Feb 10, 2025
Long-term alterations of collagen reconstruction and basement membrane regeneration after corneal full-thickness penetrating injury in rabbits
- Jingjing Chen1,
- Yuqing Luo1,
- Luting Xie1,
- Na Meng1,
- Sumei Li1,
- Shifang Xiao1,
- Xia Li1
- 1the First Affiliated Hospital of Guangxi Medical University
- Jingjing Chen: The first author
- Yuqing Luo: Co-first author
- Xia Li: Corresponding author
Protocol Citation: Jingjing Chen, Yuqing Luo, Luting Xie, Na Meng, Sumei Li, Shifang Xiao, Xia Li 2025. Long-term alterations of collagen reconstruction and basement membrane regeneration after corneal full-thickness penetrating injury in rabbits. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr9weovmk/v1
Manuscript citation:
1. Connon CJ, Meek KM. Organization of corneal collagen fibrils during the healing of trephined wounds in rabbits. Wound Repair and Regeneration. 2003;11(1):71-78. doi: 10.1046/j.1524-475X.2003.11110.x.
2. Fantes FE, Hanna KD, Waring GO III, Pouliquen Y, Thompson KP, Savoldelli M. Wound healing after excimer laser keratomileusis (photorefractive keratectomy) in monkeys. Archives of Ophthalmology. 1990;108(5):665-675. doi: 10.1001/archopht.1990.01070070051034.
3. Connon CJ, Tandon A, Sharma A, Rodier JT, Klibanov AM, Rieger FG, et al. BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo. PLoS ONE. 2013;8(6):e66434. doi: 10.1371/journal.pone.0066434.
4.Yam GHF, Riau AK, Funderburgh ML, Mehta JS, Jhanji V. Keratocyte biology. Experimental Eye Research. 2020. 196 DOI 10.1016/j.exer.2020.108062
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 08, 2025
Last Modified: February 10, 2025
Protocol Integer ID: 119834
Keywords: collagen reconstruction; anterior stroma; posterior stroma; corneal opacity; corneal wound healing; basement membrane regeneration.
Abstract
Purpose: To investigate the long-term alterations of collagen reconstruction and basement membrane (BM) regeneration after corneal full-thickness penetrating injury in rabbits.
Methods: The corneal full-thickness penetrating injury model was established in the left eye of New Zealand White rabbits using a 2.0 mm trephine. All corneas were evaluated using slit-lamp photography, hematoxylin and eosin staining, mmunofluorescent staining for collagen types I and III (Col I, III), and transmission electron microscopy for collagen fibers and basement membrane.
Results: Between 3 days and 3 weeks, Col I and III expression were documented, exhibiting a largely disorganized distribution throughout the stromal thickness. At 3 weeks, the epithelial basement membrane (EBM) partially regenerated. From 3 weeks to 2 months, Col III was undetectable in the anterior stroma but present in the posterior stroma; Col I was disorganized in the
posterior stroma. At 2 months, Descemet's membrane (DM) exhibited incomplete regeneration. From 3 to 4 months, Col I was disorganized in only a small part of the posterior stroma; Col III persisted in the posterior stroma; the EBM fully regenerated while DM exhibited incomplete regeneration.
Conclusions: Following full-thickness corneal injury, persistent fibrosis within the posterior stroma appears to be primarily responsible for the persistence of corneal scarring. Notably, regeneration of the EBM coincides with remodeling of the anterior stroma, whereas incomplete regeneration of DM is associated with posterior stromal fibrosis.
Attachments
Image Attribution
Sample
Guidelines
Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research
Materials
sterile 2.0 mm trephine (Storz, USA)
0.5% compound tropicamide eye drops (SHENYANG XINGQI PHARMACEUTICAL CO, LTD, China)
1% pentobarbital sodium (AG, Germany)
0.5% proparcaine hydrochloride ophthalmic anesthetic solution (Alcon-COUVREUR, USA)
0.3% Levofloxacin gel
0.5% Levofloxacin eye drops (YABANG Pharma, China)
4% paraformaldehyde (Solarbio, China)
3% EDTA，0.1 M phosphate-buffered saline (PBS) and permeabilized with 0.3% Triton X-100
anti-COL1A1 antibody (NB600-450, Mouse anti-Collagen I alpha 1; Novus)
anti-COL3A1 antibody (sc-271249, Mouse monoclonal antibody, 1:100, Santa
Cruz)
1% bovine serum albumin (BSA)
secondary antibody (2284614, Alexa Fluor 488 goat anti-mouse antibody; Thermo Fisher Scientific)
secondary antibody (4408S, Alexa Fluor 488 goat anti-mouse antibody; Cell Signaling Technology)、
4’,6-diamidino-2-phenylindole (DAPI) (C0065-10ml, Solarbio, China)
Leica TCS SP8 microscope (Leica, Wetzlar, Germany) equipped with a Fluoview FV1000 confocal system (Olympus, Japan).
Image analysis was performed using ImagePro software (Leica)
2.5% glutaraldehyde and 4% paraformaldehyde
1% osmium tetroxide
graded ethanol and acetone series
ultramicrotome (Leica EM UC7, Germany)
transmission electron microscope (Hitachi H-7650, Japan)
scanning electron microscopy (TESCAN VEGA3, Czech Republic)
Safety warnings
All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research (protocol number: 2024E50301), approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University.
Ethics statement
All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research (protocol number: 2024E50301), approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University.
Material and methods
Material and methods
Forty-eight New Zealand White rabbits were randomly assigned to eight groups (n = 6/group) representing different post-operative time points. The left eyes of each rabbit received a penetrating injury using a sterile 2.0 mm trephine (Storz, USA) to create the model, while the right eyes served as controls. The injury model creation followed established protocols. Briefly, each rabbit underwent left eye trephination at the central cornea using the aforementioned sterile trephine. Prior to the procedure, the experimental eyes were dilated with 0.5% compound tropicamide eye drops (SHENYANG XINGQI PHARMACEUTICAL CO, LTD, China) to prevent iris incarceration in the trephined area during the operation. Following pupil dilation in the rabbits, a precise weighing procedure was conducted. General anesthesia was subsequently administered via intravenous infusion of 1% pentobarbital sodium (at a dose of 3ml/kg) through the marginal ear vein. A 0.5% proparcaine hydrochloride ophthalmic anesthetic solution (Alcon-COUVREUR, USA) was then applied to the ocular surface of the operative eye. After confirming the effectiveness of both general and local anesthesia, a pediatric eyelid speculum was carefully positioned over the surgical eye to fully expose the rabbit's cornea. The eye was stabilized using forceps, and a sterile trephine with a 2.0 mm diameter was precisely aligned at the center of the cornea. Using the rotary trephine, the entire layer of corneal tissue was meticulously excised, revealing the aqueous humor. Next, 0.3% Levofloxacin gel was applied to the corneal wound to prevent anterior chamber collapse and iris adhesion. Notably, the rabbits regained consciousness within 30 minutes post-procedure. Within 15 minutes, fibrin clots formed on the corneal wound, effectively sealing it and re-establishing the anterior chamber. The rabbit corneal perforation injury model was successfully established. Postoperatively, one drop of 0.3% Levofloxacin gel was administered every 8 hours for the first three days, followed by 0.5% Levofloxacin eye drops (YABANG Pharma, China) every 8 hours for two weeks. Mitomycin C and topical corticosteroids were not utilized in this study. Any instances of corneal ulceration or neovascularization were excluded from the study.
Standardized broad-beam illumination corneal slit-lamp photographs were obtained at various time points: 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, and 4 months post-surgery. Euthanasia was performed by intravenous injection of pentobarbital sodium 100 mg/kg (AG, Germany). Corneoscleral rims from both wounded and unwounded eyes were excised using forceps and scissors, ensuring no contact with the cornea itself, for subsequent histopathology and corneal ultrastructural examination.
Slit-Lamp Microscopic Examination
At each time point following corneal injury, all rabbit eyes were evaluated using slit-lamp microscopy. The corneal epithelial defect area was measured using ImageJ software following the instillation of 1% sodium fluorescein solution and photography with cobalt blue light. Corneal opacity was graded on a modified Fantes' scale. (0 = no opacity, 4 = complete opacity) as previously described. Briefly, grade 0.5 indicates slight opacity with visible pupillary margin and iris details, grade 1 indicates observable opacity with both details visible, grade 2 signifies partial pupillary margin visibility, and grade 3 represents moderate opacity without a visible pupillary margin.
Histopathologic Examination
Histopathological changes in injured and control corneas were assessed using hematoxylin and eosin (H&E) staining. At each designated time point, rabbits were euthanized under deep anesthesia, and eyes were immediately enucleated. Following fixation in 4% paraformaldehyde (Solarbio, China) for 48 hours, the eyes were paraffin-embedded, sectioned into 4-μm slices, and dewaxed with xylene. These sections were then subjected to H&E staining for histological analysis.
Immunofluorescence Analysis
Immunofluorescence (IF) staining was employed to assess the expression and localization of type I collagen and type III collagen, markers for ECM protein deposition, respectively. The preparation of the wax blocks and the slicing procedures was performed in strict accordance with the previously described methods. Paraffin-embedded corneal sections were subjected to dewaxing in xylene followed by rehydration through a graded ethanol series. After rehydration, the slides were immersed in 3% EDTA and heated in a microwave oven for antigen retrieval. Following natural cooling, the slides were washed three times with 0.1 M phosphate-buffered saline (PBS) and permeabilized with 0.3% Triton X-100 at room temperature for 10 minutes. To minimize non-specific binding, the slides were blocked with 30% normal goat serum in PBS for 1 hour. The slides were then incubated overnight at 4°C with anti-COL1A1 antibody (NB600-450, Mouse anti-Collagen I alpha 1; Novus) at 1:100 dilution in 1% BSA, anti-COL3A1 antibody (sc-271249, Mouse monoclonal antibody, 1:100, Santa Cruz) at 1:30 dilution in 1% bovine serum albumin (BSA). Slides were then incubated for 30 minutes with the secondary antibody (2284614, Alexa Fluor 488 goat anti-mouse antibody; Thermo Fisher Scientific) at dilution of 1:200 (COL1A1), and with the secondary antibody (4408S, Alexa Fluor 488 goat anti-mouse antibody; Cell Signaling Technology) at dilution of 1:50 (COL3A1). Then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (C0065-10ml, Solarbio, China). Tissue sections were visualized using a Leica TCS SP8 microscope (Leica, Wetzlar, Germany) equipped with a Fluoview FV1000 confocal system (Olympus, Japan). Image analysis was performed using ImagePro software (Leica).
Transmission Electron Microscopy (TEM)
Corneal tissue processing followed previously established protocols.[32] Briefly, specimens were fixed in a 2.5% glutaraldehyde and 4% paraformaldehyde mixture for 24 hours, followed by incubation in 1% osmium tetroxide for 2 hours at 4°C. Dehydration was achieved through a graded ethanol and acetone series. Subsequently, the central corneal block was embedded in epoxy resin and sectioned using an ultramicrotome (Leica EM UC7, Germany) to 1 μm thickness. Toluidine blue staining facilitated the identification of the corneal injury area. Ultrastructural changes in the corneal tissue during the repair process were observed under a transmission electron microscope (Hitachi H-7650, Japan). Additionally, scanning electron microscopy (TESCAN VEGA3, Czech Republic) was employed to capture micrographs of the tissue.
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Acknowledgements
We are grateful to Professor Xia Li for her invaluable guidance in article writing. We also extend our appreciation to
Yuqing Luo and Jinling Wu for their technical assistance, Luxing Xu for his transmission electron microscopy expertise, Na Meng for her assistance with data organization, and Luting Xie for her immunofluorescence technical support. We acknowledge the co-equal contributions of Jingjing Chen and Yuqing Luo to this article.