Multimodal imaging and functional analysis of the chick NMDA retinal damage model

Abstract

Objectives The chick is rapidly becoming a standardized preclinical model in vision research to study mechanisms of ocular disease. We seek to comprehensively evaluate the N-methyl-D-aspartate (NMDA) model of excitotoxic retinal damage using multimodal imaging, functional, and histologic approaches in NMDA-damaged, vehicle-treated, and undamaged chicks. Methods Chicks were either left undamaged in both eyes or were injected with NMDA in the left eye and saline (vehicle) in the right eye. TUNEL assay was performed on chicks to assess levels of retinal cell death one day post-injection of NMDA or saline and on age-matched untreated chicks. Spectral domain optical coherence tomography (SD-OCT) was performed weekly on chicks and age-matched controls day 1 (D1) up to D28 post-injection. Light adapted electroretinograms (ERG) were performed alongside SD-OCT measurements on post-injection chicks along with age-matched untreated controls. Results Untreated and vehicle-treated eyes had no TUNEL positive cells while NMDA-treated eyes accumulated large numbers of TUNEL positive cells in the Inner Nuclear Layer (INL), but not other layers, at D1 post injection. Significant inner retina swelling or edema was found on SD-OCT imaging at D1 post-injection which resolved at subsequent timepoints. Both the INL and the inner plexiform layer significantly thinned by one-week post-injection and did not recover for the duration of the measurements. On ERG, NMDA-treated eyes had significantly reduced amplitudes of all parameters at D1 with all metrics improving over time. The b-wave, oscillatory potentials, and ON/OFF bipolar responses were the most affected with at least 70% reduction immediately after damage compared to the fellow eye control. Conclusion This study establishes a normative baseline on the retinal health and gross functional ability as well as intraocular pressures of undamaged, vehicle-treated, and NMDA-damaged chicks to provide a standard for comparing therapeutic treatment studies in this important animal model.

Ibudilast Protects Retinal Bipolar Cells from Excitotoxic Retinal Damage and Activates the mTOR Pathway

“…Using the ERG long flash ON/OFF response test, which allows for specific characterization of the functionality of the ON (depolarizing) and OFF (hyperpolarizing) bipolar cells, we further found that the ON bipolar cells are uniquely susceptible to NMDA damage compared to OFF bipolar cells, consistent with reports in the literature (T. Heisler-Taylor et al, 2021). Ibudilast treatment significantly rescued the function of ON bipolar cells and restored ON-wave amplitudes (Fig 4I, J).…”

“…We next assessed whether ibudilast or AV1013 could preserve retinal structure by performing SD-OCT analysis to quantitate the thickness of the retinal layers. The death of neurons from chick NMDA damage in the inner retina leads to retinal thinning, which we previously demonstrated could be measured with SD-OCT (T. Heisler-Taylor et al, 2021). We evaluated retinal layer thickness 10 days post NMDA treatment, which showed that ibudilast, but not AV1013, significantly preserved retinal thickness (Fig 4A-D, NMDA 246.5±5.044 microns vs. NMDA+ibudilast 259.6±3.215, p=0.0260).…”

“…Electrophysiology was performed as previously described (T. Heisler-Taylor et al, 2021). Briefly, light adapted ERGs were recorded with the Celeris (Diagnosys, LLC) system on subsets of chicks (n=3/condition).…”

“…SD-OCT was performed on chicks as previously described (T. Heisler-Taylor et al, 2021). Briefly, an Envisu R2200 SDOIS with a rabbit bore lens was used to capture OCT b-scans of chick retina.…”

“…Chicks are diurnal and have a cone-rich retina with a high-density region, with parallels to the human macula. These similarities to the human eye make it attractive to model damaging retinal conditions and study the underlying cellular and molecular mechanisms relevant to neuroprotection (T. Heisler-Taylor et al, 2021).…”