Imaging RGC function and dysfunction in vitro

Andrew Hartwick OD, MSc, PhD

Glaucoma culminates in the progressive degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. Experimental laboratory studies have been directed towards understanding how and why RGCs die in glaucoma and whether this death can be prevented or arrested. A key question that remains unanswered is whether RGCs undergo a period of dysfunction prior to death, as RGCs during this period may be particularly more amenable to neuroprotective strategies. With a current inability to non-invasively evaluate individual RGCs in humans, experimental studies of laboratory animal-derived RGCs in vitro may provide insight on distinguishing characteristics that separate functional and dysfunctional RGCs.

In this talk, I will discuss methods that I have used to study RGCs in vitro, with emphasis on the generation of purified RGC cultures and retinal wholemounts that are prepared from rats. Using calcium imaging techniques, the internal calcium levels were monitored in isolated RGCs that were challenged with both lethal and non-lethal exposures to the excitatory neurotransmitter glutamate. RGCs undergoing excitotoxic death display a latent loss in calcium homeostasis. While purified RGC cultures represent a pure reductionist approach to the study of these neurons, the retinal flatmount preparation can enable the study of the interactions between RGCs and other retinal cell types. As an example, calcium imaging of RGCs in flatmounts can be used to functionally assess retinal glutamate uptake. Both the advantages and limitations of in vitro RGC studies will be discussed, with the goal of stimulating debate as to the types of questions relevant to glaucoma that could be probed using these in vitro techniques.

Visual Fields and RGC Losses in Patients with Glaucoma

Ronald S. Harwerth, OD, PhD, FAAO

The lecture will describe an investigation of structure-function relationships for clinical glaucoma through an application of a model that was developed for experimental glaucoma. The model, without free parameters, produces accurate and relatively precise quantification of ganglion cell densities associated with visual field defects. Preliminary data from an extension of the model to objective imaging of retinal nerve fiber layer thickness will also be presented.

A) Introduction

1) The dogma of structure-function relationships for glaucoma

B) The structure-function model developed for experimental glaucoma

1) Visual field and histological sampling

2) Visual sensitivity as a function of ganglion cell density

3) The relationships for the parameters for linear regression of structure vs. function

4) Goodness-of-fit for sensitivity vs. ganglion cell density for experimental glaucoma

C) Application to clinical glaucoma

1) The anatomical data and perimetry data from Quigley’s laboratory

2) Prediction of age-related losses of ganglion cells from perimetry data

3) Application of the structure-function model to the group data of patients with glaucoma

4) Analysis of ganglion cell losses from perimetry for individual glaucoma patients.

D) Extension of structure-function relationships to objective imaging of retinal nerve fiber layer thickness

1) Optical coherence tomography of retinal nerve fiber layer thickness

2) The prediction of retinal ganglion cell density from nerve fiber layer thickness

E) Summary and conclusions