Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Summary

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.

Abstract

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.

Introduction

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 (Q....

Protocol

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

1. House....

Results

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.......

Discussion

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 dissection^17,28. 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........

Materials

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