Fig 1.
Local removal of internal limiting membrane and subretinal injection of balanced salt solution.
(A) Schematic drawing of local internal limiting membrane (ILM) removal showing the removed ILM (arrow), retina (asterisk), and intact ILM (arrowhead). (B) Schematic drawing of the subretinal injection procedure. Balanced salt solution (BSS) was injected with a 38-gauge cannula by placing the cannula tip in contact with the retinal nerve fiber layer. The arrow indicates the flow of the injected BSS; the asterisk and arrowhead indicate the retina and ILM, respectively. (C) Surgical photograph after local ILM removal. The arrow indicates the 38-gauge cannula; the arrowhead indicates the area of peeled ILM. (D) Surgical photograph during subretinal injection. The arrow indicates the 38-gauge cannula; arrowheads indicate the area of retinal detachment due to subretinal injection of BSS. (E) Optical coherence tomography 30 minutes after subretinal injection. The asterisk indicates retinal detachment caused by the procedure. Scale bar = 200 μm.
Fig 2.
Optical coherence tomography images of monkey retina after subretinal injection of balanced salt solution.
Optical coherence tomography (OCT) images of both control (no injection of balanced salt solution; BSS) and minimum-pressure (BSS injection at 6 psi) groups show a well-preserved retinal structure throughout the experimental period, including continuity of the ellipsoid zone (EZ) (A to F). OCT image from the high-pressure group (BSS injection at 20 psi) show EZ disruption (asterisk in G) at 1 week after the injection (G). At 3 weeks after the injection (H), OCT image from the high-pressure group shows partial recovery of the EZ (asterisk). The EZ finally became continuous (asterisk in I) at 5 weeks after the injection. Arrows in A, D, and G indicate the EZ. Scale bars = 200 μm.
Fig 3.
Light microscopy images of monkey retina after subretinal injection of balanced salt solution.
The retinal structures of both the control (no injection of balanced salt solution; BSS) and the minimum-pressure (BSS injection at 6 psi) groups are well-preserved at 1 week (A and C) and 6 weeks (B and D) after injection. The high injection pressure group (BSS injection at 20 psi) shows thinning of the photoreceptor outer segment layer (OS, arrows in E) and thickening of the retinal pigment epithelium (RPE) layer (asterisk in E) at 1 week after injection (E), while the photoreceptor cells are well-preserved. At 6 weeks after injection, the high-pressure group shows restoration of the OS (arrows in F) and flattening of the RPE (asterisk in F). Scale bars = 100 μm.
Fig 4.
Transmission electron microscopy images of monkey retina after subretinal injection of balanced salt solution.
Retinal structures of the control group (no injection of balanced salt solution; BSS) are well-preserved at 1 week (A) and 6 weeks (B) after injection. The minimum-pressure group (BSS injection at 6 psi) shows a shorter photoreceptor outer segment (OS) than the control group at 1 week after injection (C, bidirectional arrow). The OS is restored at 6 weeks after injection (D, bidirectional arrow). The retinal pigment epithelium (RPE) is well-preserved throughout the experimental period (C and D). Conversely, the high-pressure group (BSS injection at 20 psi) shows degeneration of the OS (bidirectional arrow in E) and migration of RPE cells (arrows in E), leading to multiple RPE layers at 1 week after injection. Regeneration of the OS (bidirectional arrow in F) and flattening of the RPE (arrows in F) were observed at 6 weeks after the injection. Scale bars = 10 μm.
Fig 5.
The effect of subretinal injection on photoreceptor cells.
In the minimum-pressure (injection at 6 psi with balanced salt solution; BSS) and high-pressure (BSS injection at 20 psi) groups, both light microscopy images with toluidine blue staining and transmission electron microscopy (TEM) images show normal morphology of photoreceptor cells at 1 week (A, C, G and I) and 6 weeks (D, F, J, and L) after injection. TdT-dUTP terminal nick-end labeling (TUNEL) showed no positive photoreceptor cells in either the minimum-pressure or high-pressure groups throughout the experimental period (B, E, H, and K). Black and white scale bars = 20 μm. Positive control of TUNEL staining is shown in S5 Fig.