Fig 1.
Light microscopy of developing mouse retina after birth.
The Lpcat1 deficient rd11 (lower row) and WT C57BL/6J retinas (upper row) had similar shape and thickness from P2 to P14. The ONL was completely differentiated from the NL at approximately P10. In WT retinas, the OS reached mature length with loose discs attached to the RPE at approximately P14. After P14, the thickness of the rd11 retina was markedly reduced due to RD. Age-matched WT C57BL/6J mice were used as controls. NL, neuroblastic layer; GCL, ganglion cell layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IS, inner segments; OS, outer segments; P, postnatal day; WT, wild-type.
Fig 2.
The extent of the diffusion of subretinally injected vector in rd11 mouse retinas.
In the treated rd11 eyes, 1 μL of the AAV8 (Y733F) vector was injected subretinally at postnatal day 10 (P10), 14, 18, or 22. A small amount of green fluorescein was added to help visualize the diffusion of the vector (A, black arrows). White arrows showed the area there was no accumulation of vector. *Statistical analysis (B) indicated a significant difference in the percentage of the retina covered by the vector after diffusion (P < 0.001) in the P10 group, compared to the P14, P18, and P22 groups. No statistical difference in the percentage of the retina covered by vector was found between the P14, P18, and P22 treated mice (P > 0.05). Columns and bars represent mean ± SD (n = 8 mice).
Fig 3.
Retinal sections of rd11 mice treated with gene therapy at different postnatal days.
(A) Representative images of H&E-stained retinal sections at 4 months after treatments in P10 to P22-treated groups. Due to the limited diffusion of the vector, whole retinas of the P10 group had both rescued (vector coverage) and unrescued (out of vector coverage) areas. (B) Averaged retinal thicknesses in age-matched WT C57BL/6J, P10 to P22-treated, and untreated rd11 eyes (n = 8 mice). Mice were born and reared under normal, cyclic light conditions (12 hours light, 12 hours dark), with the exception of one group of P14 treated rd11 neonates that was reared under dim red light to protect their retinas from light until 10 days after gene delivery. At an equivalent distance (0.3 mm) from the optic nerve, retinal thickness was measured from the vitreal face of the GCL to the apical face of the RPE. Age-matched C57BL/6J and untreated rd11 mice were as controls. P, postnatal day; Inj, injected; M, months. *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001, NS = no statistical difference.
Fig 4.
LPCAT1, rod rhodopsin, and cone opsin expression following AAV8 (Y733F) vector treatments on different postnatal days.
Retinal images of the posterior pole segments were collected at a distance of 0.3 mm from the optic nerve. (A) LPCAT1 immunostaining (red) of rd11 retinas at 4 months after treatment in P10 to P22-treated mice. (B) Double staining of rod rhodopsin (green) and cone opsin (red) of rd11 retinas at 4 months after treatments. Due to limited diffusion of the vector, the retinas of the P10 treated group had both rescued (vector coverage) and unrescued (out of vector coverage) areas. Age-matched wild-type C57BL/6J and untreated rd11 mice were used as controls. Nuclei were stained with DAPI (blue). P, postnatal day; Inj, injected; M, months.
Fig 5.
Dark-adapted ERGs from rd11 mice following AAV8 (Y733F) vector treatments on different postnatal days.
(A) Representative scotopic ERGs elicited at 3.0 cd∙s/m2 flash intensity in rd11 eyes 4 months after treatments at P10, 14, 18, or 22. Scotopic a-wave (B) and b-wave amplitudes (C) elicited at 3.0 cd∙s/m2 intensity were compared among age-matched wild-type C57BL/6J, P10 to P22 treated, and untreated rd11 eyes (n = 8 mice). Age-matched wild-type C57BL/6J and untreated rd11 mice were used as controls. P, postnatal day; Inj, injected; M, months. **indicates P < 0.01, ***indicates P < 0.001, NS = no statistically significant difference.
Table 1.
Dark-adapted ERG (3.0 cd∙s/m2) Amplitudes from rd11 Mice at 4 Months after Treatment.
Fig 6.
M-cone and S-cone opsins preservation in retinal whole mounts after AAV8 (Y733F) vector treatments at different postnatal days.
Retinal M-cone opsins (red) and S-cone opsins (white) of the posterior pole segments were imaged (A) at a distance of 0.3 mm from the optic nerve, and counted (B, C) within one field (44,139 μm2) at a high magnification (×40). Due to limited diffusion of the vector, the retinas of the P10 group mice had both rescued (under vector coverage) and unrescued (out of vector coverage) areas. Age-matched wild-type C57BL/6J and untreated rd11 mice were used as controls. P, postnatal day; Inj, injected; M, months. *indicates P < 0.05, ***indicates P < 0.001, NS = no statistical difference.
Fig 7.
M-cone and S-cone ERGs from rd11 mice following AAV8 (Y733F) vector treatments on different postnatal days.
(A) Photopic ERGs in rd11 eyes, 4 months after treatments at P10, 14, 18, or 22. M-cone ERGs were recorded at a green light intensity of 0.75 cd∙s/m2 while S-cone ERGs were recorded at a UV light intensity of 3.00 mW∙s/m2. M-cone (B) and S-cone ERG amplitudes (C) were compared between age-matched wild-type C57BL/6J, P10 to P22 treated, and untreated rd11 eyes (n = 8 mice). Age-matched wild-type C57BL/6J and untreated rd11 mice were used as controls. P, postnatal day; Inj, injected; M, months. **indicates P < 0.01, ***indicates P < 0.001, NS = no statistical difference.