Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

Abstract

Aims: The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits.

Methods: Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy.

Results: Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells.

Conclusions: Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.

Figure 1

Phase contrast micrograph of retinal pigment epithelial cells from rabbit embryos in primary culture used for transplantation. Bar = 100 µm.

Figure 2

Non-damaged versus damaged Bruch's membrane (b) immediately after the surgery. The denuded Bruch's membrane with the intact choriocapillaris (A), in contrast with haemorrhage (arrow in B) from a rupture of Bruch's membrane with plugged choriocapillaris (arrow in C) at one site, and the RPE basement membrane (arrowhead in D) removed from the elastic layer (arrow in D) at another site. (A), (B) PAS stain, bar = 100 µm. (C), (D) Uranyl acetate and lead citrate stain, bar = 2 µm.

Figure 3

Seven days after the surgery without transplantation. The host RPE cells without polarity layer on the damaged area (between arrows in A) of Bruch's membrane (b), while monolayered RPE cells with the normal polarity repopulate the non-damaged Bruch's membrane (b) in close contact with retinal photoreceptor outer segments (B). Uranyl acetate and lead citrate stain, bar = 2 µm.

Figure 4

RPE cells on damaged Bruch's membrane (b) 3 days after transplantation. Pigmented ( arrows in A) and non-pigmented RPE cell layer over the damaged area of Bruch's membrane. RPE cells with melanin granules cover a break (between arrows in B) in Bruch's membrane (b). Basal infoldings and microvilli are absent. (A) Azure stain, bar = 100 µm. (B) Uranyl acetate and lead citrate stain, bar = 2 µm.

Figure 5

RPE cells on moderately damaged versus severely damaged Bruch's membrane (b) 7 days after transplantation. Viable pigmented RPE cells (small arrow in A) showed apical microvilli and basal infoldings spreading on the mildly damaged Bruch's membrane. Some RPE cells lose viability as indicated by a small number of intracellular organelles (asterisks in A, B, C). Bruch's membrane (b) has such aberrant sites as a fibroblast invading its inner collagenous layer (large arrow in A), protrusion of a disrupted end of the elastic layer (arrow in B), or convoluted RPE basement membrane (arrowheads in C) with basal infoldings (arrow in C). Fibroblast-like cells form multiple layers on the severely damaged Bruch's membrane (D). Uranyl acetate and lead citrate stain, bar = 2 µm.

Figure 6

RPE cells on damaged Bruch's membrane (b) 14 days after transplantation. Subretinal fibrovascular tissue with capillary lumens (arrows in A) above PAS positive Bruch's membrane (arrowheads in A) , containing pigmented RPE cells (arrows in B) with tubuloacinar formation. A pigmented RPE cell with normal apical microvilli (arrowheads in C) and basal infoldings (arrows in C) on mildly damaged Bruch's membrane, in contrast with a pigmented cell (asterisk in D) which covers a break of full thickness of Bruch's membrane in the severely damaged area and reproduces its basement membrane (arrowheads in D). The inner nuclear layer of the retina (arrows in E) is attached to fibrous scar, and the outer nuclear layer (arrowhead in E) is in close proximity to pigmented RPE cells at some sites. (A), (E) PAS stain, bar = 100 µm. (B), (C), (D) Uranyl acetate and lead citrate stain, bar = 2 µm.

Figure 7

RPE cells on non-damaged Bruch's membrane (b) 7 days after transplantation. The retina is attached to pigmented RPE cells (arrows in A). These cells on the preserved basement membrane of the host RPE form monolayers or bilayers and show basal infoldings, apical microvilli, and tight junctions (arrow in B). (A) Azure stain, bar = 100 µm. (B) Uranyl acetate and lead citrate stain, bar = 2 µm.