Histopathological characteristics of an oral epithelial dysplasia model in rats showing a negative reaction to fluorescence visualization

Histopathological characteristics of an oral epithelial dysplasia model in rats showing a negative reaction to fluorescence visualization | 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 Histopathological characteristics of an oral epithelial dysplasia model in rats showing a negative reaction to fluorescence visualization Minori Fujikawa, Kei Nakajima, Yoshihiko Akashi, Katsutoshi Kokubun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5565034/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Vital staining has long been used to delineate the horizontal margins for resection in oral squamous cell carcinoma and in oral epithelial dysplasia (OED). In recent years, fluorescence visualization (FV), a simple and non-invasive technique, has been used to identify resection margins. However, FV occasionally fails to detect OED. The purpose of this study is to investigate why lesions are not detected using FV and to evaluate their histopathology. The tongues of an OED rat model were examined using FV, and the obtained images were objectively evaluated by quantifying the fluorescence intensity (FI) through image analysis software. Hematoxylin-eosin and immunohistochemical staining were performed to characterize FV loss (FVL) and FV retention (FVR). Some OED lesions were recognized through FVR. In FVL, the CD31-positive rate was higher than in FVR, and the CD31-positive rate negatively correlated with FI. In contrast, a positive correlation was observed between FI and the thickness of the epithelial layer. Multiple regression analysis suggested that the CD31-positivity rate and epithelial thickness may be involved in FI. These findings suggest that the accuracy of FV detection is influenced by multiple factors, including angiogenesis and epithelial layer thickness. Therefore, a combination of various testing methods should be considered. Biological sciences/Cancer/Oral cancer/Oral cancer detection Biological sciences/Biological techniques/Imaging/Fluorescence imaging fluorescence visualization oral epithelial dysplasia immunohistochemical staining pathological approach Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Surgical resection remains the treatment of choice for early-stage (T1-2) oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED) [ 1 ]. Although the prognosis of patients with these conditions is generally considered good, locoregional recurrence is possible, with the adverse pathologic features of inadequate resection margins being a major recurrence factor [ 2 , 3 ]. Conventionally, vital staining with toluidine blue or iodine is used to determine the extent of resection [ 4 ]. Normal epithelium stains with iodine solution, but abnormal areas with dysplasia do not stain well with iodine and exhibit iodine-unstained areas owing to differences in cytoplasmic glycogen content. This staining mechanism allows surgical resection using vital iodine staining to visualize the boundaries between OSCC and OED [ 5 – 8 ]. Although iodine staining is useful for defining lesion margins, fluorescence visualization (FV) has been increasingly used in recent years. FV highlights atypical epithelium, offering a less invasive alternative to vital staining and providing an option for patients with allergies [ 9 – 11 ]. FV distinguishes between normal and abnormal mucosa using visible light at wavelengths of 400–460 nm [ 11 ]. Under this light, normal mucosa emits a pale green fluorescence due to collagen crosslinking and flavin adenine dinucleotide (FAD). In contrast, abnormal areas absorb fluorescence owing to light absorption reactions caused by the disruption of collagen crosslinks, coenzyme deficiencies, and angiogenesis, resulting in dark areas with FV loss (FVL) [ 12 – 14 ]. Studies have reported no significant difference in accuracy between FV and vital staining for determining resection margins [ 15 – 17 ]. The FV device (IllumiScan®: Shofu Inc, Kyoto, Japan) used in this study was developed by our institution and approved under Japan’s Pharmaceutical and Medical Device Act. Its accuracy has been validated in multiple studies [ 11 , 15 , 18 – 21 ]. Additionally, this device allows for the measurement of fluorescence intensity (FI) in captured images using image analysis software, enhancing its utility in objective assessments. According to the 5th edition of the World Health Organization (WHO) head and neck tumor classification, OED is defined as a range of structural and cytological epithelial changes resulting from the accumulation of genetic mutations, indicating a risk of malignant transformation to squamous cell carcinoma. OED is diagnosed based on structural and cytological features [ 22 ]. Additionally, immunohistochemical staining reveals Ki67 stratification in the basal layer, decreased CK13 expression, and increased CK17 expression in the epithelial layer. Ki67 expression is positive throughout the epithelium of the lesions and is only noted in the basal cell layer in normal areas [ 23 ]. Furthermore, Sugahara et al. [ 18 ] reported that CK17 was positive in epithelial tumors and negative in normal oral mucosa, whereas the opposite was true for CK13 expression. Saitoh et al. [ 24 ] reported that the expression of CK13 and CK17 was observed in both an OED rat model and normal rats and showed that as the lesion progressed, CK17 expression increased and CK13 expression decreased. However, FV may sometimes not detect early lesions in OSCC and OED [ 15 – 17 , 25 , 26 ]. Some studies have suggested that the thickness of epithelial cells and angiogenesis may be involved, but no clear cause has been identified. This study aimed to investigate the factors contributing to FV-negative lesion detection by conducting histopathological analyses in an OED rat model, with a particular focus on the correlation between angiogenesis and epithelial thickness. Results Overview of the OED model The OED model was established in Sprague–Dawley rats as detailed in the Methods. Briefly, 20 male rats were fed a standard diet and were treated with 4-nitroquinoline 1-oxide (4NQO) in their drinking water for 10–15 weeks, while 10 untreated rats served as the control group (the study protocol is summarized in Fig. 1 a). Gross findings confirmed the presence of lesions on the tongue, and the rats were sacrificed. Their tongues were excised, and FV imaging was performed (Fig. 1 b) inside a black box to prevent light refraction and penetration (Fig. 1 c). The FVL region, which is an area darker than its surroundings, was identified in FV images by two professional oral and maxillofacial surgeons. The tongue specimens were sectioned sagittally along the left and right centers of the FVL region, and specimens without FVL were sectioned sagittally in the center. The specimens were then fixed in 10% formalin and embedded in paraffin, after which 4-µm sections were cut and stained. Sections were stained with hematoxylin and eosin (H&E) (Fig. 2 ) and subjected to immunohistochemical staining for histopathological analysis. FI and histopathological observations In all groups treated with 4NQO, macroscopic findings showed roughness and erythema of the tongue mucosa, with hyperkeratosis observed in some cases. In the FV images, the control group showed the same level of dark green fluorescence, whereas the 4NQO-treated group emitted strong green fluorescence with a mixture of bright and dark colors (Fig. 3 ). Areas of hyperkeratosis emitted bright green fluorescence. In addition, the histopathological findings of HE staining revealed cellular and structural atypia in the lower one-third of the mucosa (Fig. 4 a). Further, immunohistochemical staining showed Ki67 stratification (Fig. 4 b), decreased CK13 staining (Fig. 4 c), and increased CK17 staining (Fig. 4 d), indicating low-grade OED. Twelve areas exhibited FVL, all of which coincided with the OED area. Twenty OED areas exhibited FV retention (FVR). CD31 expression was observed in the mucosal lamina propria of all OED regions. Compared to FVR, both the FVL and control groups showed a strong trend toward CD31 expression (Fig. 4 e). Measurement of epithelial and orthokeratinized layer thickness In the control group, the mean thickness of the epithelium was 194 ± 20.58 µm, the mean FVL group was 203.39 ± 18.33 µm, and the mean FVR group was 242.90 ± 32.08 µm (Fig. 5 a). The FVR group showed significantly larger values than did the control and FVL groups. In the control group, the mean thickness of the orthokeratinized layer was 56.49 ± 7.78 µm, the mean FVL group was 65.02 ± 10.79 µm, and the mean FVR group was 70.49 ± 13.59 µm (Fig. 5 b). The control group showed significantly lower values than did the FVR group. Evaluation of immunoreactivity for Ki67, CK13, CK17, and CD31 Ki67 was weakly expressed in the basal cell layer of the control group, while in the FVL and FVR groups, Ki67 was strongly expressed in the basal and parabasal cell layers (Fig. 4 b). Ki67 expression was 4.52 ± 1.41% in the control group, 24.71 ± 4.64% in the FVL group, and 27.21 ± 5.85% in the FVR group (Fig. 6 a). The expression level of Ki67 showed a statistically significant increase in the FVL and FVR groups compared with that in the control group, but no significant differences were observed between the FVL and FVR groups. CK13 was immunoreactive in all layers of the groups except in the basal cell layer (Fig. 4 c). CK13 expression was 63.06 ± 2.30% in the control group, 37.62 ± 6.69% in the FVL group, and 34.63 ± 6.91% in the FVR group (Fig. 6 b). CK13 expression showed a statistically significant decrease in the FVL and FVR groups compared with that in the control group, but no significant differences were observed between the FVL and FVR groups. CK17 expression was rarely observed in any layer of the control group but was observed in all layers except the basal cell layer in the FVL and FVR groups (Fig. 4 d). CK17 expression was 5.50 ± 1.29% in the control group, 15.37 ± 5.09% in the FVL group, and 16.34 ± 4.53% in the FVR group (Fig. 6 c). CK17 expression levels were significantly increased in the FVL and FVR groups compared with those in the control group, but there was no significant difference between the FVL and FVR groups. The control group showed a mean CD31 expression level of 4.45 ± 0.51%. The mean CD31 expression levels in the FVL and FVR groups were 4.47 ± 0.88% and 3.48 ± 1.43%, respectively (Fig. 6 d). No significant differences were observed among any of the groups. Evaluation of the relationship between epithelial thickness, orthokeratinized layer thickness, and CD31-positivity rate with FI FI showed a positive correlation with epithelial thickness ( r = 0.706, p = 0.000000172, Fig. 7 a), a positive correlation with orthokeratinized layer thickness ( r =-0.544, p = 0.000195, Fig. 7 b), and a negative correlation with the CD31-positivity rate ( r =-0.616, p = 0.0000139) (Fig. 7 c). Multiple regression analysis showed that the CD31-positivity rate and epithelial thickness were independent factors for FVL (Table 1 ). Table 1 Results of the multiple regression analysis for factors related to FI p -value Factors involved in FI Multiple regression Thickness of the epithelium (µm) * 0.0089 Thickness of the orthokeratinized layer (µm) 0.3264 CD31-positivity rate (%) * 0.0022 FI, fluorescence intensity * Statistical significance ( p < 0.05). Discussion The pathology induced by 4NQO closely resembles carcinogenesis in a human squamous cell carcinoma model, particularly the progression of OSCC with lesions at different pathological stages. 4NQO-induced OSCC recapitulates the stepwise progression from dysplasia to invasive cancer, as observed in human disease. Tongue lesions in 4NQO-treated models have been shown to progress from hyperplasia to dysplasia, papilloma, and invasive squamous cell carcinoma [ 27 ]. Matsuhira et al. [ 28 ] reported that the characteristic expression patterns of Ki67, CK13, CK17, and p53 differ between normal tissues and OED. CK13 is expressed both in the normal epithelium and in OED, and its expression tends to decrease with advanced dysplasia. In contrast, CK17 expression is mainly observed in lesions and increases as the disease progresses to high-grade dysplasia [ 9 , 29 – 31 ]. Ki67 is a protein expressed in all phases of the cell cycle except the G0 phase and indicates cell proliferation. The Ki67 labeling index is related to the degree of dysplasia and the severity of malignancy [ 31 – 33 ]. In this study, we aimed to evaluate OED and, therefore, treated the rats with 4NQO from weeks 10 to 15. Indeed, all rats showed pathological low-grade OED, which we considered appropriate for this treatment timeframe. This study focused on CD31 expression and epithelial thickness in relation to FVL. Sekine et al. [ 34 ] reported that OED and OSCC exhibit an increase in capillary vessels with tortuosity, dilation, and aggregation. They further noted that in cases with a thick epithelium, visualization is challenging because the irradiated light does not reach the epithelium. In addition, light absorption due to angiogenesis has been suggested as a factor contributing to FVL [ 12 , 14 ]. Kikuta et al. [ 19 ] reported that FI tends to be higher in the early stages of oral cancer than in the later stages. Kosugi et al. [ 35 ] showed that low-grade OED has a high FI owing to epithelial thickening, while progression to a more aggressive stage leads to a decrease in FI, resulting in FVL. 4NQO-treated rats show a predilection for lesions on the dorsal surface of their tongues [ 36 , 37 ]. In this study, most of the OED model rats showed the leukoplakia type. OSCC occurring on keratinized mucosa is considered to have a lower FI and better lesion delineation accuracy, whereas leukoplakia results in an elevated FI [ 19 ]. Our findings showed a significantly higher CD31 expression rate in the FVL group, which was likely influenced by angiogenesis associated with OED. Lesions with macroscopic hyperkeratosis and histologically thick epithelial layers caused FVR, which is consistent with the findings of previous studies. Multiple regression analysis also showed that the thickness of the epithelium and the CD31-positivity rate were independent factors of FVL, suggesting that the thickness of the orthokeratinized layer was not associated with a negative FV result. Previous studies have confirmed the involvement of collagen crosslinks, FAD, and intercellular adhesion molecules as factors contributing to the mechanism of FV [ 18 , 38 , 39 ]. In this study, a relationship between angiogenesis and FI was suggested based on the CD31-positivity rate. However, the imaging procedure was performed after tongue resection to facilitate rapid imaging and maintain consistent conditions. As blood flow ceases post-resection, this differs from actual clinical practice. Future research should include establishment of a method for FV imaging prior to tongue resection. We believe that numerous factors contribute to negative FV results and that a multifaceted diagnostic approach—including FV, conventional biostaining, and intraoperative rapid diagnosis—will be essential for improving treatment outcomes. In conclusion, to investigate the cause of negative FV results sometimes observed in clinical practice, we examined the relationship of FI with angiogenesis and epithelial thickness. Our results suggest that the CD31-positivity rate in blood vessels and epithelial thickness correlate with FI and may play a significant role in negative FV results. Methods Ethical statement This animal study was approved by the Tokyo Dental College Review Board (approval no. 232501). All animals received humane care in accordance with the guidelines for the treatment of experimental animals. Furthermore, the acquisition and description of data in this study were carried out in accordance with ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. Animals The study protocol was based on a previous study [35]. Twenty male Sprague–Dawley (SD) rats (6 weeks old; weight, approximately 250 g; Sankyo Labo, Tokyo, Japan) were used in this study and were fed a standard diet. The rats were treated with 50 ppm solution of 4NQO in their drinking water for 10–15 weeks. The 4NQO solution was prepared as previously described [35]. A stock solution was prepared by dissolving 1.0 g 4NQO in 50 mL ethanol and 4,950 mL distilled water and stored at room temperature in the dark. The stock solution was diluted with tap water to a final concentration of 50 ppm and poured into a light-resistant water bottle. Once a week, the bottle was refilled with freshly prepared 4NQO solution. Ten untreated rats were used as the control group. The control group consisted of male SD rats (10 weeks old, approximately 250 g), which were fed a standard diet and received tap water only as drinking water (Fig. 1a). The rats were deep anesthesia with a mixture of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg) and sacrificed with an overdose of isoflurane. Experimental methods and imaging Gross findings confirmed the lesions on the tongue, and the rats were sedated using intraperitoneal injection of anesthetics combined with isoflurane inhalation. Immediately after sacrifice, the tongues were excised at the base, and FV imaging was performed using IllumiScan ® (Fig. 1b). The imaging distance was set to 5 cm. FV imaging was performed inside a black box to prevent light refraction and penetration (Fig. 1c). The FVL region was defined in images of the tongue obtained using FV. FVL, which was an area darker than its surroundings, was decided by two professional oral and maxillofacial surgeons. Preparation of specimens and histopathological observation The tongue specimens were sectioned sagittally along the left and right centers of the FVL region, and specimens without FVL were sectioned sagittally in the center. They were then fixed in 10% formalin and embedded in paraffin. Sections with a thickness of approximately 4 μm were prepared using a microtome and were stained with H&E and subjected to immunohistochemical staining for histopathological analysis. In this study, we used the following antibodies: anti-Ki67 (Abcam; ab16667, 1/200), anti-cytokeratin 13 (Abcam; ab154346, 1/500), anti-cytokeratin 17 (Abcam; ab109725, 1/200), and anti-CD31 (Abcam; ab182981, 1/2000). The sections were deparaffinized using xylol and then subjected to antigen retrieval by soaking them in distilled water prepared by diluting an immunosaver (Nisshin EM Corporation, Tokyo, Japan) with a Decloaking Chamber TM NxGen (FUNAKOSHI, Tokyo, Japan) for 15 min at 110°C for Ki67 or for 40 min at 95°C for the other antibodies. To block endogenous peroxidase activity, the sections were immersed in methanol containing 0.3% aqueous hydrogen peroxide for 30 min at room temperature. To reduce nonspecific binding, sections were blocked for 60 min with 10% goat serum at room temperature. Sections were then reacted with primary antibodies overnight at 4°C. For the negative control, 1% goat serum was used instead of the primary antibody. The sections were incubated with a peroxidase‐conjugated secondary antibody using MACH 2 Universal HRP Polymer Detection (BRR522G, Biocare Medical, Pacheco, CA) for 30 min at room temperature. Finally, the sections were stained with DAB, and counterstaining was performed with hematoxylin. The sections were observed using a light microscope (Axio Imager 2; Carl Zeiss, Oberkochen, Germany). Diagnosis Initial lesions formed in the anterior part of the half-moon structure of the tongue in the rat model; therefore, that area was observed and evaluated. All specimens were histopathologically diagnosed by three independent pathologists based on H&E and immunohistochemical staining. In this study, we used the 5th edition of the WHO head and neck tumor classification to diagnose OED [22]. In addition, immunohistochemical staining for Ki67 stratification, decreased CK13 expression, and increased CK17 expression were used to determine the lesion areas. FI and histological evaluation FV images were compared with the OED area, and only areas that matched the OED were evaluated. Images obtained with FV were analyzed using image analysis software (ImageJ; Fiji, version 1.53, National Institutes of Health, Bethesda, MD, USA). All OED areas were divided into FVL and FVR. FI was measured in the respective FVL and FVR areas of gross findings. The control group mean FI was measured over 200 pixels from the half-moon structure. To determine the thickness of the epithelial and orthokeratinized layers, we measured 20 arbitrary locations of the stained pathological histological images and calculated the average value (Fig. 2). The positivity rates of Ki67, CK13, CK17, and CD31 in each region were calculated using ImageJ software. The cell positivity rates were calculated as follows: Ki67 positivity rate (%) = (positive cells/total cells from the basal cell layer to the third layer) × 100 CK13 or CK17 positivity rate (%) = (positive cells/total epithelial cells) × 100 CD31 positivity rate (%) = (positive area/mucosal lamina propria area) × 100 Statistical analysis All data are presented as means ± standard deviation (SD) and were analyzed using Tukey's multiple comparison test, Pearson’s correlation, and multiple regression analysis. The relationships among CD31, epithelial thickness, and orthokeratinized layer thickness with FI were determined using Pearson's correlation. Additionally, multiple regression analysis was performed to identify factors significantly associated with FI. Statistical analysis was performed using EZR version 1.61 (Jichi Medical University. Saitama Medical Center), and a p -value < 0.05 was considered statistically significant. Declarations Author contribution statement M.F., K.N., K.M., M.T., A.K., K.O., K.K, and Y.A contributed to this study. K.M., M.T., and A.K. designed the study, directed its conduct, and revised the draft. M.F., K.N., Y.A., K.K., and K.O. performed specific experimental processes, analyzed data, and participated in writing the manuscript. M.F. was involved in the charting of the paper. All authors reviewed the manuscript. Competing interests statement The authors declare no competing interests. Data availability statement The data used or analyzed in this study are available from the corresponding author upon reasonable request. References Pfister, D. G. et al. Head and neck cancers, version 2.2020, NCCN Clinical Practice Guidelines in Oncology. J. Natl. Compr. Canc. Netw. 18, 873-898 (2020). Subramaniam, N. et al . Adverse pathologic features in early oral squamous cell carcinoma and the role of postoperative radiotherapy-a review. O ral Surg. Oral Med. Oral Pathol. 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Oral Maxillofac. Surg. 27, 685-700 (1981). (In Japanese). Masuda, H., Yamamoto, N. & Shibahara, T. Early detection of leukoplakic oral squamous cell carcinoma using 4NQO-induced rat tongue cancer model: study utilizing fluorescence intensity and histopathological evaluation. Bull. Tokyo Dent. Coll. 8, 1-12 (2022). Sumi, S. et al . The luminance ratio of autofluorescence in a xenograft mouse model is stable through tumor growth stages. Clin. Exp. Dent. Res. 15, 174-181 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Accepted 22 Apr, 2025 Reviewers invited by journal 14 Apr, 2025 Submission checks completed at journal 07 Apr, 2025 First submitted to journal 22 Mar, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5565034","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":442598025,"identity":"dd6d2990-e703-4e28-80b0-6b3104afee09","order_by":0,"name":"Minori Fujikawa","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Minori","middleName":"","lastName":"Fujikawa","suffix":""},{"id":442598026,"identity":"bb502217-9b4a-4f42-8fc4-4697c281c48b","order_by":1,"name":"Kei Nakajima","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3QPQrCMBiA4S8IuhRcPxd7hUgGdVCvYijUtSCI4KIU4uIBFMFjOEeEdim6Froogq4dHRxMBkEcUkfBvBBIhid/ADbbT1YGQKoGkNlJL3T0WxJS+TXRoZrhixhrViKet4KtW5uHYpKPulCdSxgGBtJe+HtEmjXWzk6k8uABJn1gSwOhcjDVhGyQi3QnJEAKwBwTOd7CuyK9jXsWgSZuIUn9SJ/C10gEaEKLydVvKeKtFjzE5OA5jYRPzW85+izDR9ZZxvE5H4+69Xq8j5jpx3QlfFuoKxHBCgSQ/HOPSxGx2Wy2v+oJcg9PJItDUCgAAAAASUVORK5CYII=","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":true,"prefix":"","firstName":"Kei","middleName":"","lastName":"Nakajima","suffix":""},{"id":442598027,"identity":"21499672-eab0-447c-b4ae-184064b9496d","order_by":2,"name":"Yoshihiko Akashi","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Yoshihiko","middleName":"","lastName":"Akashi","suffix":""},{"id":442598028,"identity":"0dd17ad1-390f-4c91-b5d8-19bb263365ac","order_by":3,"name":"Katsutoshi Kokubun","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Katsutoshi","middleName":"","lastName":"Kokubun","suffix":""},{"id":442598029,"identity":"3792fe40-5dc4-4250-8c79-65cf28b823bd","order_by":4,"name":"Keisuke Ohno","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Keisuke","middleName":"","lastName":"Ohno","suffix":""},{"id":442598030,"identity":"9d56b6d5-c737-4143-b7d1-366ba16d1cc8","order_by":5,"name":"Akira Katakura","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Katakura","suffix":""},{"id":442598031,"identity":"2b1de577-657e-4177-8985-c4627c07d49a","order_by":6,"name":"Masayuki Takano","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Masayuki","middleName":"","lastName":"Takano","suffix":""},{"id":442598032,"identity":"3f33091b-8511-4070-999f-41ec8b196a57","order_by":7,"name":"Kenichi Matsuzaka","email":"","orcid":"","institution":"Tokyo Dental College","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Matsuzaka","suffix":""}],"badges":[],"createdAt":"2024-12-02 13:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5565034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5565034/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-99797-w","type":"published","date":"2025-04-30T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80784546,"identity":"f7fee073-8dfc-4274-9b8a-e1fdcabdccd7","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1351195,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design.\u003c/p\u003e\n\u003cp\u003eFlowchart of the experimental design. After treatment with 4-nitroquinoline 1-oxide (4NQO) for 10–15 weeks, the tongue of each anesthetized rat was collected, and fluorescence visualization (FV) imaging was performed (\u003cstrong\u003ea\u003c/strong\u003e). Images of a normal rat tongue; gross observation and fluorescence images are shown (\u003cstrong\u003eb\u003c/strong\u003e). Fluorescence imaging method: the specimen is positioned at a fixed distance of 5 cm in the dark (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/0d8e41859143805cdbfc3766.png"},{"id":80784547,"identity":"16bcffee-2799-45a9-ab17-a70e85715e41","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4192458,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of the thickness of the epithelial and orthokeratinized layers.\u003c/p\u003e\n\u003cp\u003eThe thickness of the epithelial and orthokeratinized layers was measured at 20 arbitrary locations in hematoxylin and eosin (H\u0026amp;E)-stained pathological histological images to calculate the average value. Blue arrows: Thickness of the epithelium. Yellow arrows: Thickness of the orthokeratinized layer.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/418c98c4f5dc7424b1bcbd62.png"},{"id":80784568,"identity":"46eac7a3-86e3-418d-b2ab-d4d930036733","added_by":"auto","created_at":"2025-04-17 05:36:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1570225,"visible":true,"origin":"","legend":"\u003cp\u003eImages of a rat tongue subjected to fluorescence visualization (FV).\u003c/p\u003e\n\u003cp\u003eClassification based on test Results. Control group: the fluorescence image shows a slightly dark green fluorescence, except for the half-moon structure (\u003cstrong\u003ea\u003c/strong\u003e). FV loss (FVL): the fluorescence images show areas darker than the surrounding area (\u003cstrong\u003eb\u003c/strong\u003e). FVR: the fluorescence image exhibits a light green fluorescence emitted from the entire tongue (\u003cstrong\u003ec\u003c/strong\u003e). Arrowheads indicate the observed FVL regions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/2de43fee33c0e263e2b6ad34.png"},{"id":80784551,"identity":"baa5616a-2cc4-4539-b0bf-630cd91937a8","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7880857,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological observation.\u003c/p\u003e\n\u003cp\u003eHistopathological images of the control, fluorescence visualization loss (FVL) and FV retention (FVR) regions. Hematoxylin and eosin (H\u0026amp;E) staining (\u003cstrong\u003ea\u003c/strong\u003e) and immunohistochemical staining of Ki67 (\u003cstrong\u003eb\u003c/strong\u003e), CK13 (\u003cstrong\u003ec\u003c/strong\u003e), CK17 (\u003cstrong\u003ed\u003c/strong\u003e), and CD31 (\u003cstrong\u003ee\u003c/strong\u003e). In H\u0026amp;E-stained images, cellular and structural atypia are observed in the FVL and FVR groups. The percentage of positive cells within the epithelial layer was measured at each site range. Compared to the control group, CK13 expression decreased, while Ki67 and CK17 expression increased in the FVL and FVR groups. The percentage of CD31 positivity in the control, FVL, and FVR groups was measured; CD31 expression was determined by dividing the expression area of each site by the intrinsic layer area.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/4c3e1da2eee21390b9a0ce41.png"},{"id":80784550,"identity":"5953487e-e50f-4c16-9897-74fe79dafd1e","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":158000,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the thickness of the epithelial and orthokeratinized layers.\u003c/p\u003e\n\u003cp\u003eThe thickness of the epithelium was significantly greater in the fluorescence visualization retention (FVR) group than in the control and FV loss (FVL) groups (\u003cstrong\u003ea\u003c/strong\u003e). The thickness of the orthokeratinized layer differed significantly between the FVR and control groups (\u003cstrong\u003eb\u003c/strong\u003e) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Tukey's test)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/f7371b5b6c349b2a327e17b7.png"},{"id":80784553,"identity":"bc96772a-f7bb-4646-9399-271f95b4e1f5","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":255227,"visible":true,"origin":"","legend":"\u003cp\u003eExpression rates of Ki67, CK13, CK17, and CD31.\u003c/p\u003e\n\u003cp\u003eThe fluorescence visualization loss (FVL) and FV retention (FVR) groups showed significant differences from the control group in the expression of Ki67, CK13, and CK17. However, no significant differences were observed in Ki67, CK13, or CK17 expression between the FVL and FVR groups. Additionally, CD31-positivity rates did not differ significantly among any of the groups. The positivity rates of Ki67 (\u003cstrong\u003ea\u003c/strong\u003e), CK13 (\u003cstrong\u003eb\u003c/strong\u003e), CK17 (\u003cstrong\u003ec\u003c/strong\u003e), and CD31 (\u003cstrong\u003ed\u003c/strong\u003e) are shown (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Tukey's test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/09c143c10cec5454e5725a94.png"},{"id":80784552,"identity":"1686eed3-0c80-49c5-8819-977774d56eaa","added_by":"auto","created_at":"2025-04-17 05:36:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":253142,"visible":true,"origin":"","legend":"\u003cp\u003ePearson’s correlation among epithelial thickness, orthokeratinized layer thickness, and the CD31-positivity rate by fluorescence intensity (FI).\u003c/p\u003e\n\u003cp\u003eA positive correlation was observed between epithelial thickness and FI (\u003cstrong\u003ea\u003c/strong\u003e) and between orthokeratinized layer thickness and FI (\u003cstrong\u003eb\u003c/strong\u003e). A negative correlation was observed between the CD31-positivity rate and FI (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/88ce0c25bc9e464f924cb29b.png"},{"id":81987902,"identity":"00022c9a-ec03-4f3e-892a-52cfccc08ef9","added_by":"auto","created_at":"2025-05-05 16:06:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14620290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5565034/v1/14d049f7-3b83-4da1-a6ed-e056e3342e72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Histopathological characteristics of an oral epithelial dysplasia model in rats showing a negative reaction to fluorescence visualization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSurgical resection remains the treatment of choice for early-stage (T1-2) oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although the prognosis of patients with these conditions is generally considered good, locoregional recurrence is possible, with the adverse pathologic features of inadequate resection margins being a major recurrence factor [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Conventionally, vital staining with toluidine blue or iodine is used to determine the extent of resection [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Normal epithelium stains with iodine solution, but abnormal areas with dysplasia do not stain well with iodine and exhibit iodine-unstained areas owing to differences in cytoplasmic glycogen content. This staining mechanism allows surgical resection using vital iodine staining to visualize the boundaries between OSCC and OED [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although iodine staining is useful for defining lesion margins, fluorescence visualization (FV) has been increasingly used in recent years. FV highlights atypical epithelium, offering a less invasive alternative to vital staining and providing an option for patients with allergies [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFV distinguishes between normal and abnormal mucosa using visible light at wavelengths of 400\u0026ndash;460 nm [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Under this light, normal mucosa emits a pale green fluorescence due to collagen crosslinking and flavin adenine dinucleotide (FAD). In contrast, abnormal areas absorb fluorescence owing to light absorption reactions caused by the disruption of collagen crosslinks, coenzyme deficiencies, and angiogenesis, resulting in dark areas with FV loss (FVL) [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have reported no significant difference in accuracy between FV and vital staining for determining resection margins [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The FV device (IllumiScan\u0026reg;: Shofu Inc, Kyoto, Japan) used in this study was developed by our institution and approved under Japan\u0026rsquo;s Pharmaceutical and Medical Device Act. Its accuracy has been validated in multiple studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, this device allows for the measurement of fluorescence intensity (FI) in captured images using image analysis software, enhancing its utility in objective assessments.\u003c/p\u003e \u003cp\u003eAccording to the 5th edition of the World Health Organization (WHO) head and neck tumor classification, OED is defined as a range of structural and cytological epithelial changes resulting from the accumulation of genetic mutations, indicating a risk of malignant transformation to squamous cell carcinoma. OED is diagnosed based on structural and cytological features [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, immunohistochemical staining reveals Ki67 stratification in the basal layer, decreased CK13 expression, and increased CK17 expression in the epithelial layer. Ki67 expression is positive throughout the epithelium of the lesions and is only noted in the basal cell layer in normal areas [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, Sugahara et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported that CK17 was positive in epithelial tumors and negative in normal oral mucosa, whereas the opposite was true for CK13 expression. Saitoh et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported that the expression of CK13 and CK17 was observed in both an OED rat model and normal rats and showed that as the lesion progressed, CK17 expression increased and CK13 expression decreased.\u003c/p\u003e \u003cp\u003eHowever, FV may sometimes not detect early lesions in OSCC and OED [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some studies have suggested that the thickness of epithelial cells and angiogenesis may be involved, but no clear cause has been identified. This study aimed to investigate the factors contributing to FV-negative lesion detection by conducting histopathological analyses in an OED rat model, with a particular focus on the correlation between angiogenesis and epithelial thickness.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOverview of the OED model\u003c/h2\u003e \u003cp\u003eThe OED model was established in Sprague\u0026ndash;Dawley rats as detailed in the Methods. Briefly, 20 male rats were fed a standard diet and were treated with 4-nitroquinoline 1-oxide (4NQO) in their drinking water for 10\u0026ndash;15 weeks, while 10 untreated rats served as the control group (the study protocol is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Gross findings confirmed the presence of lesions on the tongue, and the rats were sacrificed. Their tongues were excised, and FV imaging was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) inside a black box to prevent light refraction and penetration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The FVL region, which is an area darker than its surroundings, was identified in FV images by two professional oral and maxillofacial surgeons. The tongue specimens were sectioned sagittally along the left and right centers of the FVL region, and specimens without FVL were sectioned sagittally in the center. The specimens were then fixed in 10% formalin and embedded in paraffin, after which 4-\u0026micro;m sections were cut and stained. Sections were stained with hematoxylin and eosin (H\u0026amp;E) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and subjected to immunohistochemical staining for histopathological analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFI and histopathological observations\u003c/h3\u003e\n\u003cp\u003eIn all groups treated with 4NQO, macroscopic findings showed roughness and erythema of the tongue mucosa, with hyperkeratosis observed in some cases. In the FV images, the control group showed the same level of dark green fluorescence, whereas the 4NQO-treated group emitted strong green fluorescence with a mixture of bright and dark colors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Areas of hyperkeratosis emitted bright green fluorescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the histopathological findings of HE staining revealed cellular and structural atypia in the lower one-third of the mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Further, immunohistochemical staining showed Ki67 stratification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), decreased CK13 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), and increased CK17 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), indicating low-grade OED. Twelve areas exhibited FVL, all of which coincided with the OED area. Twenty OED areas exhibited FV retention (FVR).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCD31 expression was observed in the mucosal lamina propria of all OED regions. Compared to FVR, both the FVL and control groups showed a strong trend toward CD31 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\n\u003ch3\u003eMeasurement of epithelial and orthokeratinized layer thickness\u003c/h3\u003e\n\u003cp\u003eIn the control group, the mean thickness of the epithelium was 194\u0026thinsp;\u0026plusmn;\u0026thinsp;20.58 \u0026micro;m, the mean FVL group was 203.39\u0026thinsp;\u0026plusmn;\u0026thinsp;18.33 \u0026micro;m, and the mean FVR group was 242.90\u0026thinsp;\u0026plusmn;\u0026thinsp;32.08 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The FVR group showed significantly larger values than did the control and FVL groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the control group, the mean thickness of the orthokeratinized layer was 56.49\u0026thinsp;\u0026plusmn;\u0026thinsp;7.78 \u0026micro;m, the mean FVL group was 65.02\u0026thinsp;\u0026plusmn;\u0026thinsp;10.79 \u0026micro;m, and the mean FVR group was 70.49\u0026thinsp;\u0026plusmn;\u0026thinsp;13.59 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The control group showed significantly lower values than did the FVR group.\u003c/p\u003e\n\u003ch3\u003eEvaluation of immunoreactivity for Ki67, CK13, CK17, and CD31\u003c/h3\u003e\n\u003cp\u003eKi67 was weakly expressed in the basal cell layer of the control group, while in the FVL and FVR groups, Ki67 was strongly expressed in the basal and parabasal cell layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Ki67 expression was 4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41% in the control group, 24.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.64% in the FVL group, and 27.21\u0026thinsp;\u0026plusmn;\u0026thinsp;5.85% in the FVR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The expression level of Ki67 showed a statistically significant increase in the FVL and FVR groups compared with that in the control group, but no significant differences were observed between the FVL and FVR groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCK13 was immunoreactive in all layers of the groups except in the basal cell layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). CK13 expression was 63.06\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30% in the control group, 37.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.69% in the FVL group, and 34.63\u0026thinsp;\u0026plusmn;\u0026thinsp;6.91% in the FVR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). CK13 expression showed a statistically significant decrease in the FVL and FVR groups compared with that in the control group, but no significant differences were observed between the FVL and FVR groups.\u003c/p\u003e \u003cp\u003eCK17 expression was rarely observed in any layer of the control group but was observed in all layers except the basal cell layer in the FVL and FVR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). CK17 expression was 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29% in the control group, 15.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.09% in the FVL group, and 16.34\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53% in the FVR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). CK17 expression levels were significantly increased in the FVL and FVR groups compared with those in the control group, but there was no significant difference between the FVL and FVR groups.\u003c/p\u003e \u003cp\u003eThe control group showed a mean CD31 expression level of 4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51%. The mean CD31 expression levels in the FVL and FVR groups were 4.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88% and 3.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). No significant differences were observed among any of the groups.\u003c/p\u003e\n\u003ch3\u003eEvaluation of the relationship between epithelial thickness, orthokeratinized layer thickness, and CD31-positivity rate with FI\u003c/h3\u003e\n\u003cp\u003eFI showed a positive correlation with epithelial thickness (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.706, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000000172, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), a positive correlation with orthokeratinized layer thickness (\u003cem\u003er\u003c/em\u003e=-0.544, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000195, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), and a negative correlation with the CD31-positivity rate (\u003cem\u003er\u003c/em\u003e=-0.616, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0000139) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Multiple regression analysis showed that the CD31-positivity rate and epithelial thickness were independent factors for FVL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eResults of the multiple regression analysis for factors related to FI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactors involved in FI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultiple regression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of the epithelium (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e*\u003c/sup\u003e 0.0089\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of the orthokeratinized layer (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD31-positivity rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e*\u003c/sup\u003e 0.0022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eFI, fluorescence intensity\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e* Statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathology induced by 4NQO closely resembles carcinogenesis in a human squamous cell carcinoma model, particularly the progression of OSCC with lesions at different pathological stages. 4NQO-induced OSCC recapitulates the stepwise progression from dysplasia to invasive cancer, as observed in human disease. Tongue lesions in 4NQO-treated models have been shown to progress from hyperplasia to dysplasia, papilloma, and invasive squamous cell carcinoma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Matsuhira et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] reported that the characteristic expression patterns of Ki67, CK13, CK17, and p53 differ between normal tissues and OED. CK13 is expressed both in the normal epithelium and in OED, and its expression tends to decrease with advanced dysplasia. In contrast, CK17 expression is mainly observed in lesions and increases as the disease progresses to high-grade dysplasia [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Ki67 is a protein expressed in all phases of the cell cycle except the G0 phase and indicates cell proliferation. The Ki67 labeling index is related to the degree of dysplasia and the severity of malignancy [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we aimed to evaluate OED and, therefore, treated the rats with 4NQO from weeks 10 to 15. Indeed, all rats showed pathological low-grade OED, which we considered appropriate for this treatment timeframe.\u003c/p\u003e \u003cp\u003eThis study focused on CD31 expression and epithelial thickness in relation to FVL. Sekine et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported that OED and OSCC exhibit an increase in capillary vessels with tortuosity, dilation, and aggregation. They further noted that in cases with a thick epithelium, visualization is challenging because the irradiated light does not reach the epithelium. In addition, light absorption due to angiogenesis has been suggested as a factor contributing to FVL [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Kikuta et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] reported that FI tends to be higher in the early stages of oral cancer than in the later stages. Kosugi et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] showed that low-grade OED has a high FI owing to epithelial thickening, while progression to a more aggressive stage leads to a decrease in FI, resulting in FVL. 4NQO-treated rats show a predilection for lesions on the dorsal surface of their tongues [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, most of the OED model rats showed the leukoplakia type. OSCC occurring on keratinized mucosa is considered to have a lower FI and better lesion delineation accuracy, whereas leukoplakia results in an elevated FI [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our findings showed a significantly higher CD31 expression rate in the FVL group, which was likely influenced by angiogenesis associated with OED. Lesions with macroscopic hyperkeratosis and histologically thick epithelial layers caused FVR, which is consistent with the findings of previous studies. Multiple regression analysis also showed that the thickness of the epithelium and the CD31-positivity rate were independent factors of FVL, suggesting that the thickness of the orthokeratinized layer was not associated with a negative FV result.\u003c/p\u003e \u003cp\u003ePrevious studies have confirmed the involvement of collagen crosslinks, FAD, and intercellular adhesion molecules as factors contributing to the mechanism of FV [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, a relationship between angiogenesis and FI was suggested based on the CD31-positivity rate. However, the imaging procedure was performed after tongue resection to facilitate rapid imaging and maintain consistent conditions. As blood flow ceases post-resection, this differs from actual clinical practice. Future research should include establishment of a method for FV imaging prior to tongue resection. We believe that numerous factors contribute to negative FV results and that a multifaceted diagnostic approach\u0026mdash;including FV, conventional biostaining, and intraoperative rapid diagnosis\u0026mdash;will be essential for improving treatment outcomes.\u003c/p\u003e \u003cp\u003eIn conclusion, to investigate the cause of negative FV results sometimes observed in clinical practice, we examined the relationship of FI with angiogenesis and epithelial thickness. Our results suggest that the CD31-positivity rate in blood vessels and epithelial thickness correlate with FI and may play a significant role in negative FV results.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis animal study was approved by the Tokyo Dental College Review Board (approval no. 232501). All animals received humane care in accordance with the guidelines for the treatment of experimental animals. Furthermore, the acquisition and description of data in this study were carried out in accordance with ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was based on a previous study [35]. Twenty male Sprague–Dawley (SD) rats (6 weeks old; weight, approximately 250 g; Sankyo Labo, Tokyo, Japan) were used in this study and were fed a standard diet. The rats were treated with 50 ppm solution of 4NQO in their drinking water for 10–15 weeks. The 4NQO solution was prepared as previously described [35]. A stock solution was prepared by dissolving 1.0 g 4NQO in 50 mL ethanol and 4,950 mL distilled water and stored at room temperature in the dark. The stock solution was diluted with tap water to a final concentration of 50 ppm and poured into a light-resistant water bottle. Once a week, the bottle was refilled with freshly prepared 4NQO solution. Ten untreated rats were used as the control group. The control group consisted of male SD rats (10 weeks old, approximately 250 g), which were fed a standard diet and received tap water only as drinking water (Fig. 1a).\u003c/p\u003e\n\u003cp\u003eThe rats were deep anesthesia with a mixture of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol (2.5 mg/kg) and sacrificed with an overdose of isoflurane.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental methods and imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGross findings confirmed the lesions on the tongue, and the rats were sedated using intraperitoneal injection of anesthetics combined with isoflurane inhalation. Immediately after sacrifice, the tongues were excised at the base, and FV imaging was performed using IllumiScan\u003csup\u003e®\u003c/sup\u003e (Fig. 1b). The imaging distance was set to 5 cm. FV imaging was performed inside a black box to prevent light refraction and penetration (Fig. 1c). The FVL region was defined in images of the tongue obtained using FV. FVL, which was an area darker than its surroundings, was decided by two professional oral and maxillofacial surgeons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of specimens and histopathological observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tongue specimens were sectioned sagittally along the left and right centers of the FVL region, and specimens without FVL were sectioned sagittally in the center. They were then fixed in 10% formalin and embedded in paraffin. Sections with a thickness of approximately 4 μm were prepared using a microtome and were stained with H\u0026amp;E and subjected to immunohistochemical staining for histopathological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we used the following antibodies: anti-Ki67 (Abcam; ab16667, 1/200), anti-cytokeratin 13 (Abcam; ab154346, 1/500), anti-cytokeratin 17 (Abcam; ab109725, 1/200), and anti-CD31 (Abcam; ab182981, 1/2000). The sections were deparaffinized using xylol and then subjected to antigen retrieval by soaking them in distilled water prepared by diluting an immunosaver (Nisshin EM Corporation, Tokyo, Japan) with a Decloaking Chamber\u003csup\u003eTM\u003c/sup\u003e NxGen (FUNAKOSHI, Tokyo, Japan) for 15 min at 110°C for Ki67 or for 40 min at 95°C for the other antibodies. To block endogenous peroxidase activity, the sections were immersed in methanol containing 0.3% aqueous hydrogen peroxide for 30 min at room temperature. To reduce nonspecific binding, sections were blocked for 60 min with 10% goat serum at room temperature. Sections were then reacted with primary antibodies overnight at 4°C. For the negative control, 1% goat serum was used instead of the primary antibody. The sections were incubated with a peroxidase‐conjugated secondary antibody using MACH 2 Universal HRP Polymer Detection (BRR522G, Biocare Medical, Pacheco, CA) for 30 min at room temperature. Finally, the sections were stained with DAB, and counterstaining was performed with hematoxylin. The sections were observed using a light microscope (Axio Imager 2; Carl Zeiss, Oberkochen, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInitial lesions formed in the anterior part of the half-moon structure of the tongue in the rat model; therefore, that area was observed and evaluated. All specimens were histopathologically diagnosed by three independent pathologists based on H\u0026amp;E and immunohistochemical staining. In this study, we used the 5th edition of the WHO head and neck tumor classification to diagnose OED [22]. In addition, immunohistochemical staining for Ki67 stratification, decreased CK13 expression, and increased CK17 expression were used to determine the lesion areas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFI and histological evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFV images were compared with the OED area, and only areas that matched the OED were evaluated. Images obtained with FV were analyzed using image analysis software (ImageJ; Fiji, version 1.53, National Institutes of Health, Bethesda, MD, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll OED areas were divided into FVL and FVR. FI was measured in the respective FVL and FVR areas of gross findings. The control group mean FI was measured over\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e200 pixels from the half-moon structure.\u003c/p\u003e\n\u003cp\u003eTo determine the thickness of the epithelial and orthokeratinized layers, we measured 20 arbitrary locations of the stained pathological histological images and calculated the average value (Fig. 2). The positivity rates of Ki67, CK13, CK17, and CD31 in each region were calculated using ImageJ software. The cell positivity rates were calculated as follows:\u003c/p\u003e\n\u003cp\u003eKi67 positivity rate (%)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e= (positive cells/total cells from the basal cell layer to the third layer) × 100\u003c/p\u003e\n\u003cp\u003eCK13 or CK17 positivity rate (%)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e= (positive cells/total epithelial cells) × 100\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD31 positivity rate (%)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e= (positive area/mucosal lamina propria area) × 100\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as means ± standard deviation (SD) and were analyzed using Tukey's multiple comparison test, Pearson’s correlation, and multiple regression analysis. The relationships among CD31,\u0026nbsp;epithelial thickness, and\u0026nbsp;orthokeratinized layer thickness with FI were determined using Pearson's correlation. Additionally, multiple regression analysis was performed to identify factors significantly associated with FI. Statistical analysis was performed using EZR version 1.61 (Jichi Medical University. Saitama Medical Center), and a\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.F., K.N., K.M., M.T., A.K., K.O., K.K, and Y.A contributed to this study. K.M., M.T., and A.K. designed the study, directed its conduct, and revised the draft. M.F., K.N., Y.A., K.K., and K.O. performed specific experimental processes, analyzed data, and participated in writing the manuscript. M.F. was involved in the charting of the paper. All authors reviewed the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used or analyzed in this study are available from the corresponding author upon reasonable request.\u003cbr\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePfister, D. G. et al. Head and neck cancers, version 2.2020, NCCN Clinical Practice Guidelines in Oncology. \u003cem\u003eJ. Natl. Compr. Canc. Netw.\u003c/em\u003e \u003cstrong\u003e18,\u003c/strong\u003e 873-898 (2020).\u003c/li\u003e\n\u003cli\u003eSubramaniam, N.\u003cem\u003e \u003c/em\u003eet al\u003cem\u003e.\u003c/em\u003e Adverse pathologic features in early oral squamous cell carcinoma and the role of postoperative radiotherapy-a review. 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Res.\u003c/em\u003e \u003cstrong\u003e15,\u003c/strong\u003e 174-181 (2018).\u003cbr\u003e \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"fluorescence visualization, oral epithelial dysplasia, immunohistochemical staining, pathological approach","lastPublishedDoi":"10.21203/rs.3.rs-5565034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5565034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVital staining has long been used to delineate the horizontal margins for resection in oral squamous cell carcinoma and in oral epithelial dysplasia (OED). In recent years, fluorescence visualization (FV), a simple and non-invasive technique, has been used to identify resection margins. However, FV occasionally fails to detect OED. The purpose of this study is to investigate why lesions are not detected using FV and to evaluate their histopathology. The tongues of an OED rat model were examined using FV, and the obtained images were objectively evaluated by quantifying the fluorescence intensity (FI) through image analysis software. Hematoxylin-eosin and immunohistochemical staining were performed to characterize FV loss (FVL) and FV retention (FVR). Some OED lesions were recognized through FVR. In FVL, the CD31-positive rate was higher than in FVR, and the CD31-positive rate negatively correlated with FI. In contrast, a positive correlation was observed between FI and the thickness of the epithelial layer. Multiple regression analysis suggested that the CD31-positivity rate and epithelial thickness may be involved in FI. These findings suggest that the accuracy of FV detection is influenced by multiple factors, including angiogenesis and epithelial layer thickness. Therefore, a combination of various testing methods should be considered.\u003c/p\u003e","manuscriptTitle":"Histopathological characteristics of an oral epithelial dysplasia model in rats showing a negative reaction to fluorescence visualization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 05:36:33","doi":"10.21203/rs.3.rs-5565034/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-23T03:12:44+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-14T09:05:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T09:56:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-22T15:54:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86d81969-4f23-47dd-aa82-59a8999f99ee","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47116648,"name":"Biological sciences/Cancer/Oral cancer/Oral cancer detection"},{"id":47116649,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"}],"tags":[],"updatedAt":"2025-05-05T16:02:47+00:00","versionOfRecord":{"articleIdentity":"rs-5565034","link":"https://doi.org/10.1038/s41598-025-99797-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-30 15:57:22","publishedOnDateReadable":"April 30th, 2025"},"versionCreatedAt":"2025-04-17 05:36:33","video":"","vorDoi":"10.1038/s41598-025-99797-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-99797-w","workflowStages":[]},"version":"v1","identity":"rs-5565034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5565034","identity":"rs-5565034","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}