This study details the progression of retinal degeneration from pre-symptomatic to end-stage disease in two distinct sheep models of NCL; CLN5 disease in Borderdale sheep and CLN6 disease in South Hampshire sheep. Total retinal thickness, individual layer thickness, inflammation and accumulation of autofluorescent storage material were assessed at 3, 6, 12, and 18 months of age in post-mortem eyes from diseased sheep and age-matched healthy controls. Sheep eye globe measurements were also determined for comparison to the dimensions of the human eye.
Total retinal thickness was assessed in both the central and peripheral retina, with the central retina showing an earlier and more distinct difference between control and affected sheep. In both breeds the central retina was thicker in affected animals compared to controls at 3 months of age. This may be due to early compensatory mechanisms in the retina of affected animals, however requires more investigation.
There was early growth of the central retina in control Borderdale sheep leading to thicker retina in control compared to CLN5−/− sheep from 6 months onwards, however growth was slower in control South Hampshire sheep. Despite initial differences in peripheral retina thickness between control and affected sheep of both breeds, the thicknesses were similar from 6 months of age until a significant drop in thickness in CLN5−/− and CLN6−/− sheep at end-stage disease.
While total retinal thickness is informative for tracking global changes in the retina, it is important to consider the structure and function of individual layers of the retina and how these change over the course of disease. An example of the importance of assessing individual layers is seen in CLN5−/− retina at end stage disease where total thickness is comparable to controls, however almost 40% of this total thickness is taken up by the NFL. Meanwhile there is severe shrinkage of the ONL and IS/OS in CLN5−/− retina, which is more likely to be the underlying cause of retinal dysfunction. The cause and physiological consequences of this increasing NFL thickness in affected animals is unknown. As stated above, in CLN5−/− sheep it is primarily the outer retinal layers (OPL, ONL, and IS/OS) which show significant degeneration over the disease course. While inner retina (GCL, IPL, INL) thicknesses do fluctuate over time, they are more comparable to control at end stage disease. In contrast, all retinal layers in CLN6−/− sheep significantly degenerate over the course of the disease, except for the RPE.
In both breeds, accumulation of lysosomal storage occurs primarily in ganglion cell bodies, however it appears earlier in the retina of CLN6−/− sheep compared to CLN5−/− sheep. Minor amounts of lysosomal storage are observed in the CLN6−/− retina at 6 months of age, which then progresses until ganglion cell bodies appear full of storage by end stage disease. The pathological findings presented here are in keeping with early studies of the CLN6−/− sheep retina, which showed atrophy of photoreceptor cell bodies and inner and outer segments, and accumulation of autofluorescent storage material primarily in ganglion cells 10,11. A reduction in ERG a- and b-waves in CLN5 and CLN6 affected sheep has previously been reported and suggests dysfunction of photoreceptor and inner retinal cells becoming significant at approximately 9 to 11 months of age, the age when clinical signs of visual deterioration (e.g. reduced menace response) also begin to occur 12,13.
Under normal conditions, quiescent astrocytes are found in the retinal NFL with an elongated morphology running in parallel with ganglion cell axons 14,15. In the control sheep retina at all ages studied, GFAP immunoreactivity was primarily confined to these horizontally elongated cell bodies and processes in the NFL. However, in affected sheep NFL resident astrocytes had an altered morphology and, from 6 months of age, GFAP-positive cells were evident throughout the retina. Müller cells typically do not express GFAP, but upregulate its expression in response to pathological processes in the retina 16. GFAP-expressing Müller cells expressing GFAP were evident in the outer retina of affected animals from 6 months of age, indicating the initiation of reactive gliosis. Early and progressive increases in GFAP immunoreactivity and activation of Müller cells has also been observed in the retina of several mouse models of NCLs 17–20.
Post-mortem data from retina tissue of NCL patients is rare, however a case study of retina from CLN5 patients showed significant loss of retinal ganglion cells, with more moderate atrophy of the INL and ONL, and high levels of lysosomal storage in surviving ganglion cells 1,21. Conversely, the retina of a CLN6 patient showed extensive cell loss throughout the retina, in particular in the inner and outer nuclear layers, and the photoreceptor layer. While the ganglion cells were less vulnerable to degeneration in the CLN6 patient, they contained high levels of storage 21. Significant accumulation of lysosomal storage in retinal ganglion cells has also been observed in canine models of CLN2 and CLN5 disease 3,22. In CLN2 dogs and CLN3 mice the inner retinal layers are more significantly affected than outer retina, whereas in CLN5 and CLN6 mice photoreceptor degeneration is the predominant retinal pathology observed 4,22−24. The results presented here from CLN6 sheep align with what is observed in CLN6 patients as all retinal layers in sheep show progressive atrophy and accumulation of lysosomal storage in ganglion cells. In CLN5 sheep no ganglion cell loss was observed as it is in human CLN5, however accumulation of lysosomal storage in and outer retinal atrophy in sheep aligns with pathology in CLN5 patients.
There are several different potential approaches for delivering corrective therapies to the retina in NCL. Intravitreal delivery is the least invasive as it involves injection of the drug to the vitreous cavity of the eye, but has less target specificity. Sub-retinal delivery involves formation of a subretinal bleb between the photoreceptor layer and RPE, leading to faster and more specific targeting of outer retinal cells. Suprachoroidal delivery consists of an injection between the sclera and the choroid, a vascular layer lying underneath, and providing nutrients to, the retina. There are advantages and limitations to each delivery route that need to be taken into consideration depending on the disease subtype being treated and the target cells. In our CLN5 sheep, the outer retinal layers are the most vulnerable to disease therefore a subretinal approach may seem like the best option to target the outer retinal cells. However, the fragility of the diseased retina needs to be taken into consideration as it will be more prone to detachment if disrupted. While the intravitreal route is not specifically targeted to the outer retina and has barriers such as the inner limiting membrane (ILM) to overcome there are several reasons to consider this less invasive approach. CLN5 is a soluble protein, therefore if only inner retinal cells were corrected through intravitreal therapy they could secrete functional protein to other retinal cells, a mechanism known as cross-correction 25. In addition, given the fragility of the diseased retina it is likely the ILM is disrupted and more permeable in diseased eyes, and in fact others have shown better viral vector transduction in diseased eyes compared to wild-type 26. This route has recently been shown to be a viable option for delivery of therapeutic products in CLN5−/− sheep as retinal dysfunction and pathology was successfully ameliorated up to 18 months of age following a single intravitreal injection of CLN5 gene therapy 27.
The outlook for treating the retinal component of CLN6 disease is more complicated as we have shown that all layers of the retina significantly degenerate in CLN6 affected sheep, and CLN6 is a membrane bound protein meaning cross-correction is unlikely to be of benefit. Indeed, a recent trial of intravitreal gene therapy in CLN6−/− sheep showed some attenuation of pathology but no rescue of visual function 27. A combination of approaches such as suprachoroidal and intravitreal may be appropriate in this case as suprachoroidal would target outer retinal cells with a lower risk of retinal detachment, and the intravitreal route would target inner retinal cells. The general consensus on timing of treatment for degenerative diseases is often ‘the earlier the better’. Our results in NCL sheep have reaffirmed this, as atrophy of retinal layers and accumulation of lysosomal storage start becoming evident by 6 months of age. In addition, early treatment is preferable if using the intravitreal approach as the ILM is still developing and therefore more permeable in younger animals 28,29.
The sheep eye is increasingly being recognised as a good model for studying human retinal disease 30–33. Previous studies report that the average adult sheep eye has an axial length of 26 mm, and the data collected in this study are in keeping with these reports 34. This is comparable to the human eye which has an axial length of approximately 24 mm 34. The human central retina is characterised by the macula and the fovea. The macula is a region in the retina which aligns with the central axis of the lens and contains the fovea, a small pit in the retina composed exclusively of closely spaced cones which produce the highest visual resolution of the retina 35. Although the sheep retina does not possess a macula or fovea, it has two cone-enriched areas instead: a streak in the central part of the retina (area centralis) and a smaller dorso-temporal area 31,36. At the cellular level, the basic ten-layered morphology of the retina is very similar between sheep and humans, although sheep only possess 2 types of cone photoreceptors (M- and S-cones) for colour vision, whereas humans possess 3 types of cones (M-, S-, and L-cones) 31,36. The similarities in eye globe size and retinal structure between the human and sheep eyes make sheep a good model for studying diseases of the retina, and for testing ocular therapies.