Quantification of Retrograde Axonal Transport in the Rat Optic Nerve by Fluorogold Spectrometry

PURPOSE: Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantified in retinal tissue lysate. METHODS: To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC) of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n = 15). In subsequent experiments axona transport was impaired by optic nerve crush (n = 3), laser-induced ocular hypertension (n = 5) or colchicine treatment to the SC (n = 10). RESULTS: Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (c(FG)), from the emission spectrum. c(FG) is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, c(FG) monotonously increases with time (p = 0.002). Optic nerve axonal damage caused a significant decrease of c(FG) (crush p = 0.029; hypertension p = 0.025; colchicine p = 0.006). Lysates are amenable to subsequent protein analysis. CONCLUSIONS: Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses

Other Procedures for Pediatric Glaucoma Surgery: New Devices and Techniques

In recent years, multiple new devices have been developed in glaucoma surgery that target the angle and can be grouped in the category of minimally invasive glaucoma surgeries (MIGS). These procedures were developed mainly for adult glaucoma surgery, but many have potential application in pediatric glaucoma patients, although there is limited experience in children for most of the MIGS. When considering new and novel techniques, it is always best to look back at the principal foundations leading to these innovations. The origin of pediatric glaucoma surgery dates back to 1938 with Barkan describing his goniotomy technique. While classical angle surgery still continues to be a first-line treatment in primary congenital glaucoma (PCG), in recent years modifications in this technique and new devices have been developed. If the first angle surgery is not successful and the angle has not been treated 360°, many surgeons will further treat the angle before performing a filtering procedure (trabeculectomy) or glaucoma drainage device. Several instruments and techniques have been developed to extend the area of angle first treated and/or to remove the trabecular meshwork (TM) from the eye or dilate Schlemm’s canal. This chapter presents new surgical techniques and devices that target the angle via an ab interno approach and are classified as MIGS: trabeculotomy with TRAB™360, viscodilation with VISCO™360, combined trabeculotomy and viscodilation with the OMNI™, gonioscopic-assisted transluminal trabeculotomy with fiber optic or suture, and TM destruction and removal with Trabectome®/Goniotome and Kahook dual blade®. These devices share indications, complications, advantages, and limitations