Cone function studied with flicker electroretinogram during progressive retinal degeneration in RCS rats

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Abstract

The Royal College of Surgeons (RCS) rat has a primary defect in retinal pigment epithelial cells that leads to the progressive loss of photoreceptors and central visual responsiveness. While most rods are lost by 90 days of age (P90), cones degenerate more slowly, and can be detected anatomically up to 2 years of age, despite massive neuronal death and retinal remodelling. To examine how this progressive degenerative process impacts on cone function, we recorded the electroretingram to white light flashes (1·37 log cd s m−2) presented at frequencies ranging from 3 to 50 Hz, under light adapted conditions (29·8 cd m−2). Pigmented dystrophic and congenic non-dystrophic RCS rats aged from 18 to 300 days were studied. In all responsive animals at all ages, maximal amplitudes were obtained at 3 Hz. In both non-dystrophic and dystrophic rats, there was an increase from P18 to P21 in response amplitude and critical fusion frequency. After P21, these two parameters declined progressively with age in dystrophic rats. Other changes included prolongation in latency, which was first detected prior to the initiation of amplitude reduction. While phase shifts were also detected in dystrophic RCS rats, they appeared at later degenerative stages. The latest age at which responses could be elicited in dystrophic rats was at P200, with positive waves being replaced by negative deflections. The effect of increments in the intensity of background illumination was tested at P50 in both groups. This caused a diminution in flicker response amplitude and critical fusion frequencies in non-dystrophics, while in dystrophic animals, response amplitudes were reduced only at low frequencies and critical fusion frequencies were unaltered.

In conclusion, although dystrophic RCS rats undergo a progressive decline in cone function with age, the flicker responsiveness at P21 is comparable to that of non-dystrophic congenic rats, suggesting normal developmental maturation of the cone system in this animal model of retinal degeneration. Flicker responses can be recorded up to P200, at which point the retina has undergone severe regressive and reactive changes in its connectivity patterns. The fact that responses at this age consist of solely negative deflections might be a reflection of the highly pathological state of the retina.

Introduction

Retinitis pigmentosa (RP) and age related macular degeneration (AMD) represent the leading causes of blindness in humans, for which no suitable treatment exists. They involve degeneration of photoreceptors as a result of defects in either the photoreceptors themselves or the associated retinal pigment epithelium (RPE). In numerous cases of RP with mutations directly affecting the rods, the loss of rod function is followed in later stage by the loss of cone function (Al-Maghtheh et al., 1993, Kajiwara et al., 1994, McLaughlin et al., 1995). In AMD, a primary RPE cell defect leads to rod followed by cone functional losses (Owsley et al., 2000, Owsley et al., 2001 Phipps et al., 2003). Animal models such as the Royal College of Surgeon (RCS) rat are used extensively to explore potential therapeutic avenues that might eventually be transfered to the clinic (LaVail, 2001, Chader, 2002).

The RCS rat (Bourne, 1938) has a recessive mutation (D'Cruz et al., 2000) which precludes RPE cells from phagocytosing shed rod outer segments effectively and this leads to the progressive death of rods followed by cones (Dowling and Sidman, 1962, Cicerone et al., 1979). Various experimental therapies have been used successfully to preserve photoreceptors and some aspects of visual responsiveness in RCS rats (for reviews see: Lund et al., 2001, Dejneka et al., 2003, Hooper and Guymer, 2003, Surace and Auricchio, 2003, Weleber et al., 2003). Recent work (Girman et al., 2003) has suggested that even when rods are rescued, they may not function normally and that their presence might be related to the functional preservation of remaining cones: how rods affect cone survival remains to be elucidated (Leveillard et al., 2004).

Considering the wide use of the RCS rat for the development of therapies, it is crucial to measure cone function at various degenerative stages as a background for evaluating efficacy of treatments regimens. A test commonly employed to evaluate cone function is the flicker electroretinogram (ERG). Cone-mediated responses can be obtained using a rod-saturating background with flashes presented at various frequencies (Goto et al., 1998). The measure of response amplitude for various stimulus frequencies and the frequency at which recordings are no longer modulated by the stimulus (critical fusion frequency) can provide useful quantitative measures for the assessment of cone functional status.

We have applied the flicker ERG approach to characterize cone function during the course of progressive degeneration in RCS rats.

Section snippets

Animals

Pigmented RCS rats, either dystrophics (RCS rdy+p+, n=36) or non-dystrophic congenics (RCS rdy p+, n=36) were studied. All animals were bred in a colony at the University of Utah, and maintained under 12 hr light dark cycle (light cycle illumination varied from 7 to 30 Lux depending on the position within respective cages); they were housed and handled with the authorization and supervision of the Institutional Animal Care and Use Committee from the University of Utah. Every procedure conformed

Results

Both dystrophic and non-dystrophic pigmented RCS rats gave stable and repeatable recordings. There was no evidence of response fatigue over time: tests done before and after presentation of flicker stimuli covering the full range of frequencies gave essentially similar results. Most responses were obtained with minimal stimulus artifacts: this was especially important to allow quantification at higher frequencies. Examples of averaged records under different stimulation conditions are shown in

Discussion

One essential condition for isolating cone ERG responses is to render rods unresponsive to light stimulation. This can be achieved by either using photopic adaptation when recording responses to single or flicker flashes (for a review in rodents: Nusinowitz et al., 2002, Peachey and Ball, 2003; and in humans: Fishman et al., 2001), or using a double flash protocol, usually under scotopic conditions (Birch et al., 1995, Lyubarsky et al., 1999, Nixon et al., 2001) in which the first flash

Acknowledgements

This work was supported by NIH (EY14038), FFB (Foundation to Fight Blindness), Wynn Foundation and Research to Prevent Blindness grants. I Pinilla was supported by grants from the Spanish Government: FIS 02/5010 and BA03/0016.

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