REPARACIÓN DEL DAÑO

The aim of RPE transplantation is to retard or restore visual loss. This is primarily achieved through restoration of photoreceptor cell function although there may be indirect beneficial effect through choroidal and retinal circulations. The Royal College of Surgeons (RCS) rat, an animal model of RPE dystrophy (Mullen and Lavail 1976), has been used extensively for in vivo experiments to demonstrate the prove of principle and mechanism of visual rescue in RPE transplantation. This dystrophic strain of rat has a recessive defect in the merTK gene (D'Cruz et al. 2000) which results in failure of RPE to phagocytose shed rod outer segments (Bok and Hall 1971). The consequences of this mutation include accumulation of subretinal debris, death of rod and later cone photoreceptor cells, secondary inner retinal degeneration, retinal vascular changes and central adaptive modulation in neural circuit. Each of these secondary changes has been shown to be prevented or reversed by RPE transplantation in the RCS rat (see Figure 2.3) (Li and Turner 1988a; Seaton et al. 1994; Lund et al. 2001a; Coffey et al. 2002;

Wang et al. 2005a).

2.2.4.1 Photoreceptor cell rescue

The first subretinal RPE grafts in RCS rats demonstrated preservation of the thickness of the outer nuclear layer, outer plexiform layer, and outer and inner segments of the photoreceptors (Li and Turner 1988a). Further more, the outer segment debris zone was reduced with increased number of phagosomes in the transplanted RPE cells (Li and Turner 1988a; Lopez et al. 1989). These rescued photoreceptor cells regenerated outer segments at normal rate (Lavail et al. 1992; Lin et al. 1996), expressed visual pigment, membrane Na⁺/K⁺ ATPase (Sheedlo et al. 1989b) and two synaptic components in the plexiform layers (Sheedlo et al. 1993a) and the outer segments were surrounded by the interphotoreceptor matrix (Lavail et al. 1992). Similar but much more transient photoreceptor rescue was seen after subretinal injection of saline (Silverman and Hughes 1990; Li and Turner 1991; Faktorovich et al. 1990).

Despite histological evidence of rescued photoreceptors with the necessary components for phototransduction, initial attempts at full-field electroretinography (ERG) failed to detect any corneal or vitreal responses to light stimulus after RPE graft (Gouras et al. 1989; Yamamoto et al. 1993). These findings may be due to inadequate number of grafted RPE cells and hence rescued photoreceptors to generate a full field response. A later study was able to demonstrate corneal ERG response in RCS rats that

had received RPE graft as small sheets in a larger area of subretinal space (Jiang and Hamasaki 1994). Corneal ERG after RPE transplantation in RCS rats has since been reported by another group (Sauve et al. 2004; Pinilla et al. 2005; Sauve et al. 2006).

These reports demonstrated a correlation between b-wave amplitude and field area rescued (Sauve et al. 2004), and the relationship between cone/rod ratio in the b-wave and the number of cone rescued (Pinilla et al. 2005). Light- and dark-adaptation study of collicular sensitivity demonstrated that only the cone threshold was prevented from deterioration after RPE graft (Girman et al. 2005).

The disparity between anatomical (Coffey et al. 2002) and functional (Girman et al. 2005) rod rescue raises intriguing questions regarding photoreceptor-donor RPE interaction (see below). One explanation for the lack of rod rescue is that cones are able to depend on Muller cells to recycle visual pigments (Mata et al. 2002) and hence are less dependent on RPE function (Girman et al. 2005).

Figure 2.3 Photoreceptor resuce in the RCS rat model

2.2.4.2 Visual pathway rescue

The function of the post photoreceptor visual pathway after RPE transplantation has been evaluated by electrophysiological and psychophysical measurements. Intraretinal and ganglion cell electrode recording by Yamamoto et al. (1993) demonstrated evidence of activity in bipolar and ganglion cells in response to light in the region of retina adjacent to the graft. Relative retinal sensitivity in the area of graft preservation has also been examined by electrode recording of single- and multi-unit receptive fields over the surface of the superior colliculus of RCS rats that received subretinal RPE allograft The central to peripheral field loss in RCS rats was shown to be limited by subretinal RPE allograft (Sauve et al. 1998) and xenograft from immortalised human RPE cell lines (Lund et al. 2001a; Sauve et al. 2002). Electrophysiological preservation of central visual pathway by RPE xenograft has been correlated with changes in retinal morphology (i.e. outer nuclear layer rescue, see Figure 2.4) and full-field ERG in the RCS rats (Lund et al. 2001a; Sauve et al. 2002; Sauve et al. 2004). In RCS rats that received spontaneously or trans-genetically modified human RPE xenograft with extended life span, single-unit electrophysiological responses to light stimuli have also been recorded in the primary visual cortex, area 17 (Coffey et al. 2002). The ability of these neurons in tuning into a range of specific stimulus parameters such as orientation, direction of movement, contrast sensitivity, spatial and temporal frequencies and complex centre-surround interactions, similar to those found in the normal rats, confirms the ability of human RPE to restore complex visual function in RCS rats (Coffey et al. 2002; Girman et al. 2003).

Non-invasive psychophysical testing, ranging from simple visual reflexes to complex visual tasks, have been used to assess the integrity of the visual system after RPE graft. Whiteley et al. (1996) demonstrated some but incomplete preservation of pupillary light reflexes. By using a water escape paradigm, Little et al. (1998) demonstrated that RCS rats that received human fetal RPE had shorter swimming pathway and time, implying an ability to see and use light as a clue for finding the escape platform. Visual acuity testing by optokinetic head-tracking to moving stripes and two-choice pattern discrimination test of vertical and horizontal stripes has also demonstrated the ability of immortalised RPE xenograft in preserving cortical visual functions for long durations (Lund et al. 2001a; Coffey et al. 2002) (see Figure 2.5).

Using the visual water task under high luminance to measure grating acuity, (McGill et al. 2004) have also demonstrated the ability of ARPE19 subretinal graft in preserving cone mediated vision in these RCS rats.

2.2.4.3 Effects on retinal and choroidal vasculature

The effects of RPE grafts extend beyond retinal and visual pathways. Seaton and colleagues (1992) demonstrated that implanted RPE cells can maintain the density and architecture of the deep retinal vasculature. Further more, in animals that had lost photoreceptors already, RPE graft not only prevented but also induced involution of retinal neovascularisation (Seaton et al. 1994). In a rabbit model of RPE autograft, Majji and De Juan E Jr (2000) demonstrated the ability of autotransplanted RPE suspension in maintaining choriocapillaris as well as photoreceptors. It was concluded that formation of a monolayer on healthy Bruch’s membrane was essential for maintaining the differentiated state of RPE and preserving adjacent choriocapillaris and photoreceptors.

2.2.4.4 Mechanisms of retinal function rescue

Although the mechanism of photoreceptor rescue by donor RPE is unknown, evidence points toward two types of interaction: (1) direct contact between RPE and photoreceptors or (2) indirect interaction through diffusible factors released by donor RPE. Electron microscopic studies suggested cell contact as a requirement for photoreceptor rescue (Lavail et al. 1992). Reduced outer segment debris zone in RCS rats correlated with increased phagocytic activity of the donor RPE (Li and Turner 1988a; Gouras et al. 1989). On the other hand, evidence of rescue extending beyond the grafted area (Lin et al. 1996; Sauve et al. 2002), photoreceptor rescue by basic fibroblast growth factor (bFGF) (Faktorovich et al. 1990), intravitreal RPE graft (Castillo, Jr. et al. 1997) and Schwann cell grafts (Lawrence et al. 2000; Wang et al.

2005b) suggest that diffusible factors such as ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) or bFGF, may be involved in cell rescue.

Variables that have been shown to positively affect the rescue of photoreceptors following RPE allograft transplantation include younger donor age (Sheedlo et al.

1993b), higher number of donor cells and fresh rather than cultured cells (Li and Turner 1991). Durlu and Tamai (1997) demonstrated photoreceptor rescue in RCS rats receiving cryopreserved bovine RPE xenograft. Fresh human fetal (Little et al. 1996), juvenile or adult (Castillo, Jr. et al. 1997) RPE as a xenograft has been shown to support photoreceptor survival and visual function (Little et al. 1998). Both spontaneously immortalised RPE and genetically modified RPE with extended lifespan have the ability to restore visual field and cortical visual function (Lund et al. 2001a). Although efficacy

of xenogeneic RPE grafts has been well documented, there has been no study which directly compares the efficacy of allogeneic to xenogeneic RPE grafts.

Figure 2.4 Functional rescue after RPE graft in the RCS rat

A comparison of retinal sensitivity and histological maps following ARPE-19

transplantation in a dystrophic RCS rat. The figure shows (a) the retinal sensitivity map from electrode recording at the superior colliculus and (b) the corresponding outer nuclear layer cell count in the retina. The grey scale coding indicates the corresponding retinal and superior collicular areas. The highest sensitivity correlates to the area of ARPE-19 transplantation (Courtesy of Professor Pete Coffey).

Figure 2.5 Optokinetic reflex testing in rats

A diagram showing the various aspects of visual acuity testing based on the optokinetic reflex. A rotating drum is used with three different square-wave grating frequencies.

The animal is placed inside the rotating drum which stimulates head turning in the test animal. The highest frequency grating that stimulates head turning indicates the visual acuity (Courtesy of Professor Pete Coffey).

Genes introduced into the donor RPE prior to transplantation have been shown to be expressed in the subretinal space (Osusky et al. 1995; Dunaief et al. 1995; Lai et al. 1999; Lai et al. 2000; Saigo et al. 2004; Abe et al. 2005). For example, Abe and colleagues (2005) demonstrated that BDNF-transfected RPE grafts resulted in significant photoreceptor rescue in both grafted and non-grafted areas. Genetically transduced donor RPE may serve as a vehicle for genetic therapy in addition to cellular therapy in retinal degeneration (Ogata et al. 1999).