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1From the Departments of Biomedical Engineering and 5Neurobiology and Physiology, Northwestern University, Evanston, Illinois; the 2Department of Biomedical Engineering and Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Chicago, Illinois; the 3Division of Ophthalmology, Evanston Northwestern Healthcare, Glenview, Illinois; and the 4Departments of Medicine and Surgery, College of Veterinary Medicine, and 6Ophthalmology, Mason Eye Institute, University of Missouri-Columbia, Columbia, Missouri.
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Abstract |
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METHODS. Vitreal and intraretinal ERGs were recorded from eight dark-adapted, anesthetized Abyssinian cats. Brief bright flashes were used to elicit vitreal a- and b-waves. Longer, weaker flashes were used to elicit intraretinal b- and c-waves. Stages 1 through 4 of the disease were characterized ophthalmoscopically. Parameters of the Lamb and Pugh a-wave model (amax, A, and teff) for the Abyssinian cats were compared with those for normal cats. Light microscopy was used to count photoreceptor nuclei.
RESULTS. The maximum a-wave amplitude, amax, was significantly smaller in stage 1, and continued to decrease (stage 1: 50% of normal, stage 2: 28%, stage 3: 27%; and stage 4: unrecordable). There was a small, but not significant, decrease in the amplification constant A from 0.24 ± 0.11 s–2 in normal cats to 0.16 ± 0.08 s–2 in Abyssinian cats. The intraretinal b- and c-wave amplitudes decreased most dramatically during the early stage of the disease. Affected animals had fewer photoreceptors than unaffected Abyssinians or control animals. The number of photoreceptors declined most rapidly in the inferior periphery.
CONCLUSIONS. The amplitudes of all ERG components were already reduced significantly by stage 1 and progressively declined. The lack of major changes in a-wave model parameters indicates that the degeneration is probably not due to a mutation in transduction proteins. Losses of photoreceptor function were larger than losses of photoreceptor nuclei.
The genetic defect in these animals is not known. To further characterize the rod photoreceptor degeneration, we investigated ERG a-waves in response to bright flashes and used the Lamb and Pugh6 model of phototransduction to examine the data. The fits of the Lamb and Pugh model allow an extraction of two a-wave model parameters: the overall amplification constant of transduction (A) and an effective delay before the response begins (teff). The parameters were obtained at various stages of the disease. We also examined vitreal and intraretinal recordings of the b- and c-waves in response to weaker flashes. The intraretinal responses were from central retina, and allowed a comparison with those generated by the entire retina. A preliminary report of these results has been presented in abstract form (Kang Derwent JJ, et al. IOVS 2000;41:ARVO Abstract 1268).
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Methods |
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Three cats were in the earliest stages of the disease (stage 1), two cats were in the middle stage (stage 2), two cats were in the late stage (stage 3), and one cat was in the final stage (stage 4). The stage 3 animals were 3 and 5 years old, and the stage 4 animal was 6 years old. Briefly, stage 1 is characterized by a subtle tapetal fundus discoloration. In stage 2, the discoloration of the tapetum is more distinct and widespread and peripheral vessels on the tapetal retina appear slightly attenuated. In stages 3 and 4, vessel attenuation is prominent, the discoloration becomes darker and more mottled, and large areas of the tapetal fundus are hyperreflective. Electroretinograms from these animals were compared to those obtained previously from normal, non-Abyssinian cats. Separate normal data sets were used for the bright flash a-wave analyses7 and for the intraretinal b- and c-wave analyses9 because the two types of recording were not available from a single set of normal animals.
Visual Stimulation
Animals were allowed to dark adapt for at least 1 hour before the ERGs were collected. Two types of visual stimulation were used to record all the signals of interest (Table 1) . First, bright flashes of diffuse white light, produced by a photoflash (photoflash model 283; Vivitar Corp., Santa Monica, CA) were used to elicit the a- and b-waves recorded with a vitreal electrode. Stimuli were reflected to the eye by a Ganzfeld hemisphere. Flash intensity was varied over 6 log units with neutral-density filters. At zero attenuation, the illumination at the cornea was 4.45 log scotopic cd-sec · m–2 measured with a photometer (model 40X; UDT, Hawthorne, CA) in photopic units and converted to scotopic units by assuming the spectral distribution of a xenon-filled flashtube).10 The conversion factor, K, from the corneal illumination to the number of photoisomerizations per rod per flash (
) followed Breton et al. (Ref. 11 , equation 13) with a few corrections for the cat eye.7 The value of K was 27.13 photoisomerizations · rod–1 · scot td–1 · s–1. The unattenuated retinal illumination was 7.93 log photoisomerizations per rod per flash.
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8.6 log quanta, 555 nm · deg–2 · s–1), which generally produced maximum b- and c-waves (e.g., Ref. 14 ). This stimulator was also used at lower illuminations, to generate intensity–response functions for the vitreal ERG.
Recording
The vitreal ERG was measured between a chlorided silver wire in the vitreous and a chlorided silver plate behind the eye. Vitreal recordings are functionally equivalent to those from the cornea, but offer more stability in long-term recordings and have slightly larger amplitude. Vitreal responses were amplified (DC to 3 kHz; model M4A; WPI, Sarasota, FL), digitized at 5 kHz, and stored in an IBM-compatible computer. The bright flashes were triggered by the computer, and ERGs were collected with EPIC-XL software (LKC, Gaithersburg, MD). After each flash, the eye was allowed to re–dark adapt for at least 3 minutes. Responses to individual flashes were analyzed.
Intraretinal ERG recordings were made by the voltage barrel of a double-barreled oxygen microelectrode8 referenced to the chlorided silver plate. The microelectrode was advanced through the vitreous in the dark until a transient voltage deflection signaled that the retina had been encountered. From this point on, the electrode was advanced in 3-µm steps, stopping every 30 µm to record the local ERG. The penetration ended when the microelectrode crossed the retinal pigment epithelium (RPE), as indicated by the transepithelial potential. After each flash, the eye was allowed to re–dark adapt for at least 1 minute.
a-Wave Analysis
The Lamb and Pugh6 a-wave model was fitted to the responses to all flash energies
 | (1) |
is the number of photoisomerizations per rod, teff is a brief delay, and A is an amplification constant. When applied to the a-waves, F(t) is equivalent to the normalized a-wave from flash onset to the peak of the a-wave
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Hill Equation Analysis
The a-, b-, and c-wave intensity series were fitted to a Hill equation
 | (3) |
is half saturation, and n is a parameter that adjusts the steepness of the function. The fitting was performed using SigmaPlot (ver. 9.0; SYSTAT Software, Inc., Point Richmond, CA).
Histologic Preparation
After measurements, both eyes were enucleated, and retinas were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer and embedded in paraffin. They were then sectioned and stained with toluidine blue for light microscopy. Measurements of outer retinal thickness and photoreceptor nuclei per section were made in three sections per retinal location at 400x and 1000x, respectively. The counts reported were made at the higher power. Measurements were made in the area centralis and far peripheral retina, in both superior and inferior regions. The same techniques were used to evaluate one eye of each of 12 additional animals. Three were stage 1 Abyssinians not used in the electrophysiological studies, to control for possible changes in counts resulting from the long duration of anesthesia and swelling of the retina. Three were from heterozygous unaffected Abyssinian cats, and six were from adult domestic short-haired cats. These latter two groups were used to determine whether the stage 1 animals had lost photoreceptors relative to the heterozygous or normal cats.
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Results |
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The parameter A, which was derived from fits similar to those in Figure 1 and reflects the gain of phototransduction, is plotted as a function of the number of photoisomerizations, , in Figure 2 for normal cats (Fig. 2A , data reproduced from Ref. 7 ), stage 1 cats (Fig. 2B) , stage 2 cats (Fig. 2C) , and one of the stage 3 cats (Fig. 2D) . Both A and teff were varied to obtain the best fits. In both normal and affected Abyssinian cats, the value of A was approximately independent of , except at the higher intensities, when A declined. This effect has been demonstrated in humans,6 11 rats,15 and normal cats.7
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Vitreal Intensity–Response Functions
Figure 4 shows the amplitudes of the a-wave (Fig. 4A) , b-wave (Fig. 4B) , and c-wave (Fig. 4C) amplitudes as a function of light intensity. The a-wave amplitudes were from data sets like those shown in Figure 1 , in response to brief bright flashes, and the b- and c-wave amplitudes were obtained using 4-second flashes of lower illumination. The 4-second flashes were used for the b-wave, because the entire stimulus–response relation was not obtained in response to the bright flashes. It can be appreciated from Figure 1 that a flash producing a very small a-wave still produces a saturated b-wave. For the c-wave, its long integration time makes the longer stimulus more appropriate.
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) as a function of the disease stage, and these were not consistent across the different ERG components. For all ERG components, it was primarily the maximum amplitude parameter, Rmax, that was affected by the disease.
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Figure 5C shows the intraretinal c-wave amplitude, recorded in the subretinal space near the RPE, and Figure 5D shows the corresponding vitreal c-wave amplitudes. Narfström et al.5 reported that the vitreal c-wave was better preserved than the a- or b-wave during the early stages of the disease. This is consistent with the present work; the difference between the normal and stage 1 animals was not significant (P = 0.47) for the vitreal responses. However, the difference in the intraretinal c-wave between normal and stage 1 animals was significant, as would be expected from the dependence of the c-wave on photoreceptor responses. The reason that the amplitude of the vitreal c-wave can give a misleading impression about the underlying intraretinal events is that the vitreal c-wave is the difference between an RPE c-wave component and a Müller cell component, slow PIII.17 18 If these decline together, as would be expected from the dependence of both events on photoreceptors,19 20 the vitreal responses can be relatively unaffected.18
Photoreceptor Counts and Histology
Figure 6 shows examples of the histology from Abyssinian cats, on which measurements of photoreceptors and retinal thickness were based. Precise correlation of the central histologic sections and the intraretinal recording location was not possible, but both were from the area centralis. Counts from the far peripheral retina were obtained because the vitreal ERG is summed across the whole retina, making counts from both central and peripheral regions relevant. Figure 7 shows photoreceptor counts as a function of the disease stage for the three investigated areas. This figure also shows data for three additional groups of animals that were not used for electrophysiological measurements: six normal domestic short-haired cats, three heterozygous Abyssinians in which the fundus appears normal, and three additional early stage 1 animals, to control for the possibility that the histology from stage 1 animals used for electrophysiology was distorted by the extended period of anesthesia or poor fixation of the tissue.
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Discussion |
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Even in stage 1, the Abyssinian cats have fewer photoreceptors than unaffected Abyssinians or normal domestic short-haired cats, which must explain some of the ERG changes. However, the ERG changes were greater than the loss of photoreceptors. In stage 1, photoreceptor numbers were reduced to approximately 70% of those in the normal retina, whereas the a-wave maximum amplitude was reduced to 50% of normal. This suggests some dysfunction in the remaining rods. In stages 2 and 3 the a-wave maximum amplitude continued to decline, but the photoreceptor count did not decrease further until stage 3, and so again the ERG changes preceded the loss of rods. Changes in the ERG measures were similar to the changes in another functional measure, photoreceptor oxygen utilization, as demonstrated in the companion work.12 Another finding of the present work was that the a-wave was reduced more than the b-wave at the earliest stages of disease (stages 1 and 2). This has also been shown recently by using component factor analysis22 23 and by graphic representation of a-and b-wave wave amplitudes in homozygous affected cats compared with heterozygous, ophthalmoscopically normal cats.24 The same trends have been observed in P23H transgenic rats.25
A new result of the present study is the small, nonsignificant changes in A and teff, which characterize the activation steps in phototransduction according to the Lamb and Pugh6 model. Another new result is the lack of substantial change in the half-saturating intensity, , of the a-wave. As has been previously found in affected Abyssinan cats up to 2 years old,4 for the b-wave was also not markedly changed. Together, these findings suggest that the decrease in amplitude of the ERG is not caused by a defect in the biochemistry of the transduction process. Caution is required, however, because cases are known where there is a mutation in rhodopsin, and the activation stage of transduction is still normal.26
The results presented herein reinforce the similarity between the slow degeneration in Abyssinian cats and that in some types of human RP. Breton et al.11 reported that amax decreased in a patient with RP, but the amplification constant A and delay teff were indistinguishable from their mean normal values. This led them to suggest that the rods in human RP were either shortened or partly absent, yet had normal phototransduction. Rods in Abyssinian cats may eventually be shortened in length or absent, as shown by earlier ultrastructural studies.3 Using an equivalent model of phototransduction, Hood and Birch27 reported that some forms of RP can alter the activation stage of transduction, but found other patients with small a-waves and normal transduction. They and Shady et al.28 suggested that patients with RP often have normal amplification early in life, but the degeneration of rods (i.e., further damage to the outer segments) may lead to reduced A. There is no evidence in cats that reduced photoreceptor numbers lead to a change in A, but based on the suggestions from human studies, A would be expected to change in cats late in stage 3; we studied too few animals at that stage to be certain on this point. Consistent with the Hill analysis performed herein, the rod a- and b-waves in patients with cone–rod degenerations were reduced in amplitude, but generally did not show reduced sensitivity.29
The present results also provide the only intraretinal recordings of the ERG in any animal model of RP and show clearly that the area centralis is affected early in the disease, even though the central fundus looks relatively normal compared with the periphery. The comparison of the intraretinal b-wave with the vitreal b-wave indicates that later in the disease the central retina is protected somewhat relative to the peripheral retina. These results provide independent confirmation of those of Seeliger and Narfström,30 who used a different technique, the multifocal ERG, to analyze the spatial distribution of disease in the Abyssinian cats. They also found a generalized loss of function in the early stage and a relative preservation of central retina late in the disease.
The intraretinal results also provide new insight on the c-wave, and show that it declines with a time course similar to that of the b-wave. This had not been apparent from previous corneal recordings4 or our vitreal recordings, which did not show a dramatic change in the c-wave (to 81% of control in the previous work and 70% of control in the current study during stage 1). We can now assert that the lack of change in the vitreal c-wave occurs because both the RPE and Müller cell components decrease, leaving the vitreal or corneal response, which is the difference between these components, relatively unchanged.
A substantial amount of the change in the ERG results from loss of photoreceptors rather than simply dysfunction. This is also true in rodent models of degeneration in which relative cell loss and ERG loss have been investigated.25 31 However, it appears that the percentage changes in the photoreceptor counts and in the a-wave amplitude can follow somewhat different time courses. In the present study, comparison of Figure 3A and 7D indicates that the a-wave was reduced to half of normal at a time when at least 70% of the photoreceptors remained in most of the retina (stage 1), and to approximately 30% of normal when there were still 40% of the photoreceptors present (stage 3). The larger decrement in the a-wave is consistent with subtle changes that are visible at the electron microscopic level. Electron microscopic changes include disorganization and vacuolization of the outer segments as well as complete degeneration of some entire photoreceptors.3 These are more prominent in the periphery, but are present at all locations. They suggest a defect in the gene for a structural protein, but this is not certain. Conceivably, this would affect the rods in several ways, at least one of which must be linked to membrane voltage responses. For instance, the a-wave would be smaller if the gene defect made the rods generally more hyperpolarized in the dark, so that when the light-dependent cation channels closed during illumination, the rod membrane potential would have less range before it reached full hyperpolarization.
Another problem that does not have a clear resolution is the relation between responses of individual rods and the size of the a-wave, which is the summation of the individual responses. One hypothesis would be that, at a particular stage of the disease, some rods produce little or no response and healthier ones produce full responses, yielding a smaller a-wave, and the other hypothesis is that the a-wave is reduced because the response of each rod is reduced to some extent. One argument weakly favors the latter interpretation, but another argument tends to favor the former hypothesis. The point in favor of the latter hypothesis is that it is now clear that there are functional defects across the entire retina, and so it cannot be that the ERG decreases because the peripheral rods are affected but the central rods are normal. This reasoning is weak because it could also be that selected cells in all locations are affected, whereas other cells in the same regions are normal. One of the arguments in favor of a patchy depression of rod responses is that the electron microscopy showed some defective cells and some apparently normal cells in all locations. There is no evidence of a general shortening of outer segments sufficient to reduce their light capturing volume uniformly, commensurate with the large change that occurs by stage 1. The disease progression would be related to an increase over time in the number of histologically abnormal cells, and presumably therefore, electrically abnormal cells.
One way to resolve some of these issues would be to obtain recordings from individual isolated photoreceptors from Abyssinian cats and reconstruct an a-wave from the population of those individual responses. Such experiments are feasible, but difficult. The other approach, which has been unsuccessful so far, would be to find the gene defect, and reason forward from that.
In summary, the ERG a-wave is attenuated, even in the earliest stage of the retinal degeneration in Abyssinian cats. The amplitude change is not caused by a defect in the transduction cascade, but rather in the number of active photoreceptors and/or the ability of each to capture light. The changes in photoreceptor signaling propagate to the b- and c-waves, although the changes in these second-order signals are somewhat smaller than those in photoreceptor responses. Although the funduscopic changes and photoreceptor counts indicate that the peripheral retina is affected earlier than the central retina, intraretinal recordings and photoreceptor counts show that major functional changes also occur in the central retina in stage 1. As far as can be determined, the results in the cat are consistent with major findings in human RP. This suggests that other attributes of the condition in the cat, which can be investigated with more invasive studies, can also provide information about the human disease.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 28, 2005; revised February 27, 2006; accepted June 6, 2006.
Disclosure: J.J. Kang Derwent, None; L. Padnick-Silver, None; M. McRipley, None; E. Giuliano, None; R.A. Linsenmeier, None; K. Narfström, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Robert A. Linsenmeier, Biomedical Engineering Department, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3107; r-linsenmeier{at}northwestern.edu.
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); Normal domestic short-haired cats (
). (D) Averages of data in (A), (B), and (C) normalized to the number of nuclei in the normal cats, where normal includes both domestic short haired and heterozygous Abyssinians. Groups are indicated in the key. (E–H) Measures of outer retinal thickness (OPL to RPE) from the same sections used to make the cell counts in (A–D).