Optical Coherence Tomography of Retinal Degeneration in Royal College of Surgeons Rats and Its Correlation with Morphology and Electroretinography

Abstract

Purpose: To evaluate the correlation between optical coherence tomography (OCT) and the histological, ultrastructural and electroretinography (ERG) findings of retinal degeneration in Royal College of Surgeons (RCS-/-) rats.

Materials and methods: Using OCT, we qualitatively and quantitatively observed the continual retinal degeneration in RCS-/- rats, from postnatal (PN) day 17 until PN day 111. These findings were compared with the corresponding histological, electron microscopic, and ERG findings. We also compared them to OCT findings in wild type RCS+/+ rats, which were used as controls.

Results: After PN day 17, the hyperreflective band at the apical side of the photoreceptor layer became blurred. The inner segment (IS) ellipsoid zone then became obscured, and the photoreceptor IS and outer segment (OS) layers became diffusely hyperreflective after PN day 21. These changes correlated with histological and electron microscopic findings showing extracellular lamellar material that accumulated in the photoreceptor OS layer. After PN day 26, the outer nuclear layer became significantly thinner (P < 0.01) and hyperreflective compared with that in the controls; conversely, the photoreceptor IS and OS layers, as well as the inner retinal layers, became significantly thicker (P < 0.001 and P = 0.05, respectively). The apical hyperreflective band, as well as the IS ellipsoid zone, gradually disappeared between PN day 20 and PN day 30; concurrently, the ERG a- and b-wave amplitudes deteriorated. In contrast, the thicknesses of the combined retinal pigment epithelium and choroid did not differ significantly between RCS-/- and RCS+/+ rats.

Conclusion: Our results suggest that OCT demonstrates histologically validated photoreceptor degeneration in RCS rats, and that OCT findings partly correlate with ERG findings. We propose that OCT is a less invasive and useful method for evaluating photoreceptor degeneration in animal models of retinitis pigmentosa.

Fig 1. Typical OCT image of a RCS^+/+ rat at PN day 33, along with a corresponding histological section of a RCS^+/+ rat at PN day 29 (hematoxylin and eosin stained).

The retinal pigment epithelium (RPE) was detached. The top yellow line indicates the retinal surface, the second line indicates the border between the inner nuclear layer and outer plexiform layer, the third line indicates the upper limit of the IS ellipsoid line, the fourth line indicates the surface of the RPE, and the bottom line indicates the bottom of the choroid. Layer A comprises the nerve fiber layer, the ganglion cell layer, the inner plexiform layer and the inner nuclear layer. Layer B comprises the outer plexiform layer and the outer nuclear layer. Layer C comprises the photoreceptor IS and OS layers. Layer D comprises the RPE and the choroid. The black and white bars represent 100μm length.

Fig 2. Characterization of typical OCT findings from RCS^+/+ rats at PN days 25 and 53, as well as the corresponding histological sections from RCS^+/+ rats at PN days 24 and 52 (hematoxylin and eosin stained, the retinal pigment epithelium was detached.).

The black and white bars represent 100μm length.

Fig 3. Chronological changes in the photoreceptor IS and OS layers during the early stages (PN days 17 to 26) of retinal degeneration in RCS^-/- rat—observed using optical coherent tomography and the corresponding histological sections (PN days 18, 23, and 28).

A yellow arrow indicates the apical hyperreflective band and a light green arrow indicates the IS ellipsoid zone. Black arrows indicate the extracellular lamellar material. Arrowheads indicate the photoreceptor inner segment. The black and white bars represent 100μm length.

Fig 4. Chronological changes in the retinal layers during the progressive stage (PN days 33 to 47) of RCS^-/- rat retinal degeneration—observed using optical coherent tomography and the corresponding histological sections (PN days 33 and 46).

Dark green arrows indicate the outer nuclear layer. An orange arrow indicates the photoreceptor IS and OS layers. Yellow arrows indicate the inner retinal layer. White arrows indicate the retinal pigment epithelium and choroid. The black and white bars represent 100μm length.

Fig 5. Chronological histological changes in the photoreceptor layer during the early stages (PN days 18 to 28; hematoxylin and eosin stained) of retinal degeneration in RCS^-/- rats.

Arrows indicate the extracellular lamellar material deposited on the apical surface of the RPE (PN days 18 and 23) and in the photoreceptor OS layer (PN days 25 and 28). Arrowheads indicate the photoreceptor IS layer (PN days 18 to 28). The black bar represents 50μm length.

Fig 6. Electron microscopic findings of the photoreceptor IS and OS layers of RCS^-/- rats at PN days 18, 23, & 28.

The semiround-shaped deposits of extracellular lamellar material had accumulated at the apical portion of the OS at PN days 18 and 23 (a). These deposits gradually increased toward the basal side of the OS until PN day 28 (a). The magnified picture of each yellow box is presented in the lower level, exhibiting the lamellar structure of the deposited material (b). The black bar represents 10μm, and the white bar represents 2μm length, respectively.

Fig 7. Chronological histological changes in the photoreceptor layer during the progressive stage (PN days 33 to 53; hematoxylin and eosin stained) of retinal degeneration.

The RPE had detached at PN day 42. The extracellular lamellar material occupied the photoreceptor IS and OS layers after PN day 33. The nuclei of the outer nuclear layer had become pycnotic. The black bar represents 50μm length.

Fig 8. Chronological changes in the thickness of each retinal sublayer (μm), A (a), B (b), C (c), and D (d).

Layer A comprises the nerve fiber layer, the ganglion cell layer, the inner plexiform layer and the inner nuclear layer. Layer B comprises the outer plexiform layer and the outer nuclear layer. Layer C comprises the photoreceptor inner and outer segments layer. Layer D comprises RPE and the choroid. Closed squares (a and d) and triangles (b and c) indicate RCS^-/- rats and open squares (a and d) and triangles (b and c) indicate RCS^+/+ rats. Bars indicate standard deviation. Statistical significance (with Bonferroni’s post hoc test): * P < 0.05; ** P < 0.01; *** P < 0.001.

Fig 9. Chronological changes in the amplitudes of the ERG a- (a) and b- (b) waves in RCS rats.

Closed circles indicate RCS^-/- rats and open circles indicate RCS^+/+ rats. Statistical significance (with Bonferroni’s post hoc test): * P < 0.05; *** P < 0.001.