Low-temperature plasma radiofrequency ablation and autologous conjunctival transplantation for pterygium excision.

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Zhen Song, Xin Jin, Rui Zhu, Jingrao Wang, Hong Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2356498/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background/Aims: To evaluate the safety and effectiveness of low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation for pterygium excision. Methods : In order to verify the safety of low-temperature plasma radiofrequency ablation, we conducted animal experiments to observe corneal structure by anterior segment photography, anterior optical coherence tomography, HE staining and immunohistochemistry(MMP-9,TNF-a) before and after corneal burning at different ablation energies (20, 15, 10, 0J/cm 2 )and different point in time(0,3d,1w,2w). Then, we observed preoperative and postoperative treatment effect of 121 eyes of 120 patients with primary pterygium from June 2018 to June 2020. 59 patients treated with pterygium excision and autologous conjunctival transplantation, 61 patients treated with pterygium excision by low-temperature plasma radiofrequency ablation and autologous conjunctival transplantation. The time to strip the pterygium, the integrity of the cornea and the number of inflammatory cell infiltration were observed and recorded. All patients were followed up for 18 months to observe whether the pterygium recurred. Results : The animal experiment confirmed that suitable low-temperature plasma radiofrequency ablation energies has no damage to the corneal tissue structure. Only one case recurred during the year of follow-up. Low-temperature plasma radiofrequency ablation can reduce the time to strip the pterygium, can better maintain the integrity of the damaged cornea and reduce the number of inflammatory cell infiltration, can reduce recurrence rate. Conclusions : Low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation is an safety and effective pterygium excision. Pterygium Low-temperature plasma radiofrequency ablation Safety Effectiveness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The pterygium is the fibrous tissue and the excessive growth of blood vessels of the Tenon capsule and the conjunctiva on the cornea, with an incidence of 0.7–31%. 1 The pathogenesis of pterygium has not yet been fully elucidated, but there is evidence that it is related to environmental factors, limbal stem cell deficiency, the imbalance of cell proliferation and apoptosis imbalance. 2–4 With the development of pterygium, the lesion gradually approaches the pupil area. In addition to affecting the appearance, it will change refractive state of the cornea. The central vision is affected, and the severe one can seriously affect the vision. 5 It is widely considered that the treatment of pterygium is surgical resection. 6 Postoperative complications of pterygium include recurrence, scleral lysis, conjunctival granuloma, sacral adhesion, etc. 7 There are many methods for the removal of pterygium. The pterygium excision combined with autologous conjunctival transplantation is one of the best treatment at present, with high safety and low recurrence rate, 8, 9 but it still doesn't solve the problem of adjacent tissue damage. Low-temperature plasma radiofrequency ablation techniques use a conductive medium (primarily H 2 O with Na and air) to form a 50–100µm plasma region around the electrode. 10 Different from the traditional electrosurgical method, the separation and solidification are realized by heating the tissue. The low-temperature plasma radiofrequency ablation is the kinetic energy generated by the rapid motion of the low temperature plasma in the electric field of 100KHZ, which decomposes the tissue and achieves the cutting effect. The cutting principle of the low-temperature plasma radiofrequency ablation is that the molecular energy of the target tissue (40–70°C) is broken by the kinetic energy of the high-speed moving ions in the plasma, and the thermal damage depth is within 0.5 mm to achieve the cutting purpose, and the trauma to the normal adjacent tissue is small. 11–13 The coagulation mechanism is that the low temperature plasma can accurately control the temperature at 60–70°C during the hemostasis process, which ensures that the helical structure of the collagen molecule shrinks and maintains the vitality of the cells. 14 Low-temperature plasma radiofrequency ablation has been studied for the treatment of tonsillectomy, urethral stricture, removal of nasopharyngeal carcinoma, mixed hemorrhoids, and sinusitis. 15–16 But the safety and efficacy of low-temperature plasma radiofrequency ablation in the treatment of pterygium was rarely reported. Studies have shown that a variety of matrix metalloproteinases can be detected in the pterygium cells, which can effectively destroy the hemyoblastic junction in the lysed cells, making the pterygium cells easy to migrate, resulting in the dissolution of the pre-corneal elastic layer, transformation. Fibroblasts are further aggravated by dissolution. The expression of MMP-9 in pterygium tissues and fibroblasts was also higher than that in normal tissues. Other studies have shown that MMP-1 expression is increased in pterygium, and MMP-2,3, 7, 9,14, TNF-a are also overexpressed. 17–20 Zidi S et al have found that IL-6, IL-17A and NO in tears and serum of patients with pterygium are associated with inflammatory infiltration and the expression of NOS2, NF-κB and Bcl-2 in pterygium biopsy. 21 Therefore, inflammatory markers play an important role in the development of pterygium. Pterygium excision causes some degree of corneal damage when the pterygium is separated from the cornea, and this process is very important for the success of the operation. In order to verify the safety of the low-temperature plasma radiofrequency ablation for cornea, we conducted animal experiments to observe corneal structure by anterior segment photography, anterior optical coherence tomography (AS-OCT), HE staining and immunohistochemistry (MMP-9, TNF-a) before and after corneal burning at different ablation energies (20, 15, 10, 0J/cm 2 ) and different point in time(0,3d,1w,2w). In order to verify the efficacy of the low-temperature plasma radiofrequency ablation, all patients were followed up for 18 months to observe whether the pterygium recurred, the integrity of the cornea and the number of inflammatory cell infiltration around cornea. Our aim is to evaluate the safety and effectiveness of low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation for pterygium excision and to provide a new method for treatment of pterygium. Materials And Methods Animal experiment Forty-eight male wistar rats were anesthetized via intraperitoneal injection of 40 mg/kg pentobarbital sodium and randomly divided into 4 groups. According to 4 groups of different ablation energies (20, 15, 10, 0J/cm 2 ), the right central corneal was treated with a 1 mm circular area for 2 min. The corneal full-thickness specimens were taken at postoperative time, postoperative 3 days, 1w, and 2w after observing by AS-OCT. The rats were sacrificed by injecting air into the cardiac apex under anesthesia, and the eyeballs were carefully harvested for use in the following experiments. The corneal tissue structure was observed by HE staining. The expression of inflammatory factors (MMP-9, TNF-a) in corneal tissue was observed by immunohistochemistry. He Staining At the end of the experiment, each group of mice was sacrificed and the eyeball tissue of the mice was removed. Frozen sections and paraffin sections were prepared. Three frozen slices of each group were proceeded to conventional HE staining, the changes of the cornea were observed and recorded under an optical microscope pathological. Immunohistochemistry Staining All specimens were fixed in 4% formaldehyde solution, embedded in paraffin, and prepared in 4 µm sections. The staining procedure was performed according to the kit instructions. Participants This is a single center prospective study that evaluated all patient charts with a pterygium removal surgery between June 2018 to June 2020 at The First Affiliated Hospital of Harbin Medical University. All patients gave informed consent to the study content. Inclusion criteria: patients diagnosed with pterygium by an experienced physician and who can tolerate surgery; Exclusion criteria: 1) Combined with severe eyelids, skin inflammation, such as blepharitis, mumps, atopic dermatitis, seborrheic dermatitis, rosacea, etc; 2) One of the following abnormalities in the ocular surface: such as trachoma, pemphigoid, chemical injury, thermal burns, etc; 3) Systemic diseases affecting the ocular surface. Surgical Procedure Levofloxacin and praprofen eye drops were applied to the patients before surgery. The topical anesthesia of the affected eye with proparacaine hydrochloride, 2.0% lidocaine, 0.1% adrenaline for subconjunctival local infiltration anesthesia in the pterygium area of the eye. The microscopic scissors were used to separate the bulbar conjunctiva from the pterygium bluntly. After the pterygium was clamped to the root of the pterygium with the Venturi pliers for 10 s, the part of the pterygium was cut off. Using a low-temperature plasma radiofrequency ablation (30-40J/cm 2 ) to remove the head of pterygium from the corneal surface and keep the corneal wound smooth as much as possible; The thick blood vessels on the surface of the sclera should be burned by low-temperature plasma radiofrequency ablation to stop bleeding, and should be avoided the injury to the internal rectus muscle and excessive damage to the sclera during the cutting process. After excision of the pterygium, the lateral conjunctival incision is used as the edge, an autologous bulbar conjunctival that matches the size of the wound that remains after removal of the pterygium were cut off. And transposed to cover the wound after the removal of the pterygium, 10 − 0 microbridge will be used to fixed the conjunctival to the cornea limbus, the remaining range of conjunctival flaps and the wound ball conjunctiva were proceeded continuous seam, then wearing a contact lens for the patient. Postoperative Care All patients were given levofloxacin eye drops, 4 times a day for 2 weeks, sodium hyaluronate eye drops, 4 times a day for 2 weeks, 0.5% prednisolone acetate eye drops, 4 times a day for 1 week, then gradually reducing. Patients were followed up for the first week,3 months,12 months and 18months. Before the operation and during the follow-up period, the patient underwent anterior segment photography, AS-OCT, and in vivo confocal microscopy (IVCM). Statistical analysis All data were statistically analyzed using SPSS 18.0 statistical software. Measurement data were expressed as mean ± standard deviation, and comparisons between groups were analyzed by analysis of variance. All tests were statistically significant at a significant level of P < 0.05. Results Safety evaluation of low-temperature plasma radiofrequency ablation by the corneal fluorescence staining and AS-OCT The corneal fluorescence staining results showed that immediately after the operation, when the energy was 0 J/cm 2 , the corneal epithelium was intact(Fig. 1 A- 1 D), and the corneal epithelium was missing when the energy was 10, 15, and 20J/cm 2 , and positive fluorescein staining was visible(Fig. 1 E, 1 I, 1 M); At three days after the operation, the integrity of the corneal epithelial cells was restored at the energy 10, 15J/cm 2 group(Fig. 1 F- 1 H, 1 J- 1 L); The corneal epithelial cells of the energy 20J/cm 2 group were restored intact one week after the operation(Fig. 1 N- 1 P); The results of AS-OCT showed that there was a small amount of exudation in the anterior chamber of each group immediately after surgery(Fig. 2B1-2D1,Table 1 ).When the energy was 0 J/cm 2 , the corneal epithelial cell layer was intact(Fig. 2A1-2A4,Table 1 ),and the corneal epithelial cell layer was partially missing with corneal edema when the energy was 10,15,20J/cm 2 (Fig. 2B1-2D1,Table 1 ),while The inflammatory response of anterior chamber disappeared in each group, corneal epithelial cells recovered and corneal edema disappeared in the rats with energy of 10,15J/cm 2 three days after surgery(Fig. 2B2-2B4,2C2-2C4,Table 1 ), but there was still loss of corneal epithelial cells and corneal edema in the rats with energy of 20J/cm 2 (Fig. 2D2,Table 1 ). The cornea of the rats with energy of 20J/cm 2 was thinner one and two weeks after operation (Fig. 2D3,2D4, Table 1 ). He Staining According to 4 groups of different ablation energies (20, 15, 10, 0J/cm 2 ), the right central corneal was treated with a 1 mm circular area for 2 min. The corneal were observed at postoperative time, postoperative 3days, 1w, and 2w. The cornea did not change significantly when the ablation energy was 0 J/cm 2 , (Fig. 3A1-3A4). A few fibroblasts infiltrated, the corneal epithelium became slightly thinner, endothelial folds were observed when the ablation energy was 10,15J/cm 2 , (Fig. 3B1,3C1). When the ablation energy is 20 J/cm 2 , more fibroblast infiltration, no inflammatory cells were seen, the corneal epithelium becomes thinner, the cornea is slightly swollen, exfoliation (Fig. 3D1), no vacuoles are seen. Three days after radiofrequency ablation, when the ablation energy was 10 J/cm 2 , the cornea did not change significantly (Fig. 3B2). When the ablation energy was 15J/cm 2 , the corneal epithelium became slightly thinner (Fig. 3C2); When the ablation energy is 20 J/cm 2 , the corneal epithelium becomes thinner, the corneal stroma is slightly swollen(Fig. 3D2), no vacuoles are seen. One week after plasma ablation, when the ablation energy was 10 J/cm 2 , the cornea did not change significantly, and the fibroblasts decreased (Fig. 3B3). When the ablation energy was 15 J/cm 2 , the corneal epithelium became slightly thinner, the other changes were not obvious, and the fibroblasts decreased (Fig. 3C3); When the ablation energy was 20 J/cm 2 , the corneal epithelium becomes thinner, the corneal stroma is slightly swollen, no vacuoles are seen, and fibroblasts are reduced (Fig. 3D3). There was no significant change in HE staining between the two weeks after surgery and one week after surgery (Fig. 3B4,3C4,3D4). Immunohistochemistry Staining Immunohistochemistry staining showed much stronger positive staining of MMP-9 and TNF-a in the corneal tissue immediately after surgery and three days after surgery when theenergy was 10,15 and 20 J/cm2(Fig. 4A1-4A3,4B1,4B2,4C1,4C2,4D1,4D2, 5A1-5A3,5B1,5B2,5C1,5C2,5D1,5D2). The expression of MMP-9, TNF-a were decreased a week after surgery (Fig. 4B3,4C3,4D3,5B3,5C3,5D3). It shows that the low-temperature plasma radiofrequency ablation is safe and does not cause inflammation of the cornea. There is no significant change two weeks after surgery and one week after surgery. Patients’ Characteristic We collected the information from 120 cases (121 eyes) of primary pterygium, 59 patients treated with pterygium excision and autologous conjunctival transplantation (control group), 61 patients treated with pterygium excision by low-temperature plasma radiofrequency ablation (experimental group). The control group included 36 males and 23 females, aged 35–72 years old, the average age was 54.6 ± 9.8 years old. The experimental group included 37 males and 24 females, aged 37–80 years old, the average age was 58.4 ± 10.2 years old. There was no statistically significant difference in age and sex ratio. Observation Of Clinical Indicators The time that the pterygium separates from the cornea was 1.52 ± 0.34 min in control group, and 1.20 ± 0.27 min in experimental group. Three patients in control group and only one patient in experimental group had pterygium recurrence. The pterygium manifested normal or slightly thickened epithelial under AS-OCT, while the epithelium showed high reflex moderately. The high-reflection signal located between the corneal epithelium and Bowman's membrane. Except for patients with recurrent pterygium, all patients had a high-reflection signal disappeared one week after surgery, and the corneal epithelium was continuous. Pterygium consists of epithelial cells in the surface layer, fibrous tissue and neovascular, a small amount of dendritic cell infiltration and scattered distribution of goblet cell by IVCM. A large number of activated Langerhans cells, fibrous tissue and vascular structures can be seen under IVCM in patients with recurrent pterygium. Compared with patients with simple autologous conjunctival transplantation, the density of inflammatory cells in the cornea of patients with low-temperature plasma radiofrequency ablation decreased (Fig. 6 A-D) and the structure of the corneal layers was clearer (Fig. 7 A-D) one week after the operation Discussion Pterygium is a common ocular surface disease, which is characterized by degeneration, hypertrophy, hyperplasia of the conjunctiva and subconjunctival tissue of interpalpebral. 22 The occurrence of pterygium is closely related to ultraviolet radiation, which has been confirmed by many studies, but the exact pathogenesis, such as the theory of apoptosis, extracellular matrix remodeling, genetic inheritance and inflammatory theory are still under investigation. 23 Resection of hyperplastic tissue is a key step in the treatment of pterygium. However, simple resection without transplantation is associated with a high recurrence rate. At present, autologous transplantation of the conjunctiva in the bare area after resection is considered to reduce the recurrence rate (4.8%-12.3%), which may be the most effective treatment for pterygium. 24–26 In the process of pterygium removal, improper excision can leave corneal scarring and damage to the surrounding normal tissue. Low-temperature plasma radiofrequency ablation have been studied for the treatment of tonsillectomy, removal of nasopharyngeal carcinoma, mixed hemorrhoids, and sinusitis. 27–29 However, there are few reports on the treatment of pterygium with low-temperature plasma radiofrequency ablation. We hope to combine the autologous conjunctival transplantation and low-temperature plasma radiofrequency ablation to treat pterygium more effectively. UVB irradiation of human epithelial cells or fibroblasts isolated from normal ocular surface tissue or surgically removed pterygium specimens stimulates the expression and secretion of several proinflammatory cytokines and chemokines, such as tumor necrosis factor TNF-α, IL-1, IL-6 and MMP-9. 17–20 Upregulation of various pro-inflammatory factors plays an important role in the pathogenesis of pterygium. MMP-9 is upregulated in pterygium tissue. 30 This cytokine has potent proinflammatory effects and also stimulates angiogenesis. 31 At present, the research of pterygium based on the level of inflammatory factors mostly through the detection of pterygium tissue and the expression of inflammatory factors in cells, little research based on the expression of inflammatory factors in corneal, we use this new way to observe postoperative outcome. In our research, compared with control, the levels of MMP-9 and TNF-a were significantly increased at different ablation energies (20, 15, 10 J/cm 2 ) at postoperative time and postoperative 3 days, and decreased at postoperative a week;This study will provide new ideas for the future pathogenesis of pterygium. In our study, the recurrence rate of autologous conjunctival transplantation combined with low-temperature plasma radiofrequency ablation in the treatment of pterygium was 1.64%. However, the best surgical method in the literature reports that the recurrence rate of pterygium after autologous conjunctival transplantation is 4.8%-12.3%. Our surgical approach greatly reduces the recurrence rate after pterygium surgery, postoperative patients did not have serious complications. Many reports that the autologous conjunctival autograft is taken from the upper conjunctival tissue, 32–33 but our study used the conjunctival tissue below the iliac crest. Considering that the patient may need to perform other operations through the upper conjunctiva, such as cataract and glaucoma, the conjunctiva is taken to prevent conjunctival scars. At the same time, we found that low-temperature plasma radiofrequency ablation can reduce the time to strip the pterygium during the operation, and can better maintain the integrity of the damaged cornea and reduce the number of inflammatory cell infiltration through the follow-up of patients for a week after surgery. Although low-temperature plasma radiofrequency ablation has been used in clinical practice, the clinical application of ophthalmology is less. Huang et al reported that low-temperature plasma is safe and efficient for pterygium surgery, but the optimal energy choice for pterygium surgery and its correlation with inflammatory factors have not yet been elucidated. 34 In our research, we have verified the safety of low-temperature plasma radiofrequency ablation for corneal tissue through animal experiments. In animal experiments, we chose the ablation energy of 20, 15, 10J/cm 2 for the safety analysis of low-temperature plasma radiofrequency ablation. We observed the corneal tissue through the same ablation time, different ablation energy, since the corneal thickness of the rat is 222–230µm and the corneal thickness of the adult is 550um-570µm, the ablation energy of the low-temperature plasma radiofrequency ablation is also inconsistent, and finally we chose energy 35 J/cm 2 for clinical pterygium surgery since the thickness of human cornea is thicker than that of rats. Our experiments confirm that the low-temperature plasma radiofrequency ablation is safe for the corneal tissue and does not cause damage to the corneal tissue structure. In our experiment, we only followed up patients for 18 months, which is somewhat intentional. In future research, we can compare the recovery time, the symptoms of self-consciousness and the difference of postoperative objective indicators of patients, providing new ideas for clinical treatment of pterygium. Declarations Acknowledgements Not applicable. Authors’ contributions Song Zhen and Jin Xin designed the project and collected clinical data of patients, Zhu Rui and Wang Jingrao conducted animal experiments and statistics, and Zhang Hong was responsible for the supervision and inspection of the project. Funding Supported by the National Natural Science Foundation of China (Grant No.U20A20363,81970776),Supported by the Natural Science Foundation of Heilongjiang Province, China(Grant No. LH2020H039) . Availability of data and materials All data generated or analysed during this study are included in this published article. Ethics approval and consent to participate All methods were carried out in accordance with research guidelines and regulations. All experimental protocols were approved by the First Afli- ated Hospital of Harbin Medical University Ethical Committee (No.18A0047) and strictly followed the Helsinki Declaration. All patients provided written informed consent before participating in the study. All experimental methods involving animals are reported following ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments and was approved by the First Afliated Hospital of Harbin Medical University animal Ethical Committee (Approval No. 2021093). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Shiroma H, Higa A, Sawaguchi S, et al. 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Thermal effects of percutaneous application of plasma/radiofrequency energy on porcine dermis and fibroseptal network.J Cosmet Dermatol. 2021 Jul;20(7):2125-2131. Di Girolamo N, Kumar RK, Coroneo MT, Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 2002;43(11):3430-3437. Nolan TM, Di Girolamo N, Sachdev NH, Hampartzoumian T, Coroneo MT, Wakefield D. The role of ultraviolet irradiation and heparin-binding epidermal growth factor-like growth factor in the pathogenesis of pterygium. Am J Pathol 2003;162(2):567-574. Nganga Ngabou CGF,Makita C,Ndalla SS,et al.[Pterygium surgery by conjunctiva autograft with autologous blood fixation].J Fr Ophtalmol 2018;41:425-432. Ghoz N,Elalfy M,Said D.Healing of autologous conjunctival grafts in pterygium surgery.Acta Ophthalmol 2018;96:e979-e988. Huang H, Li S, Zhong J, Wang B, Peng L, Deng Y, Wang M, Yuan J. Evaluation of the Safety and Efficacy of a Low-Temperature Plasma Surgical System for Pterygium. Cornea. 2020 Dec ;39(12): 1581-1587. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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-2356498","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":161176333,"identity":"5625e3ff-f72a-4cc2-bb7c-f8ba97c80d8f","order_by":0,"name":"Zhen Song","email":"","orcid":"","institution":"the First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Song","suffix":""},{"id":161176337,"identity":"001a8222-2bab-4e8e-a01c-f550753ee6bc","order_by":1,"name":"Xin Jin","email":"","orcid":"","institution":"the First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Jin","suffix":""},{"id":161176340,"identity":"89fe65ec-ac65-49f3-ac33-bf565a7ed4df","order_by":2,"name":"Rui Zhu","email":"","orcid":"","institution":"the First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Zhu","suffix":""},{"id":161176343,"identity":"1eed0961-a9e1-4934-86cf-4c098c4dd53c","order_by":3,"name":"Jingrao Wang","email":"","orcid":"","institution":"the First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingrao","middleName":"","lastName":"Wang","suffix":""},{"id":161176345,"identity":"cd515575-9a82-44d5-9b24-465716f733ff","order_by":4,"name":"Hong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCSBmbAAS7D0MzBChBGK18JwhWYtEDpFa5Gc3P3v4dYdNnnzk22OfC3MOM/Cz5xgw/NyBWwvjnGPmxrJn0ooNb+clz5657TCDZM8bA8beM7i1MEskmElLth1O3Dg7x5iZF6jF4EaOATNjG24tbBLp3yBaZp6BaLEnpIVHIsdM8iNQy3wJHqgtEgS0SEjklEkztqUlbuABOmzmtnQeiTPPCg724tEiPyN9m+TPNpvE+e1AhxVus5bjb0/e+OAnHi3gIOABEgYHoC4FEQfwawAG9A+QdQ2ElI2CUTAKRsGIBQDInE5aMjnHsQAAAABJRU5ErkJggg==","orcid":"","institution":"the First Affiliated Hospital of Harbin Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2022-12-08 06:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2356498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2356498/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30607109,"identity":"3499848d-b55e-47e0-a8e3-aff35b2d3c47","added_by":"auto","created_at":"2022-12-21 07:02:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1344105,"visible":true,"origin":"","legend":"\u003cp\u003eCorneal fluorescent staining at different time points after surgery; Corneal was not stained when the energy was 0(A-D); Positive fluorescein staining was visible at the energy 10, 15 ,but the integrity of the corneal epithelial cells was restored at three days after the operation(E-L); Positive fluorescein staining was visible at the energy 20,the integrity of the corneal epithelial cells was restored at one week after the operation(M-P);\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/e8464a5d68e47a611961870b.png"},{"id":30607108,"identity":"3b7296f2-2fc1-409e-a872-a3a04bc119d3","added_by":"auto","created_at":"2022-12-21 07:02:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1334953,"visible":true,"origin":"","legend":"\u003cp\u003eAS-OCT at different time points after surgery; The structure of each layer of the cornea was clear when the energy was 0(A1-A4); Corneal edema was visible at the energy 10, 15 at postoperative time, corneal epithelial cells recovered and corneal edema disappeared at three days after the operation(B1-B4,C1-C4); Corneal edema was visible at the energy 20 at postoperative time, three days and one week after the operation,corneal scar can be seen at two weeks after the operation(D1-D4);\u003c/p\u003e","description":"","filename":"fiugre2.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/099efe316e5f56e89f4f2f74.png"},{"id":30608290,"identity":"c5aad1bf-69b4-4009-ab22-fc49dab52208","added_by":"auto","created_at":"2022-12-21 07:18:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1304993,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining in cornea at different time points after surgery; The structure of each layer of the cornea was complete when the energy was 0(A1-A4); Corneal edema was visible(B1,C1-C3,D1-D3); Endothelial folds(C1-C3,D1-D4); Exfoliation(D1); The structure of each layer of the cornea was recovered after 3 days when the energy was 10J (B2-B4); The structure of each layer of the cornea was recovered after 2 weekss when the energy was 15J (C4);\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/b764d3fcaf79346117962a1a.png"},{"id":30607508,"identity":"e5220daa-3bbf-4822-9815-96d870af79aa","added_by":"auto","created_at":"2022-12-21 07:10:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":877163,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of MMP9 in corneal tissue; With the increase of energy, the expression of MMP9 increases, and with the extension of time, the expression of MMP9 decreases;\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/942af4038062c339fb1d2c64.png"},{"id":30607111,"identity":"5c7fb297-ffb7-436c-9a33-e15c0184a9b3","added_by":"auto","created_at":"2022-12-21 07:02:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":730539,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of TNF-α in corneal tissue; With the increase of energy, the expression of TNF-α increases, and with the extension of time, the expression of TNF-αdecreases;\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/6cdbf19e4d0efbaee1742592.png"},{"id":30607114,"identity":"cf540e3f-c48f-42d0-9d06-5b7ef8c046c4","added_by":"auto","created_at":"2022-12-21 07:02:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":761345,"visible":true,"origin":"","legend":"\u003cp\u003eIVCM in corneal by scalpel stripping and low-temperature plasma radiofrequency ablation at one week after operation; The density of inflammatory cells in the cornea of patients with low-temperature plasma radiofrequency ablation(C,D) decreased than with scalpel stripping(A,B).\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/c304b759f85c21e5000ebfd3.png"},{"id":30607510,"identity":"33ec370c-5b72-4336-beda-4a66b4bde5fe","added_by":"auto","created_at":"2022-12-21 07:10:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":423879,"visible":true,"origin":"","legend":"\u003cp\u003eAS-OCT in corneal by scalpel stripping and low-temperature plasma radiofrequency ablation at one week after operation; Preoperative eye image of the patient(A,C);Low-temperature plasma radiofrequency ablation(B) can better maintain the integrity of the damaged cornea than scalpel stripping(D).\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/07d019ef4c37f0b52c5138cb.png"},{"id":32371335,"identity":"3dc34836-65c7-47dc-8b64-cf93eae0c03f","added_by":"auto","created_at":"2023-02-02 08:59:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5815034,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2356498/v1/5109dfde-4a46-4a9b-a425-88d8e5c2389b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Low-temperature plasma radiofrequency ablation and autologous conjunctival transplantation for pterygium excision.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pterygium is the fibrous tissue and the excessive growth of blood vessels of the Tenon capsule and the conjunctiva on the cornea, with an incidence of 0.7\u0026ndash;31%.\u003csup\u003e1\u003c/sup\u003e The pathogenesis of pterygium has not yet been fully elucidated, but there is evidence that it is related to environmental factors, limbal stem cell deficiency, the imbalance of cell proliferation and apoptosis imbalance. \u003csup\u003e2\u0026ndash;4\u003c/sup\u003eWith the development of pterygium, the lesion gradually approaches the pupil area. In addition to affecting the appearance, it will change refractive state of the cornea. The central vision is affected, and the severe one can seriously affect the vision. \u003csup\u003e5\u003c/sup\u003e It is widely considered that the treatment of pterygium is surgical resection. \u003csup\u003e6\u003c/sup\u003e Postoperative complications of pterygium include recurrence, scleral lysis, conjunctival granuloma, sacral adhesion, etc. \u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThere are many methods for the removal of pterygium. The pterygium excision combined with autologous conjunctival transplantation is one of the best treatment at present, with high safety and low recurrence rate, \u003csup\u003e8, 9\u003c/sup\u003ebut it still doesn't solve the problem of adjacent tissue damage. Low-temperature plasma radiofrequency ablation techniques use a conductive medium (primarily H\u003csub\u003e2\u003c/sub\u003eO with Na and air) to form a 50\u0026ndash;100\u0026micro;m plasma region around the electrode. \u003csup\u003e10\u003c/sup\u003e Different from the traditional electrosurgical method, the separation and solidification are realized by heating the tissue. The low-temperature plasma radiofrequency ablation is the kinetic energy generated by the rapid motion of the low temperature plasma in the electric field of 100KHZ, which decomposes the tissue and achieves the cutting effect. The cutting principle of the low-temperature plasma radiofrequency ablation is that the molecular energy of the target tissue (40\u0026ndash;70\u0026deg;C) is broken by the kinetic energy of the high-speed moving ions in the plasma, and the thermal damage depth is within 0.5 mm to achieve the cutting purpose, and the trauma to the normal adjacent tissue is small. \u003csup\u003e11\u0026ndash;13\u003c/sup\u003e The coagulation mechanism is that the low temperature plasma can accurately control the temperature at 60\u0026ndash;70\u0026deg;C during the hemostasis process, which ensures that the helical structure of the collagen molecule shrinks and maintains the vitality of the cells. \u003csup\u003e14\u003c/sup\u003eLow-temperature plasma radiofrequency ablation has been studied for the treatment of tonsillectomy, urethral stricture, removal of nasopharyngeal carcinoma, mixed hemorrhoids, and sinusitis. \u003csup\u003e15\u0026ndash;16\u003c/sup\u003eBut the safety and efficacy of low-temperature plasma radiofrequency ablation in the treatment of pterygium was rarely reported.\u003c/p\u003e \u003cp\u003eStudies have shown that a variety of matrix metalloproteinases can be detected in the pterygium cells, which can effectively destroy the hemyoblastic junction in the lysed cells, making the pterygium cells easy to migrate, resulting in the dissolution of the pre-corneal elastic layer, transformation. Fibroblasts are further aggravated by dissolution. The expression of MMP-9 in pterygium tissues and fibroblasts was also higher than that in normal tissues. Other studies have shown that MMP-1 expression is increased in pterygium, and MMP-2,3, 7, 9,14, TNF-a are also overexpressed. \u003csup\u003e17\u0026ndash;20\u003c/sup\u003eZidi S et al have found that IL-6, IL-17A and NO in tears and serum of patients with pterygium are associated with inflammatory infiltration and the expression of NOS2, NF-κB and Bcl-2 in pterygium biopsy. \u003csup\u003e21\u003c/sup\u003eTherefore, inflammatory markers play an important role in the development of pterygium.\u003c/p\u003e \u003cp\u003ePterygium excision causes some degree of corneal damage when the pterygium is separated from the cornea, and this process is very important for the success of the operation. In order to verify the safety of the low-temperature plasma radiofrequency ablation for cornea, we conducted animal experiments to observe corneal structure by anterior segment photography, anterior optical coherence tomography (AS-OCT), HE staining and immunohistochemistry (MMP-9, TNF-a) before and after corneal burning at different ablation energies (20, 15, 10, 0J/cm\u003csup\u003e2\u003c/sup\u003e) and different point in time(0,3d,1w,2w). In order to verify the efficacy of the low-temperature plasma radiofrequency ablation, all patients were followed up for 18 months to observe whether the pterygium recurred, the integrity of the cornea and the number of inflammatory cell infiltration around cornea. Our aim is to evaluate the safety and effectiveness of low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation for pterygium excision and to provide a new method for treatment of pterygium.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiment\u003c/h2\u003e \u003cp\u003eForty-eight male wistar rats were anesthetized via intraperitoneal injection of 40 mg/kg pentobarbital sodium and randomly divided into 4 groups. According to 4 groups of different ablation energies (20, 15, 10, 0J/cm\u003csup\u003e2\u003c/sup\u003e), the right central corneal was treated with a 1 mm circular area for 2 min. The corneal full-thickness specimens were taken at postoperative time, postoperative 3 days, 1w, and 2w after observing by AS-OCT. The rats were sacrificed by injecting air into the cardiac apex under anesthesia, and the eyeballs were carefully harvested for use in the following experiments. The corneal tissue structure was observed by HE staining. The expression of inflammatory factors (MMP-9, TNF-a) in corneal tissue was observed by immunohistochemistry.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHe Staining\u003c/h3\u003e\n\u003cp\u003eAt the end of the experiment, each group of mice was sacrificed and the eyeball tissue of the mice was removed. Frozen sections and paraffin sections were prepared. Three frozen slices of each group were proceeded to conventional HE staining, the changes of the cornea were observed and recorded under an optical microscope pathological.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry Staining \u003c/h3\u003e\n\u003cp\u003eAll specimens were fixed in 4% formaldehyde solution, embedded in paraffin, and prepared in 4 \u0026micro;m sections. The staining procedure was performed according to the kit instructions.\u003c/p\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eThis is a single center prospective study that evaluated all patient charts with a pterygium removal surgery between June 2018 to June 2020 at The First Affiliated Hospital of Harbin Medical University. All patients gave informed consent to the study content. Inclusion criteria: patients diagnosed with pterygium by an experienced physician and who can tolerate surgery; Exclusion criteria: 1) Combined with severe eyelids, skin inflammation, such as blepharitis, mumps, atopic dermatitis, seborrheic dermatitis, rosacea, etc; 2) One of the following abnormalities in the ocular surface: such as trachoma, pemphigoid, chemical injury, thermal burns, etc; 3) Systemic diseases affecting the ocular surface.\u003c/p\u003e\n\u003ch3\u003eSurgical Procedure\u003c/h3\u003e\n\u003cp\u003eLevofloxacin and praprofen eye drops were applied to the patients before surgery. The topical anesthesia of the affected eye with proparacaine hydrochloride, 2.0% lidocaine, 0.1% adrenaline for subconjunctival local infiltration anesthesia in the pterygium area of the eye. The microscopic scissors were used to separate the bulbar conjunctiva from the pterygium bluntly. After the pterygium was clamped to the root of the pterygium with the Venturi pliers for 10 s, the part of the pterygium was cut off. Using a low-temperature plasma radiofrequency ablation (30-40J/cm\u003csup\u003e2\u003c/sup\u003e) to remove the head of pterygium from the corneal surface and keep the corneal wound smooth as much as possible; The thick blood vessels on the surface of the sclera should be burned by low-temperature plasma radiofrequency ablation to stop bleeding, and should be avoided the injury to the internal rectus muscle and excessive damage to the sclera during the cutting process. After excision of the pterygium, the lateral conjunctival incision is used as the edge, an autologous bulbar conjunctival that matches the size of the wound that remains after removal of the pterygium were cut off. And transposed to cover the wound after the removal of the pterygium, 10\u0026thinsp;\u0026minus;\u0026thinsp;0 microbridge will be used to fixed the conjunctival to the cornea limbus, the remaining range of conjunctival flaps and the wound ball conjunctiva were proceeded continuous seam, then wearing a contact lens for the patient.\u003c/p\u003e\n\u003ch3\u003ePostoperative Care\u003c/h3\u003e\n\u003cp\u003eAll patients were given levofloxacin eye drops, 4 times a day for 2 weeks, sodium hyaluronate eye drops, 4 times a day for 2 weeks, 0.5% prednisolone acetate eye drops, 4 times a day for 1 week, then gradually reducing. Patients were followed up for the first week,3 months,12 months and 18months. Before the operation and during the follow-up period, the patient underwent anterior segment photography, AS-OCT, and in vivo confocal microscopy (IVCM).\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were statistically analyzed using SPSS 18.0 statistical software. Measurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and comparisons between groups were analyzed by analysis of variance. All tests were statistically significant at a significant level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003eSafety evaluation of low-temperature plasma radiofrequency ablation by the corneal fluorescence staining and AS-OCT\u003c/h2\u003e\n \u003cp\u003eThe corneal fluorescence staining results showed that immediately after the operation, when the energy was 0 J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelium was intact(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), and the corneal epithelium was missing when the energy was 10, 15, and 20J/cm\u003csup\u003e2\u003c/sup\u003e, and positive fluorescein staining was visible(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE,\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI,\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eM); At three days after the operation, the integrity of the corneal epithelial cells was restored at the energy 10, 15J/cm\u003csup\u003e2\u003c/sup\u003e group(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH,\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eJ-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eL); The corneal epithelial cells of the energy 20J/cm\u003csup\u003e2\u003c/sup\u003e group were restored intact one week after the operation(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eN-\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eP);\u003c/p\u003e\n \u003cp\u003eThe results of AS-OCT showed that there was a small amount of exudation in the anterior chamber of each group immediately after surgery(Fig. 2B1-2D1,Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).When the energy was 0 J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelial cell layer was intact(Fig. 2A1-2A4,Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e),and the corneal epithelial cell layer was partially missing with corneal edema when the energy was 10,15,20J/cm\u003csup\u003e2\u003c/sup\u003e(Fig. 2B1-2D1,Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e),while The inflammatory response of anterior chamber disappeared in each group, corneal epithelial cells recovered and corneal edema disappeared in the rats with energy of 10,15J/cm\u003csup\u003e2\u003c/sup\u003e three days after surgery(Fig. 2B2-2B4,2C2-2C4,Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), but there was still loss of corneal epithelial cells and corneal edema in the rats with energy of 20J/cm\u003csup\u003e2\u003c/sup\u003e(Fig. 2D2,Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The cornea of the rats with energy of 20J/cm\u003csup\u003e2\u003c/sup\u003e was thinner one and two weeks after operation (Fig. 2D3,2D4, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eHe Staining\u003c/h3\u003e\n\u003cp\u003eAccording to 4 groups of different ablation energies (20, 15, 10, 0J/cm\u003csup\u003e2\u003c/sup\u003e), the right central corneal was treated with a 1 mm circular area for 2 min. The corneal were observed at postoperative time, postoperative 3days, 1w, and 2w. The cornea did not change significantly when the ablation energy was 0 J/cm\u003csup\u003e2\u003c/sup\u003e, (Fig.\u0026nbsp;3A1-3A4). A few fibroblasts infiltrated, the corneal epithelium became slightly thinner, endothelial folds were observed when the ablation energy was 10,15J/cm\u003csup\u003e2\u003c/sup\u003e, (Fig.\u0026nbsp;3B1,3C1). When the ablation energy is 20 J/cm\u003csup\u003e2\u003c/sup\u003e, more fibroblast infiltration, no inflammatory cells were seen, the corneal epithelium becomes thinner, the cornea is slightly swollen, exfoliation (Fig.\u0026nbsp;3D1), no vacuoles are seen. Three days after radiofrequency ablation, when the ablation energy was 10 J/cm\u003csup\u003e2\u003c/sup\u003e, the cornea did not change significantly (Fig.\u0026nbsp;3B2). When the ablation energy was 15J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelium became slightly thinner (Fig.\u0026nbsp;3C2); When the ablation energy is 20 J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelium becomes thinner, the corneal stroma is slightly swollen(Fig.\u0026nbsp;3D2), no vacuoles are seen. One week after plasma ablation, when the ablation energy was 10 J/cm\u003csup\u003e2\u003c/sup\u003e, the cornea did not change significantly, and the fibroblasts decreased (Fig.\u0026nbsp;3B3). When the ablation energy was 15 J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelium became slightly thinner, the other changes were not obvious, and the fibroblasts decreased (Fig.\u0026nbsp;3C3); When the ablation energy was 20 J/cm\u003csup\u003e2\u003c/sup\u003e, the corneal epithelium becomes thinner, the corneal stroma is slightly swollen, no vacuoles are seen, and fibroblasts are reduced (Fig.\u0026nbsp;3D3). There was no significant change in HE staining between the two weeks after surgery and one week after surgery (Fig.\u0026nbsp;3B4,3C4,3D4).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry Staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry staining showed much stronger positive staining of MMP-9 and TNF-a in the corneal tissue immediately after surgery and three days after surgery when theenergy was 10,15 and 20 J/cm2(Fig.\u0026nbsp;4A1-4A3,4B1,4B2,4C1,4C2,4D1,4D2, 5A1-5A3,5B1,5B2,5C1,5C2,5D1,5D2). The expression of MMP-9, TNF-a were decreased a week after surgery (Fig.\u0026nbsp;4B3,4C3,4D3,5B3,5C3,5D3). It shows that the low-temperature plasma radiofrequency ablation is safe and does not cause inflammation of the cornea. There is no significant change two weeks after surgery and one week after surgery.\u003c/p\u003e\n\u003ch3\u003ePatients’ Characteristic\u003c/h3\u003e\n\u003cp\u003eWe collected the information from 120 cases (121 eyes) of primary pterygium, 59 patients treated with pterygium excision and autologous conjunctival transplantation (control group), 61 patients treated with pterygium excision by low-temperature plasma radiofrequency ablation (experimental group). The control group included 36 males and 23 females, aged 35–72 years old, the average age was 54.6 ± 9.8 years old.\u003c/p\u003e\n\u003cp\u003eThe experimental group included 37 males and 24 females, aged 37–80 years old, the average age was 58.4 ± 10.2 years old. There was no statistically significant difference in age and sex ratio.\u003c/p\u003e\n\u003ch3\u003eObservation Of Clinical Indicators\u003c/h3\u003e\n\u003cp\u003eThe time that the pterygium separates from the cornea was 1.52 ± 0.34 min in control group, and 1.20 ± 0.27 min in experimental group. Three patients in control group and only one patient in experimental group had pterygium recurrence. The pterygium manifested normal or slightly thickened epithelial under AS-OCT, while the epithelium showed high reflex moderately. The high-reflection signal located between the corneal epithelium and Bowman's membrane. Except for patients with recurrent pterygium, all patients had a high-reflection signal disappeared one week after surgery, and the corneal epithelium was continuous. Pterygium consists of epithelial cells in the surface layer, fibrous tissue and neovascular, a small amount of dendritic cell infiltration and scattered distribution of goblet cell by IVCM. A large number of activated Langerhans cells, fibrous tissue and vascular structures can be seen under IVCM in patients with recurrent pterygium. Compared with patients with simple autologous conjunctival transplantation, the density of inflammatory cells in the cornea of patients with low-temperature plasma radiofrequency ablation decreased (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-D) and the structure of the corneal layers was clearer (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-D) one week after the operation\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePterygium is a common ocular surface disease, which is characterized by degeneration, hypertrophy, hyperplasia of the conjunctiva and subconjunctival tissue of interpalpebral. \u003csup\u003e22\u003c/sup\u003eThe occurrence of pterygium is closely related to ultraviolet radiation, which has been confirmed by many studies, but the exact pathogenesis, such as the theory of apoptosis, extracellular matrix remodeling, genetic inheritance and inflammatory theory are still under investigation. \u003csup\u003e23\u003c/sup\u003e Resection of hyperplastic tissue is a key step in the treatment of pterygium. However, simple resection without transplantation is associated with a high recurrence rate. At present, autologous transplantation of the conjunctiva in the bare area after resection is considered to reduce the recurrence rate (4.8%-12.3%), which may be the most effective treatment for pterygium. \u003csup\u003e24\u0026ndash;26\u003c/sup\u003eIn the process of pterygium removal, improper excision can leave corneal scarring and damage to the surrounding normal tissue. Low-temperature plasma radiofrequency ablation have been studied for the treatment of tonsillectomy, removal of nasopharyngeal carcinoma, mixed hemorrhoids, and sinusitis. \u003csup\u003e27\u0026ndash;29\u003c/sup\u003eHowever, there are few reports on the treatment of pterygium with low-temperature plasma radiofrequency ablation. We hope to combine the autologous conjunctival transplantation and low-temperature plasma radiofrequency ablation to treat pterygium more effectively.\u003c/p\u003e \u003cp\u003eUVB irradiation of human epithelial cells or fibroblasts isolated from normal ocular surface tissue or surgically removed pterygium specimens stimulates the expression and secretion of several proinflammatory cytokines and chemokines, such as tumor necrosis factor TNF-α, IL-1, IL-6 and MMP-9. \u003csup\u003e17\u0026ndash;20\u003c/sup\u003eUpregulation of various pro-inflammatory factors plays an important role in the pathogenesis of pterygium. MMP-9 is upregulated in pterygium tissue. \u003csup\u003e30\u003c/sup\u003e This cytokine has potent proinflammatory effects and also stimulates angiogenesis. \u003csup\u003e31\u003c/sup\u003eAt present, the research of pterygium based on the level of inflammatory factors mostly through the detection of pterygium tissue and the expression of inflammatory factors in cells, little research based on the expression of inflammatory factors in corneal, we use this new way to observe postoperative outcome. In our research, compared with control, the levels of MMP-9 and TNF-a were significantly increased at different ablation energies (20, 15, 10 J/cm\u003csup\u003e2\u003c/sup\u003e) at postoperative time and postoperative 3 days, and decreased at postoperative a week;This study will provide new ideas for the future pathogenesis of pterygium.\u003c/p\u003e \u003cp\u003eIn our study, the recurrence rate of autologous conjunctival transplantation combined with low-temperature plasma radiofrequency ablation in the treatment of pterygium was 1.64%. However, the best surgical method in the literature reports that the recurrence rate of pterygium after autologous conjunctival transplantation is 4.8%-12.3%. Our surgical approach greatly reduces the recurrence rate after pterygium surgery, postoperative patients did not have serious complications. Many reports that the autologous conjunctival autograft is taken from the upper conjunctival tissue, \u003csup\u003e32\u0026ndash;33\u003c/sup\u003e but our study used the conjunctival tissue below the iliac crest. Considering that the patient may need to perform other operations through the upper conjunctiva, such as cataract and glaucoma, the conjunctiva is taken to prevent conjunctival scars. At the same time, we found that low-temperature plasma radiofrequency ablation can reduce the time to strip the pterygium during the operation, and can better maintain the integrity of the damaged cornea and reduce the number of inflammatory cell infiltration through the follow-up of patients for a week after surgery.\u003c/p\u003e \u003cp\u003eAlthough low-temperature plasma radiofrequency ablation has been used in clinical practice, the clinical application of ophthalmology is less. Huang et al reported that low-temperature plasma is safe and efficient for pterygium surgery, but the optimal energy choice for pterygium surgery and its correlation with inflammatory factors have not yet been elucidated. \u003csup\u003e34\u003c/sup\u003eIn our research, we have verified the safety of low-temperature plasma radiofrequency ablation for corneal tissue through animal experiments. In animal experiments, we chose the ablation energy of 20, 15, 10J/cm\u003csup\u003e2\u003c/sup\u003e for the safety analysis of low-temperature plasma radiofrequency ablation. We observed the corneal tissue through the same ablation time, different ablation energy, since the corneal thickness of the rat is 222\u0026ndash;230\u0026micro;m and the corneal thickness of the adult is 550um-570\u0026micro;m, the ablation energy of the low-temperature plasma radiofrequency ablation is also inconsistent, and finally we chose energy 35 J/cm\u003csup\u003e2\u003c/sup\u003e for clinical pterygium surgery since the thickness of human cornea is thicker than that of rats. Our experiments confirm that the low-temperature plasma radiofrequency ablation is safe for the corneal tissue and does not cause damage to the corneal tissue structure.\u003c/p\u003e \u003cp\u003eIn our experiment, we only followed up patients for 18 months, which is somewhat intentional. In future research, we can compare the recovery time, the symptoms of self-consciousness and the difference of postoperative objective indicators of patients, providing new ideas for clinical treatment of pterygium.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSong Zhen and Jin Xin designed the project and collected clinical data of patients, Zhu Rui and Wang Jingrao conducted animal experiments and statistics, and Zhang Hong was responsible for the supervision and inspection of the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by the National Natural Science Foundation of China (Grant No.U20A20363,81970776),Supported by the Natural Science Foundation of Heilongjiang Province, China(Grant No. LH2020H039) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with research guidelines and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eregulations. All experimental protocols were approved by the First Afli-\u003c/p\u003e\n\u003cp\u003eated Hospital of Harbin Medical University Ethical Committee (No.18A0047)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eand strictly followed the Helsinki Declaration. All patients provided written\u0026nbsp;\u003c/p\u003e\n\u003cp\u003einformed consent before participating in the study.\u003c/p\u003e\n\u003cp\u003eAll experimental methods involving animals are reported following ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments and was approved by the First Afliated Hospital of Harbin Medical University animal Ethical Committee (Approval No. 2021093).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShiroma H, Higa A, Sawaguchi S, et al. Prevalence and risk factors of pterygium in a southwestern island of Japan: the Kumejima Study. Am J Ophthalmol 2009;148:766\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eDonepudi GD,Ramesh S,Govindarajulu M,et al.Early postoperative outcomes of pterygium surgery: Sutures versus autogenous serum in-situ fixation of limbal conjunctival autograft.Life Sci 2019;221:93-98. \u003c/li\u003e\n\u003cli\u003eMalozhen SA, Trufanov SV, Krakhmaleva DA. Pterygium: etiology, pathogenesis, treatment.Vestn Oftalmol. 2017;133(5):76-83.\u003c/li\u003e\n\u003cli\u003eYoung AL, Cao D, Chu WK, Ng TK, Yip YWY, Jhanji V, Pang CP. The Evolving Story of Pterygium.Cornea. 2018 Nov;37 Suppl 1:S55-S57.\u003c/li\u003e\n\u003cli\u003eChen X.Ologen Implantation versus Conjunctival Autograft Transplantation for Treatment of Pterygium.J Ophthalmol2018;2018:1617520.DOI:10.1155/2018/1617520\u003c/li\u003e\n\u003cli\u003eGhoz N,Elalfy M,Said D.Healing of autologous conjunctival grafts in pterygium surgery.Acta Ophthalmol 2018;96:e979-e988.\u003c/li\u003e\n\u003cli\u003eBaxter SL,Nguyen BJ,Kinori M,et al.Identification and correction of restrictive strabismus following pterygium excision surgery.Am J Ophthalmol 2019;DOI:10.1016/j.ajo.2019.02.004\u003c/li\u003e\n\u003cli\u003eAl Fayez MF. Limbal versus conjunctival autograft transplantation for advanced and recurrent pterygium. Ophthalmology 2002;109:1752\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eChu WK, Choi HL, Bhat AK, Jhanji V. Pterygium: new insights. Eye (Lond). 2020 Jun;34(6):1047-1050.\u003c/li\u003e\n\u003cli\u003eWang N, Dou Z, He L, Song L, Guo Z, Ni J. Sphenopalatine ganglion-targeted low-temperature plasma radiofrequency ablation in the treatment of refractory cluster headache.Wideochir Inne Tech Maloinwazyjne. 2020 Jun;15(2):313-318.\u003c/li\u003e\n\u003cli\u003eZhong QY, Sun Q, Liu ZH. Endoscopic Low-Temperature Plasma Radiofrequency Ablation for Primary Thyroid-Like Low-Grade Nasopharyngeal Papillary Adenocarcinoma.Ear Nose Throat J. 2021 Aug;100(7):563-564.\u003c/li\u003e\n\u003cli\u003eDai X, Wei B, He Y, Zhang Q, Xi J. Postoperative efficacy of low-temperature plasma radiofrequency ablation in elderly patients with laryngeal carcinoma and its influences on tumor markers and COX-2 and VEGF expressions in laryngeal carcinoma tissues.J BUON. 2020 Jul-Aug;25(4):1969-1975.\u003c/li\u003e\n\u003cli\u003eLi Y, Guo Y, Yang L, Ni J. Comparison of the short-term outcomes after low-temperature plasma radiofrequency ablation (coblation) in the Gasserian ganglion for the treatment of idiopathic trigeminal neuralgia.J Pain Res. 2019 Apr 17;12:1235-1242.\u003c/li\u003e\n\u003cli\u003eXu H, Chen F, Zheng Y, Li X. Efficacy and toxicities of low-temperature plasma radiofrequency ablation for the treatment of laryngomalacia in neonates and infants: a prospective randomized controlled trial.Ann Transl Med. 2020 Nov;8(21):1366. \u003c/li\u003e\n\u003cli\u003eWang HH,Liu GF,Xu JH.Comparative study on the effect of tonsillectomy with low-temperatureplasma knife, monopolar electrocoagulation and traditional operation.Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 2016;30:1564-1566.\u003c/li\u003e\n\u003cli\u003eCai W,Chen Z,Wen L,et al.Bipolar plasma vaporization using plasma-cutting and plasma-loop electrodes versus cold-knife transurethral incision for the treatment of posterior urethral stricture: a prospective, randomized study.Clinics (Sao Paulo) 2016;71:1-4.\u003c/li\u003e\n\u003cli\u003eLee H,Lee M,Lee Y,et al.Chondrocyte-derived extracellular matrix suppresses pathogenesis of human pterygium epithelial cells by blocking the NF-\u0026kappa;B signaling pathways.Mol Vis 2016;22:1490-1502. \u003c/li\u003e\n\u003cli\u003eWanzeler ACV, Barbosa IAF, Duarte B, Borges D, Barbosa EB, Kamiji D, Huarachi DRG, Melo MB, Alves M. Mechanisms and biomarker candidates in pterygium development. Arq Bras Oftalmol. 2019 Sep 30;82(6):528-536. \u003c/li\u003e\n\u003cli\u003eCui YH, Feng QY, Liu Q, Li HY, Song XL, Hu ZX, Xu ZY, Li JH, Li MJ, Zheng WL, Li ZJ, Pan HW. Posttranscriptional regulation of MMP-9 by HuR contributes to IL-1beta-induced pterygium fibroblast migration and invasion.J Cell Physiol. 2020 Jun;235(6):5130-5140.\u003c/li\u003e\n\u003cli\u003eCh\u0026aacute;vez-Mondrag\u0026oacute;n E, Palacio C, Soto-G\u0026oacute;mez A, Villanueva-N\u0026aacute;jera M, De Wit-Carter G, Su\u0026aacute;rez-Velasco R, Baiza-Duran L, Olvera-Monta\u0026ntilde;o O, Mu\u0026ntilde;oz-Villegas P. Efficacy and safety of bromfenac 0.09% and sodium hyaluronate 0.4% combination therapy, versus placebo in patients with pterygium I-III for clinical signs on ocular inflammation. Clin Ophthalmol. 2019 May 2;13:781-787.\u003c/li\u003e\n\u003cli\u003eZidi S,Bediar-Boulaneb F,Belguendouz H,et al.Local pro-inflammatory cytokine and nitric oxide responses are elevated in patients with pterygium.Int J Immunopathol Pharmacol 2017;30:395-405. \u003c/li\u003e\n\u003cli\u003eLin ZR,Wu HP,Xie ZW,et al.Efficacy of deproteinized calf blood extract eye drops on early recovery after pterygium surgery.Zhonghua Yan Ke Za Zhi 2019;55:134-140.\u003c/li\u003e\n\u003cli\u003eWanzeler A, Duarte B, de Andrade V, et al. Impact of conjunctival autograft on pterygium treatment: evaluation of related symptoms and patients\u0026rsquo; satisfaction after surgery. Clin Ophthalmol. 2018;12: 833\u0026ndash;837. \u003c/li\u003e\n\u003cli\u003eChao SC,Hu DN,Roberts J,et al.Inhibition effect of curcumin on UVB-induced secretion of pro-inflammatory cytokines from corneal limbus epithelial cells.Int J Ophthalmol 2017;10:827-833.\u003c/li\u003e\n\u003cli\u003eGhoz N, Elalfy M, Said D, Dua H. Healing of autologous conjunctival grafts in pterygium surgery. Acta Ophthalmol. 2018 Dec;96(8):e979-e988. \u003c/li\u003e\n\u003cli\u003eLiu YC, Ji AJS, Tan TE, Fuest M, Mehta JS. Femtosecond Laser-assisted Preparation of Conjunctival Autograft for Pterygium Surgery. Sci Rep. 2020 Feb 14;10(1):2674. doi: 10.1038/s41598-020-59586-z.\u003c/li\u003e\n\u003cli\u003eGuo J, Kong Q. Comparing the effect of low-temperature plasma radiofrequency and traditional method in tonsillectomy]. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2012 Apr;26(7):325-6.\u003c/li\u003e\n\u003cli\u003eZhang QF, She CP, Tong YF, Jin Y, Zhang XR. Endoscopic surgery using the low-temperature plasma radiofrequency for nasopharyngeal angiofibroma.Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2010 Jul;45(7):578-81.\u003c/li\u003e\n\u003cli\u003eRuff PG 4th. Thermal effects of percutaneous application of plasma/radiofrequency energy on porcine dermis and fibroseptal network.J Cosmet Dermatol. 2021 Jul;20(7):2125-2131.\u003c/li\u003e\n\u003cli\u003eDi Girolamo N, Kumar RK, Coroneo MT, Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 2002;43(11):3430-3437. \u003c/li\u003e\n\u003cli\u003eNolan TM, Di Girolamo N, Sachdev NH, Hampartzoumian T, Coroneo MT, Wakefield D. The role of ultraviolet irradiation and heparin-binding epidermal growth factor-like growth factor in the pathogenesis of pterygium. Am J Pathol 2003;162(2):567-574.\u003c/li\u003e\n\u003cli\u003eNganga Ngabou CGF,Makita C,Ndalla SS,et al.[Pterygium surgery by conjunctiva autograft with autologous blood fixation].J Fr Ophtalmol 2018;41:425-432. \u003c/li\u003e\n\u003cli\u003eGhoz N,Elalfy M,Said D.Healing of autologous conjunctival grafts in pterygium surgery.Acta Ophthalmol 2018;96:e979-e988. \u003c/li\u003e\n\u003cli\u003eHuang H, Li S, Zhong J, Wang B, Peng L, Deng Y, Wang M, Yuan J. Evaluation of the Safety and Efficacy of a Low-Temperature Plasma Surgical System for Pterygium. Cornea. 2020 Dec ;39(12): 1581-1587. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pterygium, Low-temperature plasma radiofrequency ablation, Safety, Effectiveness","lastPublishedDoi":"10.21203/rs.3.rs-2356498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2356498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground/Aims:\u003c/h2\u003e\n\u003cp\u003eTo evaluate the safety and effectiveness of low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation for pterygium excision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In order to verify the safety of low-temperature plasma radiofrequency ablation, we conducted animal experiments to observe corneal structure by anterior segment photography, anterior optical coherence tomography, HE staining and immunohistochemistry(MMP-9,TNF-a) before and after corneal burning at different ablation energies (20, 15, 10, 0J/cm\u003csup\u003e2\u003c/sup\u003e)and different point in time(0,3d,1w,2w). Then, we observed preoperative and postoperative treatment effect of 121 eyes of 120 patients with primary pterygium from June 2018 to June 2020. 59 patients treated with pterygium excision and autologous conjunctival transplantation, 61 patients treated with pterygium excision by low-temperature plasma radiofrequency ablation and autologous conjunctival transplantation. The time to strip the pterygium, the integrity of the cornea and the number of inflammatory cell infiltration were observed and recorded. All patients were followed up for 18 months to observe whether the pterygium recurred.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The animal experiment confirmed that suitable low-temperature plasma radiofrequency ablation energies has no damage to the corneal tissue structure. Only one case recurred during the year of follow-up. Low-temperature plasma radiofrequency ablation can reduce the time to strip the pterygium, can better maintain the integrity of the damaged cornea and reduce the number of inflammatory cell infiltration, can reduce recurrence rate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Low-temperature plasma radiofrequency ablation combined with autologous conjunctival transplantation is an safety and effective pterygium excision.\u003c/p\u003e","manuscriptTitle":"Low-temperature plasma radiofrequency ablation and autologous conjunctival transplantation for pterygium excision.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-21 07:02:23","doi":"10.21203/rs.3.rs-2356498/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a2f94ac-7496-4fe9-bab0-867f5057a3dd","owner":[],"postedDate":"December 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-02T08:59:29+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-21 07:02:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2356498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2356498","identity":"rs-2356498","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}