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The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360° thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.
Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.
Medical literature understands glaucoma as a heterogeneous group of optic neuropathies characterized by progressive degeneration of retinal ganglion cells (RGCs), dendrites, soma, and axons, resulting in structural cupping (excavation) of the optic disc and functional deterioration of the optic nerve, leading to amaurosis in uncontrolled cases by interrupting the transmission of visual information from the eye to the brain1. Glaucoma is currently the most common cause of irreversible blindness worldwide, predicted to reach approximately 111.8 million people in 20402, thus deeply affecting patients' quality of life (QoL) and leading to significant socioeconomic concerns3.
Elevated intraocular pressure (IOP) is one of the most important and the only modifiable risk factor for the development and progression of glaucoma. Among the multiple types of glaucoma, all, except for normal tension glaucoma (NTG), are associated with elevated IOP at some time in the clinical history of the disease. Despite remarkable clinical and surgical advances to target IOP and slow down or stop disease progression, patients still lose sight due to glaucoma4,5. Therefore, a thorough understanding of the complex and multifactorial pathophysiology of this disease is imperative for the development of more effective treatments, especially to provide neuroprotection to RGCs.
Among a variety of experimental approaches for the understanding of disease mechanisms, animal models based on ocular hypertension (OHT) most closely resemble human glaucoma. Rodent models are particularly useful as they are low-cost, are easy to handle, can be genetically manipulated, have a short lifespan, and present ocular anatomical and physiological features comparable to humans, such as aqueous humor production and drainage6,7,8,9,10,11,12,13. Currently used models include sclerosis of the trabecular meshwork following injection of hypertonic saline into episcleral veins14, intracameral injection of microbeads15 or viscoelastic substances16, cauterization of vortex veins17, photocoagulation of the trabecular meshwork with argon laser18, circumlimbal suture19, and use of a transgenic model of age-related OHT (DBA/2J mice)8. However, invasiveness, post-operative opacification of the cornea, anterior segment disruption, extensive learning curves, expensive equipment, and highly variable postoperative IOPs, are among few of the reported pitfalls associated with the current models, making the development of an alternative model of OHT a demand to overcome these problems20,21,22.
The present protocol formalizes a novel surgical procedure to induce OHT as a proxy to glaucoma, based on limbal plexus cauterization (LPC) in rodents23. This is an easy, reproducible, accessible, and non-invasive model that provides high efficiency and low variability of IOP elevation, associated with a uniquely high rate of full clinical recovery, therefore providing in vivo functional evaluation in a reduced number of animals used in each experiment. The surgery technique induces subacute OHT with a gradual return to baseline levels in a few days, which models the hypertensive attack seen in acute angle-closure glaucoma. Moreover, the IOP recovery in the model is followed by continuous glaucomatous neurodegeneration, which is useful for future mechanistic studies of the secondary degeneration of RGCs, which occurs in several cases of human glaucoma despite adequate control of IOP.
All procedures were performed in compliance with the Statement for the Use of Animals in Ophthalmic and Visual Research from the Association for Research in Vision and Ophthalmology (ARVO) and approved by the Ethics Committee on the Use of Animals in Scientific Experimentation from the Health Sciences Center, Federal University of Rio de Janeiro (protocol 083/17). In the present work, Lister Hooded rats of both genders were used, aged 2-3 months and weighing 180-320 g. However, the procedure can be adapted in different rat strains of various age ranges.
1. Ocular hypertension surgery and clinical follow-up
2. Optomotor response (OMR) analysis
NOTE: For this procedure, a specific system was used25.
3. Recording of pattern-electroretinogram (PERG)
NOTE: The electroretinogram was recorded using a specific system for signal processing and related software for storage and analysis of the waveforms.
4. Quantification of retinal ganglion cells somas
NOTE: The following procedure is for quantification of RGC somas, based on immunohistochemical staining of retinal flat-mounts with an antibody against the brain-specific homeobox/POU domain protein 3A (Brn3a).
5. Examination of the optic nerve
The quantitative variables are expressed as mean ± standard error of the mean (SEM). Except for the comparison of IOP dynamics between OHT and control groups (Figure 1F), statistical analysis was performed using two-way ANOVA followed by Sidak's multiple comparisons test. A p-value < 0.05 was considered statistically significant.
Figure 1 illustrates surgical steps of the full-circle limbal plexus cauterization (LPC) mode...
Limbal plexus cauterization (LPC) is a novel post-trabecular model with the advantage that it targets easily accessible vascular structures not requiring conjunctival or tenon dissection17,28. Differently from the vortex veins cauterization model, a renowned OHT model based on the surgical impairment to choroid venous drainage, venous congestion is not expected to influence IOP rise in the LPC model, as limbal veins are situated upstream in aqueous humor outflow....
The authors have nothing to disclose.
We acknowledge our laboratory technicians José; Nilson dos Santos, Daianne Mandarino Torres, José Francisco Tibúrcio, Gildo Brito de Souza, and Luciano Cavalcante Ferreira. This research was funded by FAPERJ, CNPq, and CAPES.
Name | Company | Catalog Number | Comments |
Acetone | Isofar | 201 | Used for electron microscopy tissue preparation (step 5) |
Active electrode for electroretinography | Hansol Medical Co | - | Stainless steel needle 0.25 mm × 15 mm |
Anestalcon | Novartis Biociências S/A | MS-1.0068.1087 | Proxymetacaine hydrochloride 0.5% |
Calcium chloride | Vetec | 560 | Used for electron microscopy tissue preparation (step 5) |
Cautery Low Temp Fine Tip 10/bx | Bovie Medical Corporation | AA00 | Low-temperature ophthalmic cautery |
Cetamin | Syntec do Brasil Ltda | 000200-3-000003 | Ketamine hydrochloride 10% |
DAKO | Dako North America | S3023 | Antifade mounting medium |
DAPI | Thermo Fisher Scientific | 28718-90-3 | diamidino-2-phenylindole; blue fluorescent nuclear counterstain; emission at 452±3 nm |
Ecofilm | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0298.0487 | Carmellose sodium 0.5% |
EPON Resin | Polysciences, Inc. | - | Epoxy resin used for electron microscopy, composed of a mixture of four reagents: Poly/Bed 812 Resin (CAT#08791); DDSA - Dodecenylsuccinic Anhydride (CAT#00563); NMA - Nadic Methyl Anhydride (CAT#00886); DMP-30 - 2,4,6-tris(dimethylaminomethyl)phenol (CAT#00553) |
Glutaraldehyde | Electron Microscopy Sciences | 16110 | Used for electron microscopy tissue preparation (step 5) |
Hyabak | União Química Farmacêutica Nacional S/A | MS-8042140002 | Sodium hyaluronate 0.15% |
Icare Tonolab | Icare Finland Oy | TV02 (model number) | Rebound handheld tonometer |
IgG donkey anti-mouse antibody + Alexa Fluor 555 | Thermo Fisher Scientific | A31570 | Secondary antibody solution |
LCD monitor 23 inches | Samsung Electronics Co. Ltd. | S23B550 | Model LS23B550, for electroretinogram recording |
LSM 510 Meta | Carl Zeiss | - | Confocal epifluorescence microscope |
Maxiflox | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0298.0489 | Ciprofloxacin 3.5 mg/g |
MEB-9400K | Nihon Kohden Corporation | - | System for electroretinogram recording |
monoclonal IgG1 mouse anti-Brn3a | MilliporeSigma | MAB-1585 | Brn3a primary antibody solution |
Neuropack Manager v08.33 | Nihon Kohden Corporation | - | Software for electroretinogram signal processing |
Optomotry | CerebralMechanics | - | System for optomotor response analysis |
Osmium tetroxide | Electron Microscopy Sciences | 19100 | Used for electron microscopy tissue preparation (step 5) |
Potassium ferrocyanide | Electron Microscopy Sciences | 20150 | Used for electron microscopy tissue preparation (step 5) |
Reference and ground electrodes for electroretinography | Chalgren Enterprises | 110-63 | Stainless steel needles 0.4 mm × 37 mm |
Sodium cacodylate buffer | Electron Microscopy Sciences | 12300 | Used for electron microscopy tissue preparation (step 5) |
Ster MD | União Química Farmacêutica Nacional S/A | MS-1.0497.1287 | Prednisolone acetate 0.12% |
Terolac | Cristália Produtos Químicos Farmacêuticos Ltda | MS-1.0497.1286 | Ketorolac trometamol 0.5% |
Terramicina | Laboratórios Pfizer Ltda | MS-1.0216.0024 | Oxytetracycline hydrochloride 30 mg/g + polymyxin B 10,000 U/g |
Tono-Pen XL | Reichert Technologies | 230635 | Digital applanation handheld tonometer |
TO-PRO-3 | Thermo Fisher Scientific | T3605 | Far red-fluorescent nuclear counterstain; emission at 661 nm |
Triton X-100 | Sigma-Aldrich | 9036-19-5 | Non-ionic surfactant |
Uranyl acetate | Electron Microscopy Sciences | 22400 | Used for electron microscopy tissue preparation (step 5) |
Xilazin | Syntec do Brasil Ltda | 7899 | Xylazine hydrochloride 2% |
Carl Zeiss | - | Stereo microscope for surgery and retinal dissection |
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