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. 2022 May 25;17(5):e0268763. doi: 10.1371/journal.pone.0268763

Serial histological changes in the cartilaginous eustachian tube in the rat following balloon dilation

Yehree Kim 1, Jeon Min Kang 2, Dae Sung Ryu 2, Jung-Hoon Park 2, Woo Seok Kang 1, Hong Ju Park 1,*
Editor: Ewa Tomaszewska3
PMCID: PMC9132338  PMID: 35613135

Abstract

Although balloon dilation has shown promising results in the treatment of dilatory Eustachian tube (ET) dysfunction, the histological effects of ET balloon dilation (ETBD) is unknown because histological examination of the whole human cartilaginous ET is impossible. Animal studies are needed to elucidate the effect of ETBD so we evaluated the histological changes after ETBD in a rat model. The left ET of 20 Wistar rats was dilated with a balloon catheter and the right ET was used as a control. Five rats were sacrificed immediately after ETBD, at 1, 4 and 12 weeks after the procedure for histological examination. The epithelial cells, presence of epithelial hyperplasia, and the proportion of the goblet cells in the epithelium; the vascular structures and dimensions of the submucosa; and presence of cartilage fracture and the area of the ET lumen were evaluated and compared between the groups. Desquamation of nearly all epithelial cells and the fracture of tubal cartilages were observed immediately after ETBD. At 1-week post-ETBD, the ciliated epithelial cells started to recover with epithelial hyperplasia. The goblet cells recovered by 4 weeks post-ETBD and epithelial hyperplasia decreased but was still present at 12 weeks post-ETBD. The depth of the submucosa increased and neovascularization in this region was observed at 1-week post-ETBD and persisted up to 12 weeks post-ETBD. The lumen of the cartilaginous ET increased immediately after ETBD but decreased at 1-week post-ETBD. The cartilaginous ET lumen recovered to the normal value at 4 weeks post-ETBD. This study is the first to describe the serial histological changes to the cartilaginous ET after ETBD and helps our understanding of the histological changes that occur after an ETBD intervention for intractable ET dysfunction.

Introduction

As the sole connector of the middle ear to the nasopharynx, a functioning eustachian tube (ET) is important for maintaining a healthy well-aerated middle ear [1]. The functions of the ET include middle ear ventilation, as well as the transport and secretion of pathogens and the nasopharyngeal reflux [2, 3]. A dysfunctional ET can lead to acute and chronic otitis media, one of the most common disease entities encountered in otolaryngology [4]. Conditions that interrupt the proper opening of the ET, such as an inflammatory response within its lumen caused by irritants and infectious reactions, can result in ET dysfunction (ETD). The inability of the ET to open also results in a gas equilibrium failure between the middle ear and nasopharynx. Atmospheric gases diffuse across the venous capillary cell membranes in the middle ear. So dilatory dysfunction of the ET results in a net negative pressure within the middle ear, leading to symptoms such as aural fullness or ’popping sounds’, reduced hearing, tinnitus, autophony, otalgia, and imbalance [5].

Until recent years, the understanding of ETD among otolaryngologists was limited, and few treatment options were available [6–11]. Surgical management is now available after the introduction of the ET balloon dilation (ETBD) procedure [12]. Since Ockermann et al. reported their first experience with ETBD in 2010 [13], many studies have since reported this procedure to be feasible and safe for the treatment of ETD [14–21]. The reported success rates of ETBD range from 36–80% [16, 21–24].

Although ETBD is reported to be superior to the conventional medical management of ETD, there is still a population of affected patients who do not respond to this intervention. No consensus has yet been reached regarding the further management of ETBD failure cases. This is mainly due to the limited understanding of the mechanisms underlying ETBD due to a lack of histologic studies of the ET after this surgery. A reported finding after ETBD is microtears in the cartilaginous part of the ET [12] and a resulting decrease in mucosal inflammation and reduced biofilm load [25]. A previous histological study by Kivekäs et al. reported that thinning of the mucosa, shearing of the epithelium, and a crush injury to the submucosa, particularly involving lymphocytic infiltrates, were the immediate responses to balloon dilation, and that a healthy pseudocolumnar epithelium and replacement of lymphocytic infiltrates with a thinner layer of fibrous tissue could be observed postoperatively [25]. One drawback of that prior report however was that the ET mucosal specimens were taken from the nasopharyngeal opening of the tube which does not include the true cartilaginous portion.

Histological examination of the whole human cartilaginous ET after ETBD is impossible. Therefore, an animal study is warranted to verify the histological changes of the ET after ETBD. In this study, we aimed to evaluate the serial histological changes of the ET epithelium, submucosa, and cartilage and the area of the ET lumen immediately after and at 1, 4 and 12 weeks after balloon dilation in a rat model.

Methods

Animals

This study was approved by the Institutional Animal Care and Use Committee at Asan Institute for Life Science and conformed to US National Institutes of Health guidelines for humane handling of laboratory animals and is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). Twenty male Wistar rats, 10 weeks of age (300–350 g; Orient Bio, Seongnam, Korea), were included in our study. All animals were housed at one per cage in a room with a 12-hour contrast cycle at 24 ± 1°C with a relative humidity of 55 ± 10%. Standard rodent chow and water were provided ad libitum. All animals were acclimated for at least 1 week prior to conducting the experiments. All procedures were carried out under anesthesia induced by means of an intramuscular injection of 50 mg/kg zolazepam and tiletamine (Zoletil 50; Virbac, Carros, France) and 10 mg/kg xylazine (Rompun; Bayer Healthcare, Leverkusen, Germany) and all efforts were made to minimize suffering. The left ET was dilated with a balloon catheter and the right ET was used as a normal control in all 20 rats. The rats were assigned to 4 groups, each consisting of 5 animals, for serial histological examination at 4 different time points: immediately (n = 5), at 1 week (n = 5), 4 weeks (n = 5), and 12 weeks (n = 5) after ETBD. All animals were sacrificed by placing the original housing cage in a carbon dioxide chamber and gradually increasing the concentration of carbon dioxide.

Fluoroscopic eustachian tube balloon dilation

All ETBD procedures were performed under fluoroscopic guidance by an interventional radiologist and otologist. After disinfection of the left external auditory canal with 0.05% chlorhexidine hydrochloride, the left tympanic membrane was punctured using a micro puncture needle (Cook, Bloomington, IN). The needle was directed 45 degrees anteriorly and in a horizontal plane under fluoroscopic guidance. The tip of the needle was located inside the tympanic cavity and about 0.2 ml of contrast media was injected into this cavity to visualize the tympanic orifice of the ET. The tip of the needle was then relocated close to the tympanic orifice. A 0.014-inch micro guidewire (Transend; Boston Scientific, Fremont, CA) was next gently advanced through the ET and eventually out of the nasal cavity. After removal of the micro puncture needle, a micro balloon catheter (Genoss, Suwon, Korea) was introduced over the guidewire from the nasal side and located at the middle portion of the ET to cover the whole tube structure. The balloon catheter was then inflated at a pressure of 10 atmospheres for 1 minute. During balloon inflation, waist formation and subsequent disappearance of the waist was observed. The micro balloon catheter and micro guidewire were removed after deflation of the balloon.

Histologic examinations

Surgical exploration was performed immediately after sacrifice. The mandible and anatomical structures anterior to the soft palate were then resected and the nasopharyngeal opening of the ET was identified. The soft tissue structures around the ET were dissected away whilst taking care not to disrupt the anatomy of the tube. Head samples were fixed in 10% neutral buffered formalin for 48 hours, followed by decalcification for 2 weeks (Osteosoft, Merck, Germany). The samples were then coronally sectioned from the nasopharynx to posterior wall of the tympanic cavity at intervals of 200 μm which provided 7 slides per each animal. Paraffin blocks were prepared from the sliced head samples from which 5 μm slices were obtained with an MR2258 microtome (Histoline, Pantigliate, Italy) and placed on slides. The slides were then stained with hematoxylin-eosin (H&E).

Whole slides of the ET for each rat were reviewed. The cartilage surrounding the ET was first identified. The section in which the sectioned ET cartilage was in the form of a ‘comma’ shape, showing both cartilaginous and nasopharyngeal epithelia, was selected for analysis (Fig 1). Evaluations included an assessment of changes in the epithelial cells, in the dimensions of the submucosa (degree of fibrotic changes), or in the lumen. In the epithelium, the presence of goblet cells, epithelial hyperplasia was assessed. In order to quantify the changes of these aspects of the epithelium, the length of the epithelium exhibiting goblet cells, epithelial hyperplasia were measured and divided by the whole perimeter of the ET lumen in the corresponding slide. Epithelial hyperplasia was also assessed by counting the maximum number of cell layers. These measurements were then compared among the 4 study groups (immediate, and 1, 4, and 12 weeks after ETBD) and the normal group (20 specimens from the non-dilated right ET).

Fig 1.

Fig 1

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Normal histology of the right-side rat ET sectioned from the nasopharynx (left) to the tympanic bulla (right). The tissues were sectioned coronally at 200 μm intervals, in the caudal direction from the medial to lateral sections. C, cartilage; ET, Eustachian tube; SG, submucosal glands (H&E stain). *The section in which the ET cartilage was in the form of a ‘comma’ shape, i.e. showing both the cartilaginous and nasopharyngeal epithelia, was selected for analysis.

In the submucosa, blood vessels were identified and those under the tubal cartilage were counted and analyzed as numbers per slide. After ETBD, the mucosal breaks caused by the dilatory force of the balloon were replaced by collagen tissue (S1 Fig). To evaluate the extent of fibrosis caused by ETBD, the depth of the submucosa which would correspond to the synthesis of new collagen was measured. The depth of the submucosa of the left ET (balloon) was measured at 9 different points in total in the medial, middle and lateral areas of the surrounding cartilage. The same was done for sections that were 200 μm proximal and distal to the reference slide. The 9 measurements were averaged to obtain the representative depth of the submucosa. The depth of the submucosa of the normal right ET was also measured as a control using the same points measured on the left side.

Due to the variation of the angles at which the ETs were sectioned among the slides, linear measurements alone may not accurately represent the thickness of the submucosa. The area of the submucosa was also therefore calculated. Histomorphometric analysis was conducted using CaseViewer, version 2.4 (3DHISTECH, Budapest, Hungary). The area of the lumen was also recorded. The relative proportions of the observed absolute values were compared.

Statistical analysis

The data are presented as means ± standard deviations. Statistical analyses were performed using SPSS software (version 24.0; SPSS, IBM, Chicago, IL). When comparing between two samples, significance of data was assessed by Mann-Whitney U test because samples did not pass the normality test. A p-value of less than 0.05 was considered as statistically significant for differences between groups.

Results

Histological findings of normal rat eustachian tubes

The epithelium of the rat ET is a ciliated respiratory epithelium. This ET epithelium was divided into cartilaginous and nasopharyngeal ends (Fig 2). The epithelium at the nasopharyngeal end consisted of columnar cells and also contained goblet cells (secretory mucous cells). The epithelium at the cartilaginous end consisted of cuboidal cells and contained few secretory mucous cells. The rat ET was surrounded by cartilage in the posterosuperior direction. A thin submucosa was found between the epithelium and the cartilage. The submucosa consisted of fibroblasts, collagen fibers and a few blood vessels. There was no lymphocytic involvement in the rat ET. In addition, as the rat ET extends toward the nasopharynx, the cartilage disappears and submucous glands appear, most notably in the medial portion.

Fig 2.

Fig 2

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(A) Normal histology of the rat Eustachian tube (ET) (15x power field view; H&E stain; scale bar represents 100μm). The ET consists of the cartilaginous end (*) and the nasopharyngeal end (**). (B) High-power field view of the epithelium at the cartilaginous ET (50x power field view; H&E stain; scale bar represents 50μm). (C) High-power field view of the epithelium at the nasopharyngeal end (50x power field view; H&E stain; scale bar represents 50μm).

Changes to the rat eustachian tube epithelium after balloon dilation

All 20 animals completed the whole study. The technical success rate of ETBD was 100%. The epithelial cells at the nasopharyngeal end of the rat ET were destroyed immediately after ETBD. At 1-week post-ETBD, the morphology of these epithelial cells had recovered but goblet cells were not seen. At 4 weeks post-ETBD, goblet cells could be identified but in a less ordered manner. At 12 weeks after the ETBD procedure, the columnar epithelium and goblet cells had fully recovered (Fig 3). At the cartilaginous end of the rat ET, the epithelial cells were desquamated immediately after ETBD. At 1-week post-ETBD, the epithelial cells showed a morphological change to epithelial hyperplasia. At 4 weeks post-procedure, the degree of hyperplasia decreased, and the original morphology was finally recovered at 12 weeks post-ETBD (Fig 4).

Fig 3. Serial epithelial changes at the nasopharyngeal end of the rat ET after ETBD.

Fig 3

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A 15x power field view and 50x power field sections are shown in the left and right columns, respectively. The epithelial cells were damaged (arrow) immediately after ETBD. At 1-week post-dilation, the morphology of the epithelial cells had recovered but goblet cells were not seen (black arrowhead). At 4- and 12-weeks post-dilation, the columnar epithelium and goblet cells had fully recovered (asterisk). H&E stain, scale bars represent 50μm.

Fig 4. Serial epithelial changes at the cartilaginous end of the rat ET after ETBD.

Fig 4

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A low- and high-power field of view are shown in the left and right columns, respectively. The epithelial cells were damaged (arrow) immediately after ETBD. At 1-week post-dilation, the epithelial cell morphology had changed to epithelial hyperplasia (black arrowhead). At 4 weeks after the procedure, the epithelial hyperplasia had decreased, and recovery to the original morphology (asterisk) was finally observed at 12 weeks post-ETBD. White arrowheads with black borders denote blood vessels. H&E stain, scale bars represent 50μm.

Fig 5A shows the changes in the portion of the epithelium with goblet cells in proportion to the whole perimeter of the ET lumen. The goblet cell proportion in the epithelium decreased to zero immediately after ETBD and remained at zero until 1week post-ETBD. At 4 weeks post-surgery however, the proportion of goblet cells increased significantly (28.7 ± 16.5%) to a level that was not statistically different to the normal group (32.0 ± 3.4%). At 12 weeks post-ETBD, the proportion of goblet cells remained at a similar level to the normal group (31.9 ± 6.8%).

Fig 5.

Fig 5

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(A) Serial changes in the proportion of the goblet cells in the epithelium of the nasopharyngeal end of the rat ET. (B) Serial changes of the proportion of epithelial hyperplasia. (C) Serial changes of the maximum cell layer counts present in the epithelium of the cartilaginous end of the ET. *statistically significant differences for p<0.05, Mann-Whitney U test.

Fig 5B shows the changes in the portion of the epithelium showing epithelial hyperplasia in proportion to the whole perimeter of the ET lumen. This level of epithelial hyperplasia significantly increased at 1-week post-dilation (33.7 ± 6.4%), then decreased significantly to 10.1 ± 7.7% at 4-weeks post-ETBD. At 12 weeks after ETBD, the level of epithelial hyperplasia decreased to 4.2 ± 3.7%, which was not statistically different to the normal control. Fig 5C shows the changes of the maximum cell layer count of the epithelium. Immediately after ETBD, all of the epithelia had fallen off and the maximum cell layer count was zero. A significant increase in the maximum cell layer count was evident at 1-week post-dilation (5.0 ± 0.9), which decreased to 2.8 ± 0.4 and 2.4 ± 0.5 at 4 and 12 weeks after the procedure, respectively. The maximum cell layer count was significantly increased at 12 weeks compared to the normal control (p<0.05, Mann-Whitney U test).

Serial changes in the submucosa after ETBD

Fig 6A shows the changes observed in the vascular structures in the submucosa. Immediately after ETBD, no blood vessels could be identified. The average number of blood vessels per slide was 2.4 ± 1.4, 2.2 ± 1.3 and 2.4 ± 2.0 at 1-, 4-, and 12-weeks post-ETBD, respectively and was greater at 12 weeks compared to the normal control (p<0.05, Mann-Whitney U test). Fig 6B shows the observed changes in the depth of the submucosa. No significant alterations in this depth were evident immediately after ETBD, but it was found to be increased to 157±54.3 μm at 1-week post-ETBD. The depth of the submucosa at 12 weeks was 105.2 ± 20.7 μm which was significantly thicker than the normal group (58.5 ± 21.3 μm, p<0.05, Mann-Whitney U test). The submucosa was compared in the different experimental groups by the proportion of the area calculated in each histology section. This value was increased to 23.2 ± 1.3% at 1 week, 24.4 ± 2.6% at 4 weeks, and 25.9 ± 1.8% at 12 weeks after the ETBD procedure and was significantly greater at 12 weeks compared to the normal group (18.7 ± 2.4%, p<0.05, Mann-Whitney U test, Fig 6C).

Fig 6.

Fig 6

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(A) Serial changes at the cartilaginous end of the rat ET including vascular structures; (B) submucosal depth; (C) the area of the submucosa (%) and (D) the area of the ET lumen (%). **statistically significant differences for p<0.05, Mann-Whitney U test.

The proportional area of the ET lumen was compared in each of the histology sections and was found to change throughout the 12-week time period but to reach and maintain a normal level at 12 weeks after the ETBD procedure (Fig 6D).

Serial changes in the tubal cartilage after ETBD

Out of the 20 rat ETs that were analyzed, 6 showed no frank fracture lines whereas a total of 21 fracture lines were identified in the remaining 14 ETs. These fracture lines were found at 3 distinctive locations: at the midpoint between the medial and lateral lamina of the tubal cartilage, and at the lateral 2 points that divide the lateral lamina into thirds (Fig 7). The frequency at which the fracture lines were identified were 48%, 19%, and 33% respectively, from the midpoint toward the far lateral. Fig 8 summarizes all of the changes identified in this study in the epithelium, submucosa and lumen.

Fig 7. Sites of cartilage fracture observed in 14 of the experimental rats.

Fig 7

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The cartilage tended to fracture at three distinctive locations i.e. at the midpoint between the medial and lateral lamina of the tubal cartilage (arrow), and at the lateral two points that divide the lateral lamina into thirds (arrowhead). H&E, scale bar represents 100μm.

Fig 8. Histologic changes of the lumen of the rat ET, the submucosa and the epithelium.

Fig 8

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The asterisk denotes a significant difference between the period before surgery and at 12 weeks post-ETBD (p<0.05, Mann Whitney U test).

Discussion

With the introduction of the ETBD procedure, chronic ETD could be surgically managed with a reported success rate of 36–80% [12, 16, 22, 23]. However, 20–64% of ETD patients do not respond to this ETBD treatment modality, thus necessitating further management options. There is no consensus at present about the optimal balloon catheter sizes or lengths to use in patients with ET disorders [26]. The difficulties in developing further management approaches for ETD may lie in the lack of histological studies. Hence, we speculated that a thorough investigation of the histologic changes that occur in the ET after dilation with balloons of different diameters and lengths might provide new insights into treating and managing ETD.

Kivekäs et al. have previously obtained pre- and postoperative biopsy specimens in 13 patients who underwent ETBD [25]. For practical reasons however, histologic studies of the human ET could only be carried out at the nasopharyngeal orifice. The authors suggested in their analyses that the crushing effect of the balloon on lymphocytes and lymphocytic follicles that were later replaced with a thinner fibrous scar is a possible mechanism underlying the therapeutic effects of ETBD [25]. However, the whole length of the ET needed to be investigated to properly evaluate the effects of ETBD, particularly as the cartilaginous ET is considered a crucial structure in maintaining normal ET function. Hence, a proper histological study of the whole ET using appropriate animal models became necessary. Generally, sheep and pigs are considered to be good model systems for the human middle ear [27]. However, a smaller animal model enables larger scale and higher throughput studies, making it more cost-efficient, and more suited to observing the tissue reactions after ETBD which was an important consideration [28]. In our current study, we were able to dilate the ET in the rat using commercially available micro balloon catheters.

We here investigated the serial histological changes in the rat ET after ETBD for up to 12 weeks. Immediately after ETBD, we observed desquamation of nearly all epithelial cells and fracture of the tubal cartilage. At 1-week post-ETBD, the ciliated epithelia cells started to recover via epithelial hyperplasia but goblet cells were still missing. The goblet cells recovered by 4 weeks post-ETBD, but epithelial hyperplasia (maximum cell layer count) was still decreased at 12 weeks post-ETBD. The depth of the submucosa increased and neovascularization in the submucosa was observed at 1-week post-ETBD, which persisted at 12 weeks post-ETBD. The lumen of the cartilaginous ET increased immediately after ETBD but decreased at 1-week post-ETBD due to the increased depth of the submucosa and onset of epithelial hyperplasia. The cartilaginous ET lumen recovered to normal at by 4 weeks post-ETBD.

Increased submucosal fibrosis after ETBD was the significant finding of our current study. The effect of ETBD seems to be related to the fibrosis of the submucosal connective tissue. In terms of prior histologic exams of similar tissues, Modi et al. performed ETBD of the rabbit subglottis [29]. The subglottis and ET both have a cartilaginous framework lined by a respiratory epithelium and mucosa. Thirty days after dilation, these authors observed regeneration of the normal epithelial lining and submucosal fibrosis, particularly when larger balloon sizes had been used. We speculated that submucosal fibrosis after ETBD may stiffen and keep the ET patent. Another finding in the submucosa was increased vascularity which could be attributed to the healing process after mucosal damage caused by ETBD [30, 31].

In our present study of the rat ET, epithelial damage was observed immediately after ETBD. However, the mucosal lining regenerated at 1-week post-ETBD, the goblet cells had recovered by 4 weeks post-ETBD and the epithelial hyperplasia decreased to nearly normal levels at 12 weeks after the procedure. The epithelium thus seems to recover within 12 weeks. It can be argued that a regenerated healthy mucosa will be thinner, thus facilitating ET opening, and the possibly recovered function of the cilia and of mucus production may aid the clearance of secretions from the middle ear [32]. In addition, the ET mucosa is known to generate a surfactant that reduces surface tension, which would be reduced in otitis media with effusion [33, 34], and a restored healthy mucosa may secrete surfactant, thus aiding ET opening.

The dimensions of the tubal cartilage in the rat ET did not change throughout our current study period. The cartilage healing process after fracture in general involves an equilibrium between the deposition of type I collagen (scar tissue) and expression of type II collagen (repair). Small full-thickness cartilage defects are mainly replaced by fibrocartilage, whereas partial-thickness defects heal through the deposition of fibrous scar tissue [35]. Full-thickness cartilage fractures were induced in our current rat model. Furthermore, cartilaginous structures tend to return to their original shape unless the deformation is maintained for several months [36]. These factors could account for the lack of changes to the cartilage framework following ETBD and this may suggest that this is a safe procedure. If the thickness of the cartilage did change, we would not be able to exclude the possibility of cartilage contracture leading to further narrowing of the luminal stricture.

The main limitation of our current study was that no functional assessment of the ET could be done. The ET is a dynamic organ that remains closed at rest and opens during certain movements such as swallowing or yawning. The compliance of the ET is a known characteristic of its proper function [37]. Hence, we cannot yet ascertain how the submucosal fibrosis induced by ETBD affects the ET function. Another limitation was that our analyses were carried out on rats with a normal ET and we could not therefore analyze lymphocytic involvement. Moreover, ETBD was found to induce a thicker submucosa but no obvious changes in the lumen. In a chronically inflamed ET, the lumen would be narrower, and further studies in a rat model of otitis media and ETD might therefore provide better insights into the histologic changes caused by ETBD.

Conclusion

Increased submucosal fibrosis and maintenance of the cartilage framework despite cartilage fractures after ETBD in the rat were the significant findings of our current study. At 12-weeks post-dilation, the observed findings were increased epithelial hyperplasia and increased depth of submucosa accompanied by neovascularization. This study is the first to describe serial histological changes after ETBD and these observations will assist with the planning of future histological studies of the middle ear in animal models after the placement of various types of balloons and stents that are typically used to treat intractable ET dysfunction in humans.

Supporting information

S1 Fig. Histology of the left-side rat ET sectioned immediately, at 1 week, 4weeks, and 12 weeks after ETBD.

Mucosal breaks are observed immediately after ETBD (arrow). Mucosal wound is healed by 1 week and fibroblasts (asterisk) are seen. At 4 and 12 weeks, the submucosa is replaced with collagen tissue (arrowheads). Scale bars represent 100μm.

(TIF)

Click here for additional data file. (9.9MB, tif)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) (2020R1F1A1049412 to Park H.J.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Ewa Tomaszewska

1 Dec 2021

PONE-D-21-31219Serial Histological Changes in the Cartilaginous Eustachian Tube in the Rat Following Balloon DilationPLOS ONE

Dear Dr. Hong Ju Park,

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

**********

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Reviewer #1: No

Reviewer #2: I Don't Know

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript presented for the review the Authors describe the histological changes in the Eustachian tube (ET) after balloon dilation procedure (ETBD) at different intervals of time. I find the subject interesting and purposeful in the aspect of understanding processes that occur in the tissues after ETBD, which may result in the improvement of methodology and success rate of the procedure. A basic histological technique was used in this study to assess the epithelium, submucosa and the lumen of the ET. A series of morphometric analyses were also conducted to shed a light on the significance of the histological observations.

However, the manuscript was prepared carelessly and thus this article could be suitable for publication in PlosOne journal after major revisions.

Firstly, the Authors did not use the continuous line numbering which should be included according to a submission guideline and it would be helpful in preparing the review.

The English language should be improved throughout the whole manuscript. For example: “A proposed underlying mechanism of ETBD is microtears in the…” – a mechanism is the process. Microtears are not a mechanism. “The rats were randomly assigned to four groups of each five for serial…” - I guess there were 5 rats in each of four groups but the sentence is incorrect, “All rats were sacrificed immediately, 1 week, 4 weeks and 12 weeks.” – maybe adding “after ETBD” would make the sentence more understandable. “Normal Histological Findings for the Rat Eustachian Tube” – Were the histological findings normal or were they conducted in normal tissue?. “… worsening of the luminal stricture” – Can luminal stricture get worse? It is difficult to point out all the language discrepancies without line numbers.

ABSTRACT and INTRODUCTION. The aim of the study should be clearly pointed and precise. The statement that some issues “remain unclear” is insufficient and does not include the purpose of the study. The last sentence in the “introduction” should include not only the histological observations but also the morphometry and time intervals in which the alterations were observed.

MATERIALS AND METHODS. In “Animals” the sentences referring to conditions of animal housing and adaptation should be put before the description of the study. The method of anaesthesia should be described as it was done in the “Fluoroscopic Eustachian tube balloon dilation”. If the method is the same, it should be described when it appears in the text for the first time. Moreover, the Authors divide 20 animals into 4 groups but in “Histopathologic examination” section there are five groups mentioned with normal (before) group (probably from the right ear, but from which rats?). The division into study groups should be explained more precisely.

“Histopathologic examinations” should be changed to Histological examinations in accordance with the title of the manuscript. It should be also stated, how many slides were used from every animal for the morphological observations and how many microphotographs were taken for the morphometry (number of data taken into statistics). It should be explained how the depth of the submucosa relates to the fibrosis. Changes in the dimensions of the submucosa may be caused by different factors. Histopathological lesions should be marked on the figures if there were such alterations. Then they may be analysed morphometrically. In this case, the presence of fibrosis is a speculation.

“Statistical analysis”. The Mann Whitney U test is a nonparametric test. It should be explained why such test was used for the analyses. It should be mentioned if all the data did not meet the assumptions for the parametric test? For example normality of the distribution or homogeneity of variance. “A p-value of < 0.05 was considered statistically significant.” Is a p-value significant or the differences between groups? (Another unfortunate language expression).

RESULTS. If the Authors decide to emphasize the significance of the changes by adding “(p<0,05)” at the end of sentence they should follow this rule everywhere and not only in selected places. After some of the sentences, the Authors put “p=0,03”… does it mean that the result is ambiguous with p-value close to 0,05? After p-value, the name of the test should also be mentioned (also in legends to figures).

In “Serial changes in the tubal cartilage after ETBD” the Authors stated that a fracture lines were identified in 14 rats’ ET. After that the Authors write: “All the cartilage fracture lines were observed immediately after ETBD” which gives only 5 rats (immediately group) and not 14. It should be explained more precisely.

DISCUSSION. How the paragraph “In the urethra,…” relates to the changes in ET?

REFERENCES. The names of the Journals should be abbreviated in references: 24, 25, 29, 34, 37 and 41.

FIGURES AND FIGURE LEGENDS. The figures should contain markers of different structures of interest explained in the legends e.g. ET lumen, epithelium, Goblet cells, blood vessels, cartilage etc. for the readers who are not familiar with histology. The values on the scale bars in Fig 1 A,B,C and Fig 6 are not visible. The Authors should decide if they put the values on the scale bar or in the legends and follow their rule in all figures. In the Legends to figures, the Authors should avoid descriptions which are intended to be in the Results section. The H&E abbreviation should be used in all figures instead of full names. The letters “A,B,C” should be put at the beginning or at the end of the particular legend (the same arrangement in all figures). In the figure 7, the order of the parameters should be changed according to the legend to figure 7. The significant changes in the submucosa should be marked on the slides (e.g. fibrotic changes).

Another minor comments. After “Seongnam, Korea” there are 2 parentheses. “Ad libitum” should be in italics. In every “Fig.” there should be no dot. Abbreviation “BD” was not explained in the text. There was only ETBD.

Reviewer #2: The subject of this manuscript is interesting, as well as the obtained results. Unfortunately, the manuscript is not well written. Especially the methodological part requires major improvements. The manuscript cannot be published in this form.

Material and Methods

-It is said: In order to quantify changes in any of these aspects of the epithelium, the length of the epithelium exhibiting goblet cells (why the number of cells was not calculated?), and the level ( what does level mean – area, depth or length?) of squamous metaplasia or epithelial hyperplasia were measured and divided by the whole perimeter of the ET lumen in the corresponding slide (the charts in Figure 4 give percentages).

The authors did not clearly describe the methodology for quantifying these data. It is not clear to the reader how the authors made these calculations.

- The authors should use -blood vessels rather than vessels

- other staining should be used to identify fibrosis in the submucosa, e.g. Masson-Goldner's trichrome staining - This method would clearly visualize collagen fibers in the tissue

In the statistical analysis section there is no information on the presentation of data (as mean± SEM or SD?)

Results

It is said: the epithelial cells at the nasopharyngeal end were columnar and contained goblet cells. The epithelial cells at the cartilaginous end were more cuboidal in shape and contained few secretory mucous cells - These sentences are imprecise as its suggest that the epithelial cells contained secretory cells (the same applies to the description of Figure 1). Rather it should be: for example, the cartilaginous end epithelium consists of cuboidal and secretory cells.

- The authors write: The epithelial cells at the nasopharyngeal end of the ET rat were desquamated immediately after ETBD. It should be written - they were destroyed or damaged.

- It is said: At 4 weeks post-procedure, the epithelial hyperplasia decreased, and the original morphology was finally recovered at 12 weeks post-ETBD (Fig. 3). But in the earlier description, the authors did not mention anything about hyperplasia (here it appears for the first time, so it is difficult to understand when the authors write that the hyperplasia has decreased).

- The higher magnifications of the photos in Figure 2 are not sharp

- The authors should mark in Figure 3 the places where squamous metaplasia and hyperplasia occur.

- the data shown in the graphs (especially Figures 4 and 5A) have large SEM or standard deviations??- which proves the large dispersion of data

Discussion

-Based on the analysis of the depth of the submucosa, the authors conclude that there was an increase in fibrosis, however, the authors did not perform any analyzes confirming the fibrosis. It is worth using other staining that will confirm the fibrosis (e.g. the mentioned Masson-Goldner staining).

-the authors did not discuss the observed increase in vascularity in the submucosa. How to explain increased angiogenesis?

- Linguistic proofreading needed!!

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2022 May 25;17(5):e0268763. doi: 10.1371/journal.pone.0268763.r002

Author response to Decision Letter 0

2 Feb 2022

We appreciate all the reviewers’ valuable and insightful comments that potentially improve our manuscript. We made our best effort to reflect the suggestions in the revised manuscript as well as to address all the concerns that the reviewers presented. Below are our line-by-line responses to the reviewers’ comments.

Reviewer #1: In the manuscript presented for the review the Authors describe the histological changes in the Eustachian tube (ET) after balloon dilation procedure (ETBD) at different intervals of time. I find the subject interesting and purposeful in the aspect of understanding processes that occur in the tissues after ETBD, which may result in the improvement of methodology and success rate of the procedure. A basic histological technique was used in this study to assess the epithelium, submucosa and the lumen of the ET. A series of morphometric analyses were also conducted to shed a light on the significance of the histological observations.

However, the manuscript was prepared carelessly and thus this article could be suitable for publication in PlosOne journal after major revisions.

Firstly, the Authors did not use the continuous line numbering which should be included according to a submission guideline and it would be helpful in preparing the review.

→ Thankyou for pointing this out and we are sorry for omitting the numbering. We included the continuous line numbering in the revised manuscript.

The English language should be improved throughout the whole manuscript. For example: “A proposed underlying mechanism of ETBD is microtears in the…” – a mechanism is the process. Microtears are not a mechanism. “The rats were randomly assigned to four groups of each five for serial…” - I guess there were 5 rats in each of four groups but the sentence is incorrect, “All rats were sacrificed immediately, 1 week, 4 weeks and 12 weeks.” – maybe adding “after ETBD” would make the sentence more understandable. “Normal Histological Findings for the Rat Eustachian Tube” – Were the histological findings normal or were they conducted in normal tissue?. “… worsening of the luminal stricture” – Can luminal stricture get worse? It is difficult to point out all the language discrepancies without line numbers.

→ Thankyou for the comments. “A proposed underlying mechanism of ETBD” was changed to “A reported finding after ETBD”. “The rats were randomly assigned to four groups of each five for serial…” was changed to “The rats were randomly assigned to 4 groups, each consisting of 5 animals, for serial histological examination at 4 different time points: immediately (n = 5), at 1 week (n = 5), 4 weeks (n = 5), and 12 weeks (n = 5) after ETBD”. “Normal Histological Findings for the Rat Eustachian Tube” was changed to “Histological Findings of Normal Rat Eustachian Tubes”. ““… worsening of the luminal stricture” was changed to ““… further narrowing of the luminal stricture””

ABSTRACT and INTRODUCTION. The aim of the study should be clearly pointed and precise. The statement that some issues “remain unclear” is insufficient and does not include the purpose of the study. The last sentence in the “introduction” should include not only the histological observations but also the morphometry and time intervals in which the alterations were observed.

→ Thankyou for the comments. The first 2 sentences in the ABSTRACT were changed to “Although balloon dilation has shown promising results in the treatment of dilatory Eustachian tube (ET) dysfunction, the histological effects of ET balloon dilation (ETBD) is unknown because histological examination of the whole human cartilaginous ET is impossible. Animal studies are needed to elucidate the effect of ETBD so we evaluated the histological changes after ETBD in a rat model.” The last sentence in the “introduction” was changed to “In this study, we aimed to evaluate the serial histological changes of the ET epithelium, submucosa, and cartilage and the area of the ET lumen immediately after and at 1, 4 and 12 weeks after balloon dilation in a rat model”

MATERIALS AND METHODS. In “Animals” the sentences referring to conditions of animal housing and adaptation should be put before the description of the study. The method of anaesthesia should be described as it was done in the “Fluoroscopic Eustachian tube balloon dilation”. If the method is the same, it should be described when it appears in the text for the first time. Moreover, the Authors divide 20 animals into 4 groups but in “Histopathologic examination” section there are five groups mentioned with normal (before) group (probably from the right ear, but from which rats?). The division into study groups should be explained more precisely.

→ Thankyou for the comments. The sentences referring to conditions of animal housing and adaptation were brought before the description of the study in the manuscript. Method of anesthesia was explained in the “Animals” section. Descriptions of the 4 groups and the how the right ear were used as normal control ear were rephrased for clearer communication. The revised version now reads “The left ET was dilated with a balloon catheter and the right ET was used as a normal control in all 20 rats. The rats were randomly assigned to 4 groups, each consisting of 5 animals, for serial histological examination at 4 different time points: immediately (n = 5), at 1 week (n = 5), 4 weeks (n = 5), and 12 weeks (n = 5) after ETBD.” and “These measurements were then compared among the 4 study groups (immediate, and 1, 4, and 12 weeks after ETBD) and the normal group (20 specimens from the non-dilated right ET”.

“Histopathologic examinations” should be changed to Histological examinations in accordance with the title of the manuscript. It should be also stated, how many slides were used from every animal for the morphological observations and how many microphotographs were taken for the morphometry (number of data taken into statistics). It should be explained how the depth of the submucosa relates to the fibrosis. Changes in the dimensions of the submucosa may be caused by different factors. Histopathological lesions should be marked on the figures if there were such alterations. Then they may be analysed morphometrically. In this case, the presence of fibrosis is a speculation.

→ “Histopathologic examinations” was changed to “Histological examinations”. We made 7 slides per animal however one slide where ET cartilage was in the form of a ‘comma’ shape, showing both cartilaginous and nasopharyngeal epithelia was used for analysis. As provided in supplementary Figure 1, all 7 slides were reviewed and the section with the ‘comma’ shaped cartilage (slide with asterisk) was selected. How the depth of the submucosa relates to the fibrosis was also added “After ETBD, the mucosal breaks caused by the dilatory force of the balloon were replaced by collagen tissue. To evaluate the extent of fibrosis caused by ETBD, the depth of the submucosa which would correspond to the synthesis of new collagen was measured” and added a supplementary figure (S1_Fig) to show the histologic changes corresponding to the replacement with collagen tissue.

“Statistical analysis”. The Mann Whitney U test is a nonparametric test. It should be explained why such test was used for the analyses. It should be mentioned if all the data did not meet the assumptions for the parametric test? For example normality of the distribution or homogeneity of variance. “A p-value of < 0.05 was considered statistically significant.” Is a p-value significant or the differences between groups? (Another unfortunate language expression).

→ The whole “Statistical analysis” section was rewritten as “Statistical analyses were performed using SPSS software (version 24.0; SPSS, IBM, Chicago, IL). When comparing between two samples, significance of data was assessed by Mann-Whitney test because samples did not pass the normality test. A p-value of less than 0.05 was considered as statistically significant for differences between groups.”

RESULTS. If the Authors decide to emphasize the significance of the changes by adding “(p<0,05)” at the end of sentence they should follow this rule everywhere and not only in selected places. After some of the sentences, the Authors put “p=0,03”… does it mean that the result is ambiguous with p-value close to 0,05? After p-value, the name of the test should also be mentioned (also in legends to figures).

→ Thankyou for the comment. All the p-values were written as p<0.05 and the name of the test was written both in the results section and the legends to figures.

In “Serial changes in the tubal cartilage after ETBD” the Authors stated that a fracture lines were identified in 14 rats’ ET. After that the Authors write: “All the cartilage fracture lines were observed immediately after ETBD” which gives only 5 rats (immediately group) and not 14. It should be explained more precisely.

→ We have erased the sentence as it may not be necessary and only cause confusion.

DISCUSSION. How the paragraph “In the urethra,…” relates to the changes in ET?

→ Our original intention was to discuss other examples of balloon dilation in non-vascular structures but decided to erase the paragraph as it may be irrelevant. Thankyou.

REFERENCES. The names of the Journals should be abbreviated in references: 24, 25, 29, 34, 37 and 41.

→ Thankyou for pointing this out. The correct journal abbreviations were used in the revised manuscript.

FIGURES AND FIGURE LEGENDS. The figures should contain markers of different structures of interest explained in the legends e.g. ET lumen, epithelium, Goblet cells, blood vessels, cartilage etc. for the readers who are not familiar with histology. The values on the scale bars in Fig 1 A,B,C and Fig 6 are not visible. The Authors should decide if they put the values on the scale bar or in the legends and follow their rule in all figures. In the Legends to figures, the Authors should avoid descriptions which are intended to be in the Results section. The H&E abbreviation should be used in all figures instead of full names. The letters “A,B,C” should be put at the beginning or at the end of the particular legend (the same arrangement in all figures). In the figure 7, the order of the parameters should be changed according to the legend to figure 7. The significant changes in the submucosa should be marked on the slides (e.g. fibrotic changes).

→ Thankyou for the comments. A new Fig1 (previously submitted as supplementary figure 1) was shown explaining the low power field view of the ET lumen and the cartilage at the materials&methods section. The epithelium, Goblet cells, blood vessels were marked in the new figures 3 and 4. The values of the scale bars were put in the legends and not the figures themselves. H&E abbreviations were used throughout all figures. The letters “A,B,C” were put at the beginning in all figure legends. The order of the legends of the new Fig8 (previous Fig7) were changed to fit the order of the figure.

Another minor comments. After “Seongnam, Korea” there are 2 parentheses. “Ad libitum” should be in italics. In every “Fig.” there should be no dot. Abbreviation “BD” was not explained in the text. There was only ETBD.

→ Thankyou for the comments. One of the 2 parentheses was erased. ‘Ad libitum’ was changed to italics. The dot after the ‘Fig.” were all removed. BD was changed to ETBD.

Reviewer #2: The subject of this manuscript is interesting, as well as the obtained results. Unfortunately, the manuscript is not well written. Especially the methodological part requires major improvements. The manuscript cannot be published in this form.

Material and Methods

-It is said: In order to quantify changes in any of these aspects of the epithelium, the length of the epithelium exhibiting goblet cells (why the number of cells was not calculated?), and the level ( what does level mean – area, depth or length?) of squamous metaplasia or epithelial hyperplasia were measured and divided by the whole perimeter of the ET lumen in the corresponding slide (the charts in Figure 4 give percentages). The authors did not clearly describe the methodology for quantifying these data. It is not clear to the reader how the authors made these calculations.

→ Thankyou for the comment. Because there may be some variations in the angles at which the ETs were sectioned (although we did try to keep the sections as coronal as possible checking with the hard palate and brain which were large enough to be grossly observed), we thought that proportions would better represent the changes rather than absolute numbers and counts. The sentences were rephrased and the revised manuscript now reads “In the epithelium, the presence of goblet cells, squamous metaplasia and epithelial hyperplasia was assessed. In order to quantify the changes of these aspects of the epithelium, the length of the epithelium exhibiting goblet cells, squamous metaplasia or epithelial hyperplasia were measured and divided by the whole perimeter of the ET lumen in the corresponding slide. Epithelial hyperplasia was also assessed by counting the maximum number of cell layers. These measurements were then compared among the 4 study groups (immediate, and 1, 4, and 12 weeks after ETBD) and the normal group (20 specimens from the non-dilated right ET).”

- The authors should use -blood vessels rather than vessels

→ All “vessels” were changed to “blood vessels”.

- other staining should be used to identify fibrosis in the submucosa, e.g. Masson-Goldner's trichrome staining - This method would clearly visualize collagen fibers in the tissue

→ Thankyou for the kind suggestion. In this study, we wanted to outline an overview of serial histologic changes after ETBD. Unfortunately, special stains were not considered when we were planning the experiments and only H&E sections were done. So collagen fibers had to be identified through eosin stain by looking at its morphology. In future studies, we plan to include special stains.

In the statistical analysis section there is no information on the presentation of data (as mean± SEM or SD?)

→ The sentence “The data are presented as means ± standard deviations” were added to the beginning of the statistical analysis section

Results

It is said: the epithelial cells at the nasopharyngeal end were columnar and contained goblet cells. The epithelial cells at the cartilaginous end were more cuboidal in shape and contained few secretory mucous cells - These sentences are imprecise as its suggest that the epithelial cells contained secretory cells (the same applies to the description of Figure 1). Rather it should be: for example, the cartilaginous end epithelium consists of cuboidal and secretory cells.

→ Thankyou for your kind suggestion. The sentences were rewritten as “The epithelium at the nasopharyngeal end consisted of columnar cells and also contained goblet cells (secretory mucous cells). The epithelium at the cartilaginous end consisted of cuboidal cells and contained few secretory mucous cells.”

- The authors write: The epithelial cells at the nasopharyngeal end of the ET rat were desquamated immediately after ETBD. It should be written - they were destroyed or damaged.

→ The word “desquamated” was changed to “destroyed” as suggested.

- It is said: At 4 weeks post-procedure, the epithelial hyperplasia decreased, and the original morphology was finally recovered at 12 weeks post-ETBD (Fig. 3). But in the earlier description, the authors did not mention anything about hyperplasia (here it appears for the first time, so it is difficult to understand when the authors write that the hyperplasia has decreased).

→ The term “epithelial hyperplasia” was erased and changed to “squamous metaplasia”.

- The higher magnifications of the photos in Figure 2 are not sharp

→ The figures were recaptured (759dpi) for better resolution

- The authors should mark in Figure 3 the places where squamous metaplasia and hyperplasia occur.

→ Thankyou for your suggestion. Squamous metaplasia and hyperplasia as well as blood vessels and Goblet cells etc were marked in the figures which was also suggested by reviewer 1.

- the data shown in the graphs (especially Figures 4 and 5A) have large SEM or standard deviations??- which proves the large dispersion of data

→ For some variables (especially where absolute counts of small numbers were used) we did see large variance among animals.

Discussion

-Based on the analysis of the depth of the submucosa, the authors conclude that there was an increase in fibrosis, however, the authors did not perform any analyzes confirming the fibrosis. It is worth using other staining that will confirm the fibrosis (e.g. the mentioned Masson-Goldner staining).

→ Thankyou for the suggestion. Again, in this particular study, only H&E sections were done. We tried to include explanations for measuring the depth of the submucosa for representation of newly formed collagen in the material and methods section under “histologic examinations”. “After ETBD, the mucosal breaks caused by the dilatory force of the balloon were replaced by collagen tissue. To evaluate the extent of fibrosis caused by ETBD, the depth of the submucosa which would correspond to the synthesis of new collagen was measured” and added supplementary Fig 2 to show the histologic changes corresponding to the replacement with collagen tissue.”

-the authors did not discuss the observed increase in vascularity in the submucosa. How to explain increased angiogenesis?

→ Increased vascularity was discussed in the 4th paragraph of the Discussion section. “Another finding in the submucosa was increased vascularity which could be attributed to the healing process after mucosal damage caused by ETBD [31,32].”

- Linguistic proofreading needed!!

→ Thankyou for all your considerate suggestions. Certificate of language editing is included.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (24.5KB, docx)

Decision Letter 1

Ewa Tomaszewska

15 Mar 2022

PONE-D-21-31219R1Serial histological changes in the cartilaginous Eustachian tube in the rat following balloon dilationPLOS ONE

Dear Dr. Hong Ju Park,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Ewa Tomaszewska, DVM Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled: “Serial histological changes in the cartilaginous Eustachian tube in the rat following balloon dilation” is interesting and purposeful in the aspect of understanding processes that occur in the tissues after Eustachian tube ballon dilation. The Authors have assessed the epithelium, submucosa and the lumen of the ET using a basic histological technique enriched with morphometric analyses.

The Authors have put a great effort to improve their manuscript after first revision. My comments and suggestions were taken into consideration, however there are still some minor issues that should be revised:

1. In References section, literature positions 21 and 24 are the same. After deleting one of them the numbers in the text should be corrected.

2. In References some names of journals are still not abbreviated:

Reference 25: J Vasc Interv Radiol

Reference 29: J Pharm Bioallied Sci

Reference 34: Int J Pediatr Otorhinolaryngol

Reference 38: Am J Otolaryngol

3. Figures and Figure legends

In Fig 3 and 4 the white arrowheads are barely visible

After * in fig 5 and 6 there should be: *statistically significant differences for p<0.05, Mann-Whitney U test.

In S1 Fig. there should be a scale bar or magnification indicated in the legend.

In my opinion, the manuscript is suitable for publication in PLOS ONE after minor revision.

Reviewer #2: The work has been corrected in accordance with the comments of the reviewer, but I do have some remark, however. I see ambiguity in the description of Figure 4 (lines 457-460) and the description of the Results (lines 190-192) - what exactly did the authors want to write - epithelial hyperplasia or squamous metaplasia?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2022 May 25;17(5):e0268763. doi: 10.1371/journal.pone.0268763.r004

Author response to Decision Letter 1

28 Apr 2022

We appreciate the reviewers’ valuable comments that potentially improve our manuscript. We made our best effort to reflect the suggestions in the revised manuscript. Below in blue are our line-by-line responses to the reviewers’ comments.

Reviewer #1: The manuscript entitled: “Serial histological changes in the cartilaginous Eustachian tube in the rat following balloon dilation” is interesting and purposeful in the aspect of understanding processes that occur in the tissues after Eustachian tube ballon dilation. The Authors have assessed the epithelium, submucosa and the lumen of the ET using a basic histological technique enriched with morphometric analyses.

The Authors have put a great effort to improve their manuscript after first revision. My comments and suggestions were taken into consideration, however there are still some minor issues that should be revised:

1. In References section, literature positions 21 and 24 are the same. After deleting one of them the numbers in the text should be corrected.

→ Thank you for pointing this out. Reference 24 was removed and changed to 21.

2. In References some names of journals are still not abbreviated:

Reference 25: J Vasc Interv Radiol

Reference 29: J Pharm Bioallied Sci

Reference 34: Int J Pediatr Otorhinolaryngol

Reference 38: Am J Otolaryngol

→ Thank you for pointing this out. The abbreviations were corrected as suggested.

3. Figures and Figure legends

In Fig 3 and 4 the white arrowheads are barely visible

→ For Fig 4, the white arrowheads were changed to “white arrowheads with black borders” for better visualization. For Fig 3, there was a mistake with the legends so the sentence reading “White arrowheads denote blood vessels” was removed from the manuscript.

After * in fig 5 and 6 there should be: *statistically significant differences for p<0.05, Mann-Whitney U test.

→ Thank you for the suggestion. The suggested corrections were made to the manuscript.

In S1 Fig. there should be a scale bar or magnification indicated in the legend.

→ Scale bars were added to the figure and indicated in the legend

In my opinion, the manuscript is suitable for publication in PLOS ONE after minor revision.

→ Thank you for your positive feedback.

Reviewer #2: The work has been corrected in accordance with the comments of the reviewer, but I do have some remark, however. I see ambiguity in the description of Figure 4 (lines 457-460) and the description of the Results (lines 190-192) - what exactly did the authors want to write - epithelial hyperplasia or squamous metaplasia?

→ There was confusion with the choice of terminology in the original manuscript. Thank you for pointing this out. In the new manuscript, the finding was uniformly described as epithelial hyperplasia.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (16.8KB, docx)

Decision Letter 2

Ewa Tomaszewska

9 May 2022

Serial histological changes in the cartilaginous Eustachian tube in the rat following balloon dilation

PONE-D-21-31219R2

Dear Dr. Hong Ju Park,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ewa Tomaszewska, DVM Ph.D

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The work has been corrected in accordance with the comments of the reviewer. This manuscript is accepted for publication.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Acceptance letter

Ewa Tomaszewska

16 May 2022

PONE-D-21-31219R2

Serial histological changes in the cartilaginous Eustachian tube in the rat following balloon dilation

Dear Dr. Park:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Ewa Tomaszewska

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Histology of the left-side rat ET sectioned immediately, at 1 week, 4weeks, and 12 weeks after ETBD.

    Mucosal breaks are observed immediately after ETBD (arrow). Mucosal wound is healed by 1 week and fibroblasts (asterisk) are seen. At 4 and 12 weeks, the submucosa is replaced with collagen tissue (arrowheads). Scale bars represent 100μm.

    (TIF)

    Click here for additional data file. (9.9MB, tif)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (24.5KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (16.8KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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