkey anti-rabbit IgG (Thermo Fisher Scientific). In addition, 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. After washing with PBS, the samples were mounted with glycerol and observed. The LSM-700 confocal microscope (Carl Zeiss, Jena, Germany) was used to observe the immunohistochemical results.\u003c/p\u003e\u003cp\u003eHematoxylin and eosin (HE) staining was performed by conventional methods. The BX-53 upright microscope (Olympus) was used to observe HE staining.\u003c/p\u003e\u003cp\u003eFor evaluation of inner ear ototoxicity, cochlear specimens were harvested from the decalcified temporal bone at 2 weeks postoperatively and prepared for the whole-mount method to evaluate ototoxicity due to retinoids. Whole-mount cochlear tissue in which the organ of Corti was well preserved was observed using ImageJ software and the response regions at 4, 8, 16 and 32 kHz were analysed.\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis of the cochlear specimens was performed exactly as described above for the temporal bone sections, with the exception of the antibodies used. The primary antibodies used were rabbit anti-myosin7a (1:500; Proteus Biosciences, Waltham, MA, USA) \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, as a hair cell marker, and rabbit anti-NF200 (1:1000; Sigma-Aldrich, St. Louis, MO, USA) \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, as a spiral ganglion marker. The secondary antibodies used were Alexa568 donkey anti-rabbit IgG (Thermo Fisher Scientific) and Alexa488 donkey anti-rabbit IgG (Thermo Fisher Scientific).\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor evaluation of lineage epithelial regeneration, the HE staining results were scored as follows: 2, healing; 1, improvement; 0, unchanged. The differences in the HE scores between the retinoid and control groups were statistically analyzed using the two-tailed Mann–Whitney U test. Similarly, in the ciliary motility study, CBF was compared between the retinoid and control groups using the two-tailed Mann-Whitney U test. The results are presented as means ± standard error of the mean. All statistical analyses were performed using Prism 5 (Graph Pad Software, Inc., La Jolla, CA, USA). Statistical significance was set at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by the Ministry of Education, Science, Sports, and Culture (Grant in Aid for Basic Research, C).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and data analysis were performed by S.T. and T.I. The first draft of the manuscript was written by S.T., and all authors commented on the previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number 23K08955.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data associated with this study are present in the paper or the Supplementary Information. All relevant data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGaihede, M., et al. Middle ear pressure regulation\u0026ndash;complementary active actions of the mastoid and the eustachian tube. Otol Neurotol 31, 603\u0026ndash;611 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, W., et al. Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity. 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Front Cell Neurosci 14, 572434 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cholesteatoma, cilia, middle ear mucosa, mucosal regeneration, retinoids","lastPublishedDoi":"10.21203/rs.3.rs-6273919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6273919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMiddle ear dysfunction can lead to hearing loss and diseases, most commonly cholesteatoma. Although radical treatment of cholesteatoma involves complete removal of the lesion, incomplete regeneration of the middle ear mucosa can lead to hearing loss and recurrence of cholesteatoma. Recently, several middle ear mucosal regeneration therapies have been reported; however, none have been widely used clinically. Therefore, we focused on retinoids that have been reported to have regenerative effects on the nasal mucosal epithelium. We investigated the efficacy of retinoids in an animal model of middle ear mucosal damage using guinea pigs. Our results suggest that the regenerated ciliated epithelium has histological and functional characteristics similar to those of the normal epithelium. We additionally investigated the ototoxicity of retinoids and found no evidence of such. Retinoids may be useful as a novel therapy for middle ear mucosal regeneration.\u003c/p\u003e","manuscriptTitle":"Regenerative treatment of middle ear mucosa with topical retinoid administration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 06:41:35","doi":"10.21203/rs.3.rs-6273919/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e65a6270-05d4-4ffc-9905-389b75b6f136","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46049877,"name":"Health sciences/Diseases"},{"id":46049878,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-04-23T12:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-02 06:41:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6273919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6273919","identity":"rs-6273919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}