A Comparative Study of Peripheral Capsular Linear Incision and Circular Micro-Tear Capsulotomy in the In Situ Regeneration of Rabbit Eyes

A Comparative Study of Peripheral Capsular Linear Incision and Circular Micro-Tear Capsulotomy in the In Situ Regeneration of Rabbit Eyes | 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 Research Article A Comparative Study of Peripheral Capsular Linear Incision and Circular Micro-Tear Capsulotomy in the In Situ Regeneration of Rabbit Eyes Ming Zhang, Tengyu Xv, Menghan Wang, Wenwen Hou, Yan Wang, Liming Cai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8087513/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Lens regeneration is regarded as a promising strategy for restoring vision in cataract patients after surgery. However, conventional small curvilinear capsulorhexis often results in the loss of anterior capsular lens epithelial cells, thereby compromising the integrity and transparency of the regenerated lens. Building on the concept of small capsulorhexis, this study introduces a 1.5-mm linear incision in the peripheral capsule to maximally preserve epithelial cells and enhance regenerative outcomes. Methods Forty 4-week-old New Zealand white rabbits were randomly assigned to two groups: the peripheral capsular linear incision group (Group A) and the group (Group B), with 20 rabbits in each group. Lens removal was performed via irrigation/aspiration (I/A). Postoperatively, regenerated lenses were evaluated for morphology, size, weight, transparency, and capsular healing, followed by histological analysis. Results Successful lens regeneration was observed in both groups. Group A demonstrated superior regenerative outcomes, characterized by intact lens morphology, higher transparency, and significantly greater lens weight and thickness compared with Group B (P < 0.05). Histological analysis showed that epithelial cells in Group A differentiated into well-organized lens fibers with a more orderly arrangement, shorter capsular healing time, smaller scars, and smoother capsular surfaces than those in Group B. Conclusions Compared with small curvilinear capsulorhexis, peripheral capsular linear incision offers clear advantages in promoting morphological and functional lens regeneration, as well as capsular healing, in rabbit eyes. These findings provide novel strategies for developing surgical strategies for lens regeneration in children with congenital cataracts. Lens regeneration Cataract surgery peripheral capsule linear incision small curvilinear capsulorhexis rabbit model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Currently, the primary treatment for cataracts is the removal of the opaque lens followed by intraocular lens (IOL) implantation [ 1 , 2 ]. However, IOL implantation in children under 2 years of age with congenital cataracts remains controversial. Since the eyeballs of infants are still developing, unpredictable myopic shifts may occur postoperatively. On the other hand, the absence of the lens greatly affects the recovery of visual function. After cataract removal in children, residual lens cell growth disorders can cause visual axis opacity (VAO) and even secondary blindness due to inflammation and proliferation [ 3 ]. In addition, IOL displacement or loss of accommodative function can seriously compromise the visual prognosis in young children. Previous studies have reported that early IOL implantation in infants significantly increases the risk of reoperation [ 4 , 5 ]. Thus, the search for novel treatment strategies for congenital cataracts remains of considerable clinical importance. Recent studies have demonstrated that children with congenital cataracts may develop unorganized “donut-shaped” transparent lens tissue, suggesting the potential for human lens regeneration [ 6 , 7 ]. Liu proposed a small curvilinear capsulorhexis technique for congenital cataract surgery to promote in situ lens regeneration. Although this approach involves a smaller capsulorhexis incision, it is still associated with challenges such as capsular defects, epithelial cell loss, and insufficient regeneration quality, resulting in lenses that fall short of normal standards. To address these limitations, we designed a novel peripheral capsular linear incision technique. Compared with small curvilinear capsulorhexis, postoperative observations revealed significant differences in capsular opening healing, as well as in the weight, transparency, and fiber arrangement of regenerated lenses. Materials and methods Experimental animals A total of forty healthy 4-week-old New Zealand white rabbits (average weight: ~0.6 kg) were obtained from the Xuzhou Medical University Animal Experiment Center. Grouping and surgical procedures The 40 rabbits were randomly divided into two groups (n = 20 each): Group A (peripheral capsular linear incision) and Group B (small curvilinear capsulorhexis). In each rabbit, one eye was randomly selected for surgery. Pupillary dilation was achieved with three administrations of compound tropicamide eye drops (0.5%, Shenyang Sinqi). Anesthesia was induced via subcutaneous injection of dexmedetomidine hydrochloride (0.02 mg/kg) combined with a mixture of tiletamine hydrochloride and zolazepam hydrochloride (15 mg/kg). Lens contents were aspirated with the Legacy 20000 phacoemulsification system (Alcon, Fort Worth, USA) as follows: A 2.4-mm corneal tunnel incision was made at the 12 o’clock position, followed by capsulotomy and capsulorhexis near the lens equator. Group A received a 1.5-mm linear incision, whereas Group B underwent a 1.5-mm capsulorhexis. Incision length or capsular opening diameter was confirmed using a 1-mm scale calibration needle (Xinkeling, Shanghai, China) before and after capsulorhexis. The lens nucleus and residual cortex were aspirated using an irrigation/aspiration (I/A) system. The corneal incision was closed with interrupted 10 − 0 nylon sutures. All surgeries were performed by the same senior surgeon. Postoperative examination Postoperative care included TobraDex eye drops (tobramycin 0.3%, dexamethasone 0.1%; Alcon, USA) four times daily, TobraDex eye ointment (tobramycin 0.3%, dexamethasone 0.1%; Alcon, USA), and atropine sulfate eye ointment (1%, Shenyang Sinqi, China) once daily, continued for two weeks. Anterior segment slit-lamp images were captured with an anterior segment camera (Chongqing Kanghua Ruiyu, China) to assess corneal, anterior capsule, and lens regeneration of both groups on the day of surgery and at 4, 8, and 12 weeks postoperatively. Lens thickness was measured using optical coherence tomography (TowardPi, Tianjin, China). Lens morphology, weight, and fundus status were also evaluated. Healing of the anterior capsule opening The anterior capsule opening area (ACOA), healing time, and scar formation were observed and compared between the two groups. Sample collection and histological examination At designated time points, rabbits were euthanized by intravenous injection of 10 ml of air into the ear vein. The regenerated and contralateral lenses were carefully separated. Regenerated lenses were immediately immersed in phosphate-buffered saline (PBS, 37°C) and photographed under a microscope (Stemi DV4, Carl Zeiss, Oberkochen, Germany). Lens weight was measured three times, and the mean value was used to calculate the weight percentage (WP): WP = weight of regenerated lens×100​/weight of contralateral intact lens. Lenses were then fixed in 10% neutral buffered formalin for 2 hours, dehydrated in graded ethanol, cleared in xylene, embedded in paraffin, sectioned at 4 µm (MiDiII, 3DHISTECH, Hungary), and stained with hematoxylin and eosin (H&E). Sections were examined under a microscope (Eclipse 50i, Nikon, Japan). Statistical analysis Statistical analyses were conducted using SPSS software (version 17.0, SPSS Inc., Chicago, USA). Data are expressed as mean ± standard deviation (SD). Paired t-tests were used to compare regenerated lens thickness and weight percentage between the two groups. All tests were two-tailed, and P < 0.05 was considered statistically significant. Results Morphology of regenerated lenses Slit-lamp examination was performed to assess the morphology of regenerated lenses. At 12 weeks postoperatively, the capsules in Group A were completely filled with regenerated lenses. Except for mild central opacity, the lenses were generally transparent (Fig. 1 A, anterior view). Fundus examination revealed clear visualization of the optic disc, retina, and choroidal vessels, indicating no impairment of retinal function. In contrast, Group B showed incomplete lens regeneration that did not fully fill the capsules, with localized defects in the equatorial region and reduced thickness compared with Group A. Central opacity was also observed, and the optic disc, retina, and choroidal vessels could not be clearly visualized during fundus examination. In some cases, anterior and posterior capsule adhesions resulted in an “8-shaped” regenerated lens. Overall, Group A demonstrated superior regeneration, with a larger lens size, higher transparency, and intact morphology that is increasingly aligned with the normal lens. Notably, all 20 eyes in Group A exhibited well-healed capsular openings without scar adhesions, whereas 13 of 20 eyes in Group B showed anterior–posterior capsule adhesions Changes in lens weight during regeneration Lens regeneration was quantitatively assessed by calculating the WP, defined as the ratio of regenerated lens weight to that of the contralateral intact lens. WP gradually increased in both groups over time (Figure). At 4 weeks, WP was significantly higher in Group A (24.53 ± 1.17%) than in Group B (16.67 ± 1.32%). At 8 weeks, WP values were 76.48 ± 3.61% in Group A and 72.57 ± 3.67% in Group B. At 12 weeks, WP in Group A reached 90.57 ± 6.37%, approaching the normal contralateral lens weight, while Group B reached only 83.81 ± 6.07%. These findings suggest that the peripheral linear incision preserved more lens epithelial cells, thereby enhancing regeneration efficiency compared with the small curvilinear capsulorhexis, which may enhance endothelial cell loss. The form and thickness of the regenerated lens Lens thickness was measured using OCT. Group A consistently exhibited greater lens thickness compared with Group B at all time points: 4 weeks: 2.64 ± 0.08 mm vs. 2.42 ± 0.06 mm (P < 0.05); 8 weeks: 4.62 ± 0.10 mm vs. 4.57 ± 0.09 mm (P < 0.05); 12 weeks: 5.37 ± 0.11 mm vs. 5.18 ± 0.12 mm (P 0.05). Both groups exhibited rapid growth between weeks 4 and 8, followed by slower growth thereafter. ACOA contact and healing Slit-lamp imaging was used to evaluate capsular healing between Groups A and B after pupillary dilation. In Group A, the linear incision exhibited nearly complete healing within 1 week and full closure by 2 weeks postoperatively (Figure). Scars were minimal, with fine punctate wrinkles and no adhesions in the capsular cavity. By week 8, the scars had stabilized and further contracted. In Group B, the capsular opening showed near-complete healing by 2 weeks, forming a linear scar and a closed cavity. However, several cases exhibited anterior–posterior capsule adhesions. Scar morphology stabilized by week 8. Collectively, linear incisions promoted shorter healing times, smaller scars, and reduced epithelial cell loss compared with circular incisions. Histological characteristics of regenerated lenses Histological examination at 12 weeks revealed that in Group A, most LECs had differentiated into lens fibers (Fig. 8 A). Near the equatorial region, LECs retained forward-moving nuclei, forming a characteristic crescent-shaped “lens bow” structure (Fig. 8 A-a, d, black arrows). A single layer of LECs was present beneath the anterior capsule (Fig. 8 A-b), while the intermediate nuclear layer was absent, and no LECs were observed under the posterior capsule (Fig. 8 A-c). In certain regions, multilayered fibroblast-like LECs were detected, resembling developmental processes of the normal lens. In Group B, the regenerated lenses also displayed a lens bow structure (Fig. 8 B-a, d, black arrows), but disorganized lens fiber arrangements were more frequent. Taken together, these findings confirm at the cellular level that the peripheral linear incision preserved more functional LECs, thereby promoting orderly fiber differentiation, reducing fibroblast-like transformation, and ultimately improving the growth rate and transparency of regenerated lenses, making them more comparable to normal lenses. Intraoperative and postoperative complications All 40 rabbits (40 eyes) successfully underwent lens extraction, and postoperative intraocular pressure (IOP) remained within the normal range in all groups. On the first postoperative day, fibrinous exudation was observed in one eye of Group B, and two eyes showed mild corneal edema. In Group A, only one eye exhibited mild corneal edema. All symptoms resolved spontaneously within one week. At 8 weeks postoperatively, posterior synechiae (IPS) developed in 2 eyes in Group B, whereas no such complication occurred in Group A. By 12 weeks, the overall complication rate was 5% in Group A and 20% in Group B, representing a statistically significant difference between the groups. Discussion Current clinical management of cataracts primarily involves removal of the opaque lens and implantation of an IOL. However, IOL implantation in infants under 2 years of age carries a high risk of complications, underscoring the urgent need for alternative treatment strategies. In recent years, ophthalmologists have reported cases in which children with congenital cataracts developed “donut-shaped” regenerated lenses following capsulotomy and cortical aspiration. Although these lenses exhibited scarring and opacity at the capsule opening, the capsular sac itself remained largely transparent. Such postoperative proliferation suggests that the human lens retains regenerative potential. Notably, in 2016, Liu et al. [ 8 ] introduced a minimally invasive technique employing a 1.5-mm capsulorhexis incision displaced from the central visual axis to the periphery, thereby improving postoperative transparency. While this method achieved partial in situ lens regeneration in infants under 2 years of age, several limitations remain. First, the regenerated lens often reached only approximately 50% of the thickness of a normal lens, and the biconvex shape was disrupted by capsular scarring. Adhesions between the anterior and posterior capsules, as well as anterior subcapsular epithelial cell loss, frequently resulted in localized opacities. Second, even small curvilinear capsulorhexis caused loss of LECs, thereby diminishing the regenerative capacity of the lens. To address these limitations, we proposed a new strategy: removal of the lens cortex while preserving capsular integrity through a peripheral linear incision. This approach aims to minimize capsular defects, preserve a larger pool of viable LECs, and foster in situ lens regeneration. Importantly, linear incisions appear to reduce the release of inflammatory cytokines such as TGF-β, thereby lowering the risk of fibroblast-like differentiation of LECs. The healing of the ACOA is a critical determinant of successful regeneration. Prior studies have shown that smaller capsulorhexis tears heal more rapidly and yield more transparent regenerated lenses [ 9 – 11 ], whereas larger tears heal slowly and incompletely. Tan et al. [ 15 ] demonstrated in animal experiments that capsulorhexis size is a key factor influencing transparency and integrity, with smaller incisions favoring ACOA healing. The advantages of the linear incision in our study include: (i) rapid healing with minimal loss of capsule and LECs [ 12 ], (ii) effective sealing of the capsule opening, creating a microenvironment conducive to regeneration, and (iii) maximal preservation of equatorial LECs. Our findings confirm these benefits. In Group A, the linear incision stretched into an elliptical opening under intraocular pressure but realigned after cortical aspiration, resulting in nearly perfect closure with minimal tissue loss, resulting in rapid healing. However, in Group B, although complete healing of the capsule was ultimately achieved, there was still a loss of capsule tissue and lens epithelial cells compared with conventional ACOA. This led to slightly slower healing of the ACOA and the formation of larger scars. Moreover, fibrin proliferation around the torn capsule edges impeded the migration of new lens fibers from the periphery toward the center. Second, Gwon et al. [ 13 ] demonstrated that sealing the ACOA with collagen patches and inflating the posterior capsule for separation can accelerate lens regeneration and improve lens clarity. Similarly, the linear incision technique adopted in this study enabled complete closure of the capsule opening, thereby establishing a sealed microenvironment favorable for lens regeneration. This closure also restricted the entry of inflammatory cytokines known to interfere with regeneration [ 14 , 15 ]. Furthermore, a linear incision design allows maximal preservation of LECs beneath the anterior capsule. Previous studies have shown that LECs located in the equatorial zone function as stem cells with the capacity to proliferate and differentiate into new lens fibers [ 16 , 17 ]. Thus, the greater the number of residual LECs retained after surgery, the higher the regenerative potential of the lens. Excessive loss of LECs during previous capsulorhexis procedures may be one reason why the human lens often fails to regenerate. The reason for selecting an incision length of 1.5 mm can be obtained from the results of the previous studies. In our preliminary experiments, larger incisions (> 1.5 mm) were technically easier to perform but resulted in greater surgical trauma, whereas smaller incisions (< 1.5 mm) increased the risk of capsule tearing during lens aspiration. With future advances in microsurgical instruments, smaller incisions may become feasible, and we are actively exploring this possibility. Although earlier studies reported that small curvilinear capsulorhexis can produce regenerated lenses that are morphologically and functionally more similar to normal lenses, our findings revealed significant differences between the two approaches. Specifically, lens thickness in Group A was consistently greater than in Group B at 4, 8, and 12 weeks. A plausible explanation is that Group A, which underwent a linear 1.5 mm incision, retained capsule integrity and avoided defects, thereby supporting the regeneration of lenses with morphology closer to that of normal lenses. By contrast, Group B developed incision adhesions that disrupted capsule continuity, resulting in irregular lens shapes and compromised integrity. By limiting the size of the capsular incision and minimizing the removal of the anterior capsule, our method reduces damage to lens epithelial cells while preserving most of the lens epithelium. This creates an optimal environment for lens regeneration. In this study, regenerated lenses in Group A demonstrated superior integrity, transparency, capsule healing, capsule size, and reduced scar hyperplasia compared with those in Group B. These findings suggest that the proposed peripheral linear capsulotomy confers advantages over circular micro-tear capsulotomy and may offer new insights into strategies for in situ lens regeneration. This study has several limitations. First, the relatively small sample size limited the statistical power of our findings. Second, the follow-up period was restricted to 12 weeks, which precluded long-term evaluation of the functional and optical properties of the regenerated lenses. Although the regenerated lenses were morphologically complete and transparent in the early stages, by 12 weeks, we observed one case of mild sublenticular opacity. This may have been caused by residual viscoelastic material or inflammatory responses that disrupted the proliferation and migration of lens epithelial cells. Further investigation is warranted to clarify these mechanisms. In addition, even though the regenerated lenses in Group A closely resembled normal lenses, a small central deficit was noted during epithelial cell migration. Whether this phenomenon is attributable to insufficient numbers of lens epithelial cells or to intrinsic limitations in their regenerative capacity remains unclear. Conclusion In summary, this study demonstrates that a peripheral capsular linear incision can promote favorable conditions for lens regeneration by preserving lens epithelial cells and enabling rapid closure of the capsular opening. Compared with the circular micro-incision technique, this approach resulted in regenerated lenses with superior integrity, transparency, and morphology, while also reducing scar formation and accelerating healing. These findings highlight the potential of peripheral linear capsulotomy as a promising surgical strategy for in situ lens regeneration and provide a foundation for developing improved treatments for children with congenital cataracts. Declarations Author contributions Ming Zhang contributed to the entire experimental work of the participating articles and the writing of the articles;Tengyu Xv;Menghan Wang;Wenwen Hou;Yan Wang;Liming Cai;Wei Wang,all of them participated in the experimental work and article image creation.Suchang Wang was responsible for the design of the experiment and the final review. Founding This study was supported by the Xuzhou Science and Technology Plan Fund (Grant No. KC23171) and the Development Fund of the Affiliated Hospital of Xuzhou Medical University (Grant No. XYFM202319). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate All experimental procedures were approved by the Animal Ethics Committee of Xuzhou Medical University (Ethics Number: 202404T024) and adhered to the guidelines of the Association for Research in Vision and Ophthalmology on the Care and Use of Laboratory Animals. Competing interests The authors declare no competing interests References Tan X, et al. Impact of cataract screening integrated into establishment of resident health record on surgical output in a rural area of south China. Ann Transl Med. 2020;8(19):1222. Khairallah M, et al. Number of People Blind or Visually Impaired by Cataract Worldwide and in World Regions, 1990 to 2010. Invest Ophthalmol Vis Sci. 2015;56(11):6762–9. Tan X, et al. Capsular Outcomes After Pediatric Cataract Surgery Without Intraocular Lens Implantation: Qualitative Classification and Quantitative Measurement. Med (Baltim). 2016;95(10):e2993. Solebo AL, Cumberland P, Rahi JS. 5-year outcomes after primary intraocular lens implantation in children aged 2 years or younger with congenital or infantile cataract: findings from the IoLunder2 prospective inception cohort study. Lancet Child Adolesc Health. 2018;2(12):863–71. Mandal AK, Gollakota R. Soemmering's Ring Ophthalmol. 2017;124(7):1064. Luo L, et al. In-the-bag intraocular lens placement via secondary capsulorhexis with radiofrequency diathermy in pediatric aphakic eyes. PLoS ONE. 2013;8(4):e62381. Bhattacharjee H, Deshmukh S. Soemmering's ring. Indian J Ophthalmol. 2017;65(12):1489. Lin H, et al. Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2016;531(7594):323–8. Lin H, et al. Capsular Outcomes Differ with Capsulorhexis Sizes after Pediatric Cataract Surgery: A Randomized Controlled Trial. Sci Rep. 2015;5:16227. Lin H, et al. Corrigendum: Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2017;541(7638):558. Lin H, et al. Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2016;531(7594):323–8. Zhou M, et al. A hierarchy of proliferative cells exists in mouse lens epithelium: implications for lens maintenance. Invest Ophthalmol Vis Sci. 2006;47(7):2997–3003. Gwon A, Gruber LJ, Mantras C. Restoring lens capsule integrity enhances lens regeneration in New Zealand albino rabbits and cats. J Cataract Refract Surg. 1993;19(6):735–46. Fisus AD, Findl O. Capsular fibrosis: a review of prevention methods and management. Eye (Lond). 2020;34(2):256–62. Liu J, Finkel T. Stem cell aging: what bleach can teach. Nat Med. 2006;12(4):383–4. Beebe DC, Holekamp NM, Shui YB. Oxidative damage and the prevention of age-related cataracts. Ophthalmic Res. 2010;44(3):155–65. Smith AN, et al. Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis. Development. 2009;136(17):2977–85. Additional Declarations No competing interests reported. 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10:02:11","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14225,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/8b94678ed91e703b54d8492a.png"},{"id":99318972,"identity":"d5e28479-7c96-4ccd-82d8-6d1ab1ce3815","added_by":"auto","created_at":"2025-12-31 16:35:49","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342292,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/7645cc5d7aa8ddab3c2209d2.png"},{"id":99223834,"identity":"bc291aa9-0b20-4d96-bd5e-b387fa7e70ac","added_by":"auto","created_at":"2025-12-30 10:02:11","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":284553,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/36cdb17d1bd4a28ca815f445.png"},{"id":99319200,"identity":"98c8eaff-18db-490e-9186-76d7d0ffd59f","added_by":"auto","created_at":"2025-12-31 16:36:39","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":480988,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/cc1cfd1766d82e7fdd07a9f0.png"},{"id":99223836,"identity":"3a5554ca-e111-4bc4-9c41-ad916a7219ae","added_by":"auto","created_at":"2025-12-30 10:02:11","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":55159,"visible":true,"origin":"","legend":"","description":"","filename":"550cd3a2874645988bb2c91fdf2e2af51structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/901f290f53613d2d259db1e6.xml"},{"id":99319017,"identity":"da3aa49d-d921-4d8e-a507-23094c1dd9e2","added_by":"auto","created_at":"2025-12-31 16:36:00","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":62550,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/184dc634358ae9cb597b57c3.html"},{"id":99317666,"identity":"e32097d7-8e0d-4a93-9adb-a9268b0945ad","added_by":"auto","created_at":"2025-12-31 16:30:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196751,"visible":true,"origin":"","legend":"\u003cp\u003eTraditional capsulotomy exhibits extensive loss of lens epithelial cells (LEC), along with epithelial-to-mesenchymal transition (EMT) and fibrosis developing around the surgical site. The small curvilinear capsulorhexis creates a 1.5 mm opening in the anterior capsule, leading to partial capsule loss, LEC loss, fibrosis, adhesion, and scarring around the incision. In contrast, the peripheral linear incision minimizes LEC loss, promotes rapid wound healing, reduces scarring, and yields a more transparent regenerated lens.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/c680ffe171fa07b1163695fe.png"},{"id":99223818,"identity":"c23a2728-c185-4fda-bd36-178262aa9cac","added_by":"auto","created_at":"2025-12-30 10:02:10","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":390374,"visible":true,"origin":"","legend":"\u003cp\u003eIn Group A, the blue arrow highlights the regenerated lens, which appears relatively intact with high transparency, resembling the normal lens shown in Figure C. In contrast, the blue arrow in Group B points to visible damage at the edge of the regenerated lens, with poorer transparency.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/6f4656ce7a2d0a50ccc61476.jpeg"},{"id":99318978,"identity":"460b0425-c98a-4469-ba84-6154dd344e8c","added_by":"auto","created_at":"2025-12-31 16:35:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":718249,"visible":true,"origin":"","legend":"\u003cp\u003eIn the linear incision group, the regenerated lens grew uniformly and completely, as indicated by arrow in Figure 3 A2, showing high transparency and coverage that filled the capsule bag. In contrast, the small curvilinear capsulorhexis, marked by arrow in Figure 3 B2, displayed incision adhesion, with the regenerated lens demonstrating a figure-8 shape and exhibiting poor transparency.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/be387087ed07ce20ad1fe89b.jpeg"},{"id":99223815,"identity":"94de2a75-77a3-44cb-9047-df7eeb1bc978","added_by":"auto","created_at":"2025-12-30 10:02:10","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54256,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the weight of regenerated lenses in two groups. At weeks 4, 8, and 12, three rabbits from each group were euthanized, and the regenerated and contralateral lenses were carefully separated and weighed. The weight percentage was calculated as follows: (weight of regenerated lens × 10) / weight of the intact contralateral lens. All data are presented as mean ± standard deviation.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/4383fa91844c0118da91a476.jpeg"},{"id":99317436,"identity":"14802fae-7096-455a-9fcf-95c2402d0e62","added_by":"auto","created_at":"2025-12-31 16:30:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":197433,"visible":true,"origin":"","legend":"\u003cp\u003eThickness of the regenerated lens at 4, 8, and 12 weeks in groups A and B, measured using whole-field OCT. In Figure 5A, the red arrow indicates the thickness of the regenerated lens, which continuously increased from 4 to 12 weeks, maintaining a regular shape. In Figure 5B, the red arrow highlights the “8”-shaped morphology of the regenerated lens, which resulted from adhesion at the capsule opening.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/c2ba459a17e30a80fbec810b.jpeg"},{"id":99223827,"identity":"57a2b2a2-85ce-41b3-a310-99dc40d2b652","added_by":"auto","created_at":"2025-12-30 10:02:11","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43905,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the thickness of regenerated lenses at different time points. From 4 to 12 weeks postoperatively, the thickness of regenerated lenses in both groups A and B gradually increased. Compared to group B, the regenerated lenses in group A exhibited superior thickness at all three time points, with statistically significant differences (**p \u0026lt; 0.01, *p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/5b5db79fc32719cbdc54891f.jpeg"},{"id":99223839,"identity":"67e6c73f-c083-4a87-8af8-6cd467c1c6b9","added_by":"auto","created_at":"2025-12-30 10:02:11","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1157924,"visible":true,"origin":"","legend":"\u003cp\u003eHealing of Incisions in Groups A and B. The arrow in Figure 7A indicates that the linear incision was nearly healed at 2 weeks and completely healed by 8 weeks, exhibiting minimal scarring and a smooth capsule. In contrast, the arrow in Figure 7B shows that the circular micro-tear incision displayed more pronounced scarring than that of Group A at the same time points, with the presence of scar hyperplasia contributing to capsule contraction.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/20093d8952bece22b3d0f4c9.jpeg"},{"id":99319026,"identity":"d36a6752-4f04-4cfb-b1bd-6f39fc8d48dc","added_by":"auto","created_at":"2025-12-31 16:36:03","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":986098,"visible":true,"origin":"","legend":"\u003cp\u003eFigures 8A and 8B provide an overview of the 12-week regenerated lens histology (HE) in both groups. Panels A-a, A-d, B-a, and B-d illustrate typical crescent-shaped IECs, known as \"lens bow,\" which consist of a single layer of lens epithelial cells located beneath the anterior capsule (as shown in A-b and B-b).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/5dcc944496798d78e16c67c0.jpeg"},{"id":99323861,"identity":"d5d49bd8-5221-484b-b088-d81d5f898729","added_by":"auto","created_at":"2025-12-31 16:46:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4365947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8087513/v1/557f925f-ad01-4aa2-88ae-ba12b3f331f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA Comparative Study of Peripheral Capsular Linear Incision and Circular Micro-Tear Capsulotomy in the In Situ Regeneration of Rabbit Eyes\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eCurrently, the primary treatment for cataracts is the removal of the opaque lens followed by intraocular lens (IOL) implantation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, IOL implantation in children under 2 years of age with congenital cataracts remains controversial. Since the eyeballs of infants are still developing, unpredictable myopic shifts may occur postoperatively. On the other hand, the absence of the lens greatly affects the recovery of visual function. After cataract removal in children, residual lens cell growth disorders can cause visual axis opacity (VAO) and even secondary blindness due to inflammation and proliferation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, IOL displacement or loss of accommodative function can seriously compromise the visual prognosis in young children. Previous studies have reported that early IOL implantation in infants significantly increases the risk of reoperation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, the search for novel treatment strategies for congenital cataracts remains of considerable clinical importance. Recent studies have demonstrated that children with congenital cataracts may develop unorganized \u0026ldquo;donut-shaped\u0026rdquo; transparent lens tissue, suggesting the potential for human lens regeneration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Liu proposed a small curvilinear capsulorhexis technique for congenital cataract surgery to promote in situ lens regeneration. Although this approach involves a smaller capsulorhexis incision, it is still associated with challenges such as capsular defects, epithelial cell loss, and insufficient regeneration quality, resulting in lenses that fall short of normal standards. To address these limitations, we designed a novel peripheral capsular linear incision technique. Compared with small curvilinear capsulorhexis, postoperative observations revealed significant differences in capsular opening healing, as well as in the weight, transparency, and fiber arrangement of regenerated lenses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eA total of forty healthy 4-week-old New Zealand white rabbits (average weight: ~0.6 kg) were obtained from the Xuzhou Medical University Animal Experiment Center.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrouping and surgical procedures\u003c/h3\u003e\n\u003cp\u003eThe 40 rabbits were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;20 each): Group A (peripheral capsular linear incision) and Group B (small curvilinear capsulorhexis). In each rabbit, one eye was randomly selected for surgery. Pupillary dilation was achieved with three administrations of compound tropicamide eye drops (0.5%, Shenyang Sinqi). Anesthesia was induced via subcutaneous injection of dexmedetomidine hydrochloride (0.02 mg/kg) combined with a mixture of tiletamine hydrochloride and zolazepam hydrochloride (15 mg/kg). Lens contents were aspirated with the Legacy 20000 phacoemulsification system (Alcon, Fort Worth, USA) as follows: A 2.4-mm corneal tunnel incision was made at the 12 o\u0026rsquo;clock position, followed by capsulotomy and capsulorhexis near the lens equator. Group A received a 1.5-mm linear incision, whereas Group B underwent a 1.5-mm capsulorhexis. Incision length or capsular opening diameter was confirmed using a 1-mm scale calibration needle (Xinkeling, Shanghai, China) before and after capsulorhexis. The lens nucleus and residual cortex were aspirated using an irrigation/aspiration (I/A) system. The corneal incision was closed with interrupted 10\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon sutures. All surgeries were performed by the same senior surgeon.\u003c/p\u003e\n\u003ch3\u003ePostoperative examination\u003c/h3\u003e\n\u003cp\u003ePostoperative care included TobraDex eye drops (tobramycin 0.3%, dexamethasone 0.1%; Alcon, USA) four times daily, TobraDex eye ointment (tobramycin 0.3%, dexamethasone 0.1%; Alcon, USA), and atropine sulfate eye ointment (1%, Shenyang Sinqi, China) once daily, continued for two weeks. Anterior segment slit-lamp images were captured with an anterior segment camera (Chongqing Kanghua Ruiyu, China) to assess corneal, anterior capsule, and lens regeneration of both groups on the day of surgery and at 4, 8, and 12 weeks postoperatively. Lens thickness was measured using optical coherence tomography (TowardPi, Tianjin, China). Lens morphology, weight, and fundus status were also evaluated.\u003c/p\u003e\n\u003ch3\u003eHealing of the anterior capsule opening\u003c/h3\u003e\n\u003cp\u003eThe anterior capsule opening area (ACOA), healing time, and scar formation were observed and compared between the two groups.\u003c/p\u003e\n\u003ch3\u003eSample collection and histological examination\u003c/h3\u003e\n\u003cp\u003eAt designated time points, rabbits were euthanized by intravenous injection of 10 ml of air into the ear vein. The regenerated and contralateral lenses were carefully separated. Regenerated lenses were immediately immersed in phosphate-buffered saline (PBS, 37\u0026deg;C) and photographed under a microscope (Stemi DV4, Carl Zeiss, Oberkochen, Germany). Lens weight was measured three times, and the mean value was used to calculate the weight percentage (WP): WP\u0026thinsp;=\u0026thinsp;weight of regenerated lens\u0026times;100​/weight of contralateral intact lens. Lenses were then fixed in 10% neutral buffered formalin for 2 hours, dehydrated in graded ethanol, cleared in xylene, embedded in paraffin, sectioned at 4 \u0026micro;m (MiDiII, 3DHISTECH, Hungary), and stained with hematoxylin and eosin (H\u0026amp;E). Sections were examined under a microscope (Eclipse 50i, Nikon, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using SPSS software (version 17.0, SPSS Inc., Chicago, USA). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Paired t-tests were used to compare regenerated lens thickness and weight percentage between the two groups. All tests were two-tailed, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMorphology of regenerated lenses\u003c/h2\u003e \u003cp\u003eSlit-lamp examination was performed to assess the morphology of regenerated lenses. At 12 weeks postoperatively, the capsules in Group A were completely filled with regenerated lenses. Except for mild central opacity, the lenses were generally transparent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, anterior view). Fundus examination revealed clear visualization of the optic disc, retina, and choroidal vessels, indicating no impairment of retinal function. In contrast, Group B showed incomplete lens regeneration that did not fully fill the capsules, with localized defects in the equatorial region and reduced thickness compared with Group A. Central opacity was also observed, and the optic disc, retina, and choroidal vessels could not be clearly visualized during fundus examination. In some cases, anterior and posterior capsule adhesions resulted in an \u0026ldquo;8-shaped\u0026rdquo; regenerated lens. Overall, Group A demonstrated superior regeneration, with a larger lens size, higher transparency, and intact morphology that is increasingly aligned with the normal lens. Notably, all 20 eyes in Group A exhibited well-healed capsular openings without scar adhesions, whereas 13 of 20 eyes in Group B showed anterior\u0026ndash;posterior capsule adhesions\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChanges in lens weight during regeneration\u003c/h2\u003e \u003cp\u003eLens regeneration was quantitatively assessed by calculating the WP, defined as the ratio of regenerated lens weight to that of the contralateral intact lens. WP gradually increased in both groups over time (Figure). At 4 weeks, WP was significantly higher in Group A (24.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17%) than in Group B (16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32%). At 8 weeks, WP values were 76.48\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61% in Group A and 72.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67% in Group B. At 12 weeks, WP in Group A reached 90.57\u0026thinsp;\u0026plusmn;\u0026thinsp;6.37%, approaching the normal contralateral lens weight, while Group B reached only 83.81\u0026thinsp;\u0026plusmn;\u0026thinsp;6.07%. These findings suggest that the peripheral linear incision preserved more lens epithelial cells, thereby enhancing regeneration efficiency compared with the small curvilinear capsulorhexis, which may enhance endothelial cell loss.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe form and thickness of the regenerated lens\u003c/h2\u003e \u003cp\u003eLens thickness was measured using OCT. Group A consistently exhibited greater lens thickness compared with Group B at all time points: 4 weeks: 2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mm vs. 2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); 8 weeks: 4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mm vs. 4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); 12 weeks: 5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mm vs. 5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By week 12, the regenerated lens thickness in Group A closely approximated that of the contralateral normal lens (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Both groups exhibited rapid growth between weeks 4 and 8, followed by slower growth thereafter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eACOA contact and healing\u003c/h2\u003e \u003cp\u003eSlit-lamp imaging was used to evaluate capsular healing between Groups A and B after pupillary dilation. In Group A, the linear incision exhibited nearly complete healing within 1 week and full closure by 2 weeks postoperatively (Figure). Scars were minimal, with fine punctate wrinkles and no adhesions in the capsular cavity. By week 8, the scars had stabilized and further contracted. In Group B, the capsular opening showed near-complete healing by 2 weeks, forming a linear scar and a closed cavity. However, several cases exhibited anterior\u0026ndash;posterior capsule adhesions. Scar morphology stabilized by week 8. Collectively, linear incisions promoted shorter healing times, smaller scars, and reduced epithelial cell loss compared with circular incisions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistological characteristics of regenerated lenses\u003c/h2\u003e \u003cp\u003eHistological examination at 12 weeks revealed that in Group A, most LECs had differentiated into lens fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Near the equatorial region, LECs retained forward-moving nuclei, forming a characteristic crescent-shaped \u0026ldquo;lens bow\u0026rdquo; structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-a, d, black arrows). A single layer of LECs was present beneath the anterior capsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-b), while the intermediate nuclear layer was absent, and no LECs were observed under the posterior capsule (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-c). In certain regions, multilayered fibroblast-like LECs were detected, resembling developmental processes of the normal lens. In Group B, the regenerated lenses also displayed a lens bow structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-a, d, black arrows), but disorganized lens fiber arrangements were more frequent. Taken together, these findings confirm at the cellular level that the peripheral linear incision preserved more functional LECs, thereby promoting orderly fiber differentiation, reducing fibroblast-like transformation, and ultimately improving the growth rate and transparency of regenerated lenses, making them more comparable to normal lenses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIntraoperative and postoperative complications\u003c/h2\u003e \u003cp\u003eAll 40 rabbits (40 eyes) successfully underwent lens extraction, and postoperative intraocular pressure (IOP) remained within the normal range in all groups. On the first postoperative day, fibrinous exudation was observed in one eye of Group B, and two eyes showed mild corneal edema. In Group A, only one eye exhibited mild corneal edema. All symptoms resolved spontaneously within one week. At 8 weeks postoperatively, posterior synechiae (IPS) developed in 2 eyes in Group B, whereas no such complication occurred in Group A. By 12 weeks, the overall complication rate was 5% in Group A and 20% in Group B, representing a statistically significant difference between the groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrent clinical management of cataracts primarily involves removal of the opaque lens and implantation of an IOL. However, IOL implantation in infants under 2 years of age carries a high risk of complications, underscoring the urgent need for alternative treatment strategies.\u003c/p\u003e \u003cp\u003eIn recent years, ophthalmologists have reported cases in which children with congenital cataracts developed \u0026ldquo;donut-shaped\u0026rdquo; regenerated lenses following capsulotomy and cortical aspiration. Although these lenses exhibited scarring and opacity at the capsule opening, the capsular sac itself remained largely transparent. Such postoperative proliferation suggests that the human lens retains regenerative potential. Notably, in 2016, Liu et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] introduced a minimally invasive technique employing a 1.5-mm capsulorhexis incision displaced from the central visual axis to the periphery, thereby improving postoperative transparency. While this method achieved partial in situ lens regeneration in infants under 2 years of age, several limitations remain. First, the regenerated lens often reached only approximately 50% of the thickness of a normal lens, and the biconvex shape was disrupted by capsular scarring. Adhesions between the anterior and posterior capsules, as well as anterior subcapsular epithelial cell loss, frequently resulted in localized opacities. Second, even small curvilinear capsulorhexis caused loss of LECs, thereby diminishing the regenerative capacity of the lens.\u003c/p\u003e \u003cp\u003eTo address these limitations, we proposed a new strategy: removal of the lens cortex while preserving capsular integrity through a peripheral linear incision. This approach aims to minimize capsular defects, preserve a larger pool of viable LECs, and foster in situ lens regeneration. Importantly, linear incisions appear to reduce the release of inflammatory cytokines such as TGF-β, thereby lowering the risk of fibroblast-like differentiation of LECs. The healing of the ACOA is a critical determinant of successful regeneration. Prior studies have shown that smaller capsulorhexis tears heal more rapidly and yield more transparent regenerated lenses [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], whereas larger tears heal slowly and incompletely. Tan et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] demonstrated in animal experiments that capsulorhexis size is a key factor influencing transparency and integrity, with smaller incisions favoring ACOA healing. The advantages of the linear incision in our study include: (i) rapid healing with minimal loss of capsule and LECs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], (ii) effective sealing of the capsule opening, creating a microenvironment conducive to regeneration, and (iii) maximal preservation of equatorial LECs. Our findings confirm these benefits. In Group A, the linear incision stretched into an elliptical opening under intraocular pressure but realigned after cortical aspiration, resulting in nearly perfect closure with minimal tissue loss, resulting in rapid healing. However, in Group B, although complete healing of the capsule was ultimately achieved, there was still a loss of capsule tissue and lens epithelial cells compared with conventional ACOA. This led to slightly slower healing of the ACOA and the formation of larger scars. Moreover, fibrin proliferation around the torn capsule edges impeded the migration of new lens fibers from the periphery toward the center. Second, Gwon et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] demonstrated that sealing the ACOA with collagen patches and inflating the posterior capsule for separation can accelerate lens regeneration and improve lens clarity. Similarly, the linear incision technique adopted in this study enabled complete closure of the capsule opening, thereby establishing a sealed microenvironment favorable for lens regeneration. This closure also restricted the entry of inflammatory cytokines known to interfere with regeneration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, a linear incision design allows maximal preservation of LECs beneath the anterior capsule. Previous studies have shown that LECs located in the equatorial zone function as stem cells with the capacity to proliferate and differentiate into new lens fibers [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, the greater the number of residual LECs retained after surgery, the higher the regenerative potential of the lens. Excessive loss of LECs during previous capsulorhexis procedures may be one reason why the human lens often fails to regenerate. The reason for selecting an incision length of 1.5 mm can be obtained from the results of the previous studies. In our preliminary experiments, larger incisions (\u0026gt;\u0026thinsp;1.5 mm) were technically easier to perform but resulted in greater surgical trauma, whereas smaller incisions (\u0026lt;\u0026thinsp;1.5 mm) increased the risk of capsule tearing during lens aspiration. With future advances in microsurgical instruments, smaller incisions may become feasible, and we are actively exploring this possibility.\u003c/p\u003e \u003cp\u003eAlthough earlier studies reported that small curvilinear capsulorhexis can produce regenerated lenses that are morphologically and functionally more similar to normal lenses, our findings revealed significant differences between the two approaches. Specifically, lens thickness in Group A was consistently greater than in Group B at 4, 8, and 12 weeks. A plausible explanation is that Group A, which underwent a linear 1.5 mm incision, retained capsule integrity and avoided defects, thereby supporting the regeneration of lenses with morphology closer to that of normal lenses. By contrast, Group B developed incision adhesions that disrupted capsule continuity, resulting in irregular lens shapes and compromised integrity. By limiting the size of the capsular incision and minimizing the removal of the anterior capsule, our method reduces damage to lens epithelial cells while preserving most of the lens epithelium. This creates an optimal environment for lens regeneration. In this study, regenerated lenses in Group A demonstrated superior integrity, transparency, capsule healing, capsule size, and reduced scar hyperplasia compared with those in Group B. These findings suggest that the proposed peripheral linear capsulotomy confers advantages over circular micro-tear capsulotomy and may offer new insights into strategies for in situ lens regeneration.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the relatively small sample size limited the statistical power of our findings. Second, the follow-up period was restricted to 12 weeks, which precluded long-term evaluation of the functional and optical properties of the regenerated lenses. Although the regenerated lenses were morphologically complete and transparent in the early stages, by 12 weeks, we observed one case of mild sublenticular opacity. This may have been caused by residual viscoelastic material or inflammatory responses that disrupted the proliferation and migration of lens epithelial cells. Further investigation is warranted to clarify these mechanisms. In addition, even though the regenerated lenses in Group A closely resembled normal lenses, a small central deficit was noted during epithelial cell migration. Whether this phenomenon is attributable to insufficient numbers of lens epithelial cells or to intrinsic limitations in their regenerative capacity remains unclear.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study demonstrates that a peripheral capsular linear incision can promote favorable conditions for lens regeneration by preserving lens epithelial cells and enabling rapid closure of the capsular opening. Compared with the circular micro-incision technique, this approach resulted in regenerated lenses with superior integrity, transparency, and morphology, while also reducing scar formation and accelerating healing. These findings highlight the potential of peripheral linear capsulotomy as a promising surgical strategy for in situ lens regeneration and provide a foundation for developing improved treatments for children with congenital cataracts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMing Zhang contributed to the entire experimental work of the participating articles and the writing of the articles;Tengyu Xv;Menghan Wang;Wenwen Hou;Yan Wang;Liming Cai;Wei Wang,all of them participated in the experimental work and article image creation.Suchang Wang was responsible for the design of the experiment and the final review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFounding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Xuzhou Science and Technology Plan Fund (Grant No. KC23171) and the Development Fund of the Affiliated Hospital of Xuzhou Medical University (Grant No. XYFM202319).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Animal Ethics Committee of Xuzhou Medical University (Ethics Number: 202404T024) and adhered to the guidelines of the Association for Research in Vision and Ophthalmology on the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTan X, et al. Impact of cataract screening integrated into establishment of resident health record on surgical output in a rural area of south China. Ann Transl Med. 2020;8(19):1222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhairallah M, et al. Number of People Blind or Visually Impaired by Cataract Worldwide and in World Regions, 1990 to 2010. Invest Ophthalmol Vis Sci. 2015;56(11):6762\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan X, et al. Capsular Outcomes After Pediatric Cataract Surgery Without Intraocular Lens Implantation: Qualitative Classification and Quantitative Measurement. Med (Baltim). 2016;95(10):e2993.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolebo AL, Cumberland P, Rahi JS. 5-year outcomes after primary intraocular lens implantation in children aged 2 years or younger with congenital or infantile cataract: findings from the IoLunder2 prospective inception cohort study. Lancet Child Adolesc Health. 2018;2(12):863\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMandal AK, Gollakota R. Soemmering's Ring Ophthalmol. 2017;124(7):1064.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo L, et al. In-the-bag intraocular lens placement via secondary capsulorhexis with radiofrequency diathermy in pediatric aphakic eyes. PLoS ONE. 2013;8(4):e62381.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharjee H, Deshmukh S. Soemmering's ring. Indian J Ophthalmol. 2017;65(12):1489.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin H, et al. Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2016;531(7594):323\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin H, et al. Capsular Outcomes Differ with Capsulorhexis Sizes after Pediatric Cataract Surgery: A Randomized Controlled Trial. Sci Rep. 2015;5:16227.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin H, et al. Corrigendum: Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2017;541(7638):558.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin H, et al. Lens regeneration using endogenous stem cells with gain of visual function. Nature. 2016;531(7594):323\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou M, et al. A hierarchy of proliferative cells exists in mouse lens epithelium: implications for lens maintenance. Invest Ophthalmol Vis Sci. 2006;47(7):2997\u0026ndash;3003.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGwon A, Gruber LJ, Mantras C. Restoring lens capsule integrity enhances lens regeneration in New Zealand albino rabbits and cats. J Cataract Refract Surg. 1993;19(6):735\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFisus AD, Findl O. Capsular fibrosis: a review of prevention methods and management. Eye (Lond). 2020;34(2):256\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Finkel T. Stem cell aging: what bleach can teach. Nat Med. 2006;12(4):383\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeebe DC, Holekamp NM, Shui YB. Oxidative damage and the prevention of age-related cataracts. Ophthalmic Res. 2010;44(3):155\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith AN, et al. Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis. Development. 2009;136(17):2977\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\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":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lens regeneration, Cataract surgery, peripheral capsule linear incision, small curvilinear capsulorhexis, rabbit model","lastPublishedDoi":"10.21203/rs.3.rs-8087513/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8087513/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLens regeneration is regarded as a promising strategy for restoring vision in cataract patients after surgery. However, conventional small curvilinear capsulorhexis often results in the loss of anterior capsular lens epithelial cells, thereby compromising the integrity and transparency of the regenerated lens. Building on the concept of small capsulorhexis, this study introduces a 1.5-mm linear incision in the peripheral capsule to maximally preserve epithelial cells and enhance regenerative outcomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eForty 4-week-old New Zealand white rabbits were randomly assigned to two groups: the peripheral capsular linear incision group (Group A) and the group (Group B), with 20 rabbits in each group. Lens removal was performed via irrigation/aspiration (I/A). Postoperatively, regenerated lenses were evaluated for morphology, size, weight, transparency, and capsular healing, followed by histological analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSuccessful lens regeneration was observed in both groups. Group A demonstrated superior regenerative outcomes, characterized by intact lens morphology, higher transparency, and significantly greater lens weight and thickness compared with Group B (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Histological analysis showed that epithelial cells in Group A differentiated into well-organized lens fibers with a more orderly arrangement, shorter capsular healing time, smaller scars, and smoother capsular surfaces than those in Group B.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCompared with small curvilinear capsulorhexis, peripheral capsular linear incision offers clear advantages in promoting morphological and functional lens regeneration, as well as capsular healing, in rabbit eyes. These findings provide novel strategies for developing surgical strategies for lens regeneration in children with congenital cataracts.\u003c/p\u003e","manuscriptTitle":"A Comparative Study of Peripheral Capsular Linear Incision and Circular Micro-Tear Capsulotomy in the In Situ Regeneration of Rabbit Eyes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 10:02:06","doi":"10.21203/rs.3.rs-8087513/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-21T04:52:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-12T06:18:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14592651589554805550800944814252176848","date":"2026-01-05T12:54:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-04T14:02:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229242542932731866879923670123031334784","date":"2026-01-01T22:56:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T09:50:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226810496850037004268665101626884312957","date":"2025-12-26T09:34:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-24T08:25:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T07:06:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-02T06:50:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T14:07:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ophthalmology","date":"2025-12-01T13:31:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"boph","sideBox":"Learn more about [BMC Ophthalmology](http://bmcophthalmol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/boph","title":"BMC Ophthalmology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f47133c2-cfe6-47ef-95c3-02295856e2d1","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-10T04:09:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 10:02:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8087513","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8087513","identity":"rs-8087513","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}