April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Cone Transplantation
Author Affiliations & Notes
  • Sher A Aslam
    Moorfields Eye Hospital, London, United Kingdom
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Alun R Barnard
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Sumathi Sekaran
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Mandeep S Singh
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Robert E MacLaren
    Moorfields Eye Hospital, London, United Kingdom
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships Sher Aslam, None; Alun Barnard, None; Sumathi Sekaran, None; Mandeep Singh, None; Robert MacLaren, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3981. doi:
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      Sher A Aslam, Alun R Barnard, Sumathi Sekaran, Mandeep S Singh, Robert E MacLaren; Cone Transplantation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3981.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

Previous studies have shown that rod transplantation is feasible. However, there are no reports that investigate the transplantation of cones when isolated from rods. We show that cone transplantation and restoration of cone function is possible using a new approach to enrich cone photoreceptor donors.

 
Methods
 

Donor cells were derived from post-natal day (P)1 dissociated rd1, DsRed+, Opn1-EGFP+ retinas that exhibit ubiquitous red fluorescence on an rd1 background and in which enhanced green fluorescent protein (EGFP) expression is restricted to medium wavelength-sensitive cones. These were transplanted into P1 hosts of the following strains: C57BL/6 and Opn4-/-, Gnat1-/-, Cnga3-/- [or TKO (triple knockout)]. The functional response of transplanted cones in TKO mice was confirmed ex vivo by calcium imaging, using the specific mGluR8 agonist, (S)-3,4-dicarboxyphenylglycine (DCPG), in order to detect a similar response in donor to host cells, the latter serving as positive controls. Behavioural light aversion (BLA) was tested using a light-dark box.

 
Results
 

At three weeks after transplantation, cones integrated into the host retina and displayed an atypical morphology (Figure 1). Calcium imaging showed that the application of DCPG resulted in an expected decrease in intracellular calcium concentration in 70.6 ± 1.4% of GFP+ (donor) cells vs. 70.2 ± 0.9% of control (TKO) cells (t12 = 0.67, P = 0.52, N = 4, paired t-test). BLA testing showed that treated TKO (N = 11) and wild type (N = 6) mice spent more time (66.3 ± 5.0% and 79.0 ± 3.5% respectively) in the dark compartment compared to sham-injected mice (N = 10) (53.7 ± 2.3%, F3,34 = 10.5, P = 0.031 and 0.0002 respectively, one-way ANOVA with Dunnett’s post-hoc test).

 
Conclusions
 

These results demonstrate that cone transplantation and restoration of cone-mediated visual behaviour is possible, with calcium imaging demonstrating similar responses in transplanted to host cells. Importantly, as opposed to the findings of previous studies of photoreceptor integration, an atypical morphology excludes cell fusion artefact.

 
 
Integration of cone photoreceptors. Three examples are shown of integrated rd1.DsRed+, Opn1-EGFP+ cones, each with its cell body within the subretinal space and a process extending towards the outer plexiform layer. The identity of each cone is confirmed by the presence of GFP and DsRed fluorescence (A-C) and by anti- arrestin (D-F) and anti-recoverin staining (G-I). Scale bar: 20µm.
 
Integration of cone photoreceptors. Three examples are shown of integrated rd1.DsRed+, Opn1-EGFP+ cones, each with its cell body within the subretinal space and a process extending towards the outer plexiform layer. The identity of each cone is confirmed by the presence of GFP and DsRed fluorescence (A-C) and by anti- arrestin (D-F) and anti-recoverin staining (G-I). Scale bar: 20µm.
 
Keywords: 648 photoreceptors • 698 retinal development • 741 transplantation  
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