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) model, with important landmarks such as 360° thermal-induced disappearance of limbal vessels, as well as the mild to moderate mydriasis in the operated eye at the end of the procedure.
In the present series of 131 rats, full-circle limbal plexus cauterization (LPC) induced IOP elevation immediately after surgery from 13.0 ± 0.2 mmHg at baseline to 22.7 ± 0.4 mmHg. Peak postoperative IOP was observed on the first day after surgery (25.3 ± 0.6 mmHg), followed by a gradual return to baseline levels at the 6th postoperative day (statistical analysis: multiple t-test corrected for multiple comparisons using the Holm-Sidak method; Figure 1F). Corneal fibrosis or edema were clinical intercurrences that could potentially compromise accurate IOP measurement. The first, on one hand, was rare, affecting 3.92% of animals and noticed late during the postoperative follow-up, thus sparing the first 5 days of ocular hypertension and preserving the subacute OHT profile described23. Corneal edema, on the other hand, was a more common complication seen during the first few days after surgery (1-3 days), but mostly mild and temporary, thus did not robustly affect IOP23.
Retinal function was evaluated both behaviorally and electrophysiologically using the optomotor reflex and pattern-ERG, respectively (Figure 1G–J). Both parameters showed two phases of impairment: an ocular hypertension acute phase at 3rd day post-surgery, and a secondary degeneration phase at the 30th day post-surgery (Figure 1H, Figure J, and Table 1). In between, a period of functional recovery was detected, as previously discussed elsewhere23.
Compared with control fellow optic nerves, axonal counts in semi-thin transversal optic nerve sections showed progressive decrease after surgery (3rd day: 68.3% ± 0.9%; 7th day: 59.2% ± 2.6%; 14th day: 45.4% ± 2.2%; 30th day: 28.2 % ± 3.0%; two-way ANOVA: p < 0.0001; Figure 2A). Ultra-structurally, optic nerves from control eyes presented densely packed myelinated fibers, separated by thin glial cells processes, and evident axonal microtubules and neurofilaments (Figure 2B). In contrast, at 3 days after OHT, we found focal disruption of axon bundles, a few degenerated fibers, cytoplasmic vacuolation in glial cells processes, and condensed chromatin in glial cell nuclei. After 7 days of OHT induction, there was an increase in degenerated axonal fibers, hypertrophic glial cell processes, and swelling and voids in individual axonal fibers. At 14 days, one of the most prominent changes was a greater disarrangement of the optic nerve fibers, associated with the invasion of glial cell processes among the axons. Filament bundles filled these processes and dark degenerated fibers, and myelin breakdown was more common, associated with detached and vacuolized lamellae (Figure 2B).
The density of Brn3a+ profiles decreased along time (Figure 2C), mainly in the dorsal and temporal retinal quadrants, down to 32.4% ± 9.6% and 35.7% ± 9.1% after 30 days, respectively (Figures 2D–G and Table 2).
Figure 1: Thermal cauterization of limbal vascular plexus and consequences to retinal function in vivo. (A) Alternative methods to measure IOP in rats: rebound tonometry (superior), and applanation tonometry (inferior). (B–E) Surgical procedure; arrowhead: limbal vascular plexus; arrow: curved forceps used to expose anterior surface of the eyeball and optimize surgical assessment to limbal vessels; hash: the round tip of the low-temperature ophthalmic cautery; asterisk: cauterization mark. Scale bar: 2 mm. Inset in (D) shows in higher magnification the limbal vasculature to be cauterized. (F) Time course of IOP measurements in OHT (red) and control (black) eyes (n = 131). Vertical downward arrow: LPC surgery. * = p < 0.05 (G) Arena for optomotor response analysis, comprised of four computer monitors arranged in a quadrangle, with a platform in the middle. The monitors display the image of a virtual cylinder composed of vertical alternate black and white stripes moving around the animal with constant rotational speed and variable spatial frequencies. The red crosshair corresponds to the center of the virtual cylinder. (H) Optomotor responses. Two distinct phases are distinguished upon surgical follow-up: the OHT phase (0-5 days) and the secondary degeneration phase (6-30 days). **** = p < 0.0001. (I) Electrodes and animal positioning for pattern-ERG (PERG) acquisition: the active electrode at the temporal periphery of the cornea, and the reference and ground electrodes into the subcutaneous tissue of the ipsilateral temporal canthus and one of the hind limbs, respectively. The animal is positioned at 20 cm from the stimulus screen. (J) PERG amplitude upon stimuli with different spatial frequencies. Similar to optomotor response, PERG evaluation also shows two distinct phases of responses after surgery: ocular hypertensive at 3 days post-surgery, followed by recovery at day 7 and 14, although still statistically lower than naïve responses, and a subsequent decrease at day 30 after surgery. c/d = cycles per degree. Naïve group: eyes of animals unexposed to any previous experimental manipulation. (H) and (J) show mean ± SEM, plus individual replicates in (H). Please click here to view a larger version of this figure.
Figure 2: Structural assessment of retina and optic nerve after limbal vascular plexus cauterization (LPC) with low-temperature ophthalmic cautery. (A) Axon counts at distinct times after OHT (n = 3). *** = p = 0.0005, **** = p < 0.0001. (B) Electron micrographs of optic nerve degeneration following OHT. The left image shows the control optic nerve, and the following images illustrate the progressive degeneration after 3, 7, and 14 days of OHT. Arrowhead: normal myelinated fibers; thin arrows: degenerated fibers; asterisk: cytoplasmic vacuolation; hashes: glial cells process; and Nu: glial cell nucleus. (C) Photomicrographs of representative counting fields of RGCs labeled with an antibody to Brn3a (red) and TO-PRO3 labeled nuclei (blue); scale bar: 50µm. (D–G) Distribution of average Brn3a+ cell density in the four quadrants of the retina after 3-30 days of surgery. The graphs show individual averages of RGC densities for 3-11 animals per time after the procedure. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Quantitative data is mean ± SEM. Please click here to view a larger version of this figure.
Table 1: Statistical analysis of PERG data. Two-way ANOVA followed by Sidak's multiple comparisons test. P-value less than 0.05 was considered statistically significant. c/d = cycles per degree. Please click here to download this Table.
Table 2: Regional loss of RGC after LPC. SEM: standard error of the mean. Control: fellow eye. P-values less than 0.05 were considered statistically significant (*). Please click here to download this Table.
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. Also, it is technically easy to learn and low-cost, requiring mostly a low-temperature thermic cautery. Moreover, it is associated with a unique rate of OHT induction and full clinical recovery (> 90%, as previously reported)23, which reduces the number of animals necessary for experiments and allows for both electrophysiological and behavioral analysis. Finally, glaucomatous degeneration is present in both the RGC layer and optic nerve in a relatively short time frame after surgery, enabling either short-term or mid-term experimental designs23. Future studies are necessary to further elucidate the impact of full-circle limbal vasculature cauterization on individual retinal layers.
Critical steps in the surgical protocol are: (1) the cauterization tip must gently touch the scleral limbus parallel to the vessel axis; (2) corneal tissue must be spared, not only during cauterization but also during animal manipulation. Regularly perform eyedrop instillation and removal of excess solution from eye surface with a cotton swab (be careful not to scrub the cotton swab on the corneal surface while removing any liquid, as it leads to corneal epithelial abrasion and eventual post-surgery complications); (3) a full-circle of contiguous cauterization marks should be visualized; (4) do not neglect postoperative clinical follow-up with a non-steroidal anti-inflammatory drug and antibiotic ointment, at least until the 5th day after surgery.
The subacute IOP elevation seen in the described model differs from the pressure dynamics of open-angle glaucoma but is akin to acute angle-closure glaucoma, neovascular glaucoma, or multiple types of post-trabecular glaucoma with elevated episcleral venous pressure29. This is a major limitation of this method, as open-angle glaucoma is the most prevalent phenotype of the disease, characterized by chronic OHT and slowly progressive RGC degeneration. Nevertheless, the association of progressive normalization of IOP with continuous RGC degeneration represents a unique opportunity to study, in the same animal model, both the biological mechanisms that link ocular hypertension with the development and progression of glaucoma, as well as the secondary degenerative process mostly observed in open-angle glaucoma cases, whereupon patients present with glaucoma progress despite clinical or surgical success in reaching target IOP30. Thus, this model represents an opportunity to better elucidate this phenomenon through short-term or mid-term experimental designs, and eventually develop pressure-independent neuroprotective therapies to benefit patients that still lose sight despite best IOP control treatment.
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.
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 |