BROTES DE CRECIMIENTO

Short term survival of donor RPE depends on how the cells were delivered. As cell suspension, the RPE will need to attach to a suitable basement membrane to prevent apoptosis (Tezel and Del Priore 1997). Autologous RPE-choroid patch graft, however, requires establishment of choroidal perfusion through vascular re-connection with adjacent or underlying choroid at the recipient site. Longer term survival of donor RPE largely depends on their sources. Allogeneic graft faces the barrier of immunological rejection within the first 6 months whereas autologous equatorial RPE may not be able to support foveal cone functions for long durations. Furthermore, delayed patch graft ischaemia may lead to late visual loss. The study of donor RPE survival and integration during the first months after grafting has been facilitated by the use of silicone oil as tamponade. Graft survival, structure and perfusion have been studied directly using AF imaging, OCT and high-speed ICG angiography, and indirectly through mapping of the preferred retinal locus for fixation and microperimetry. The following sections will address RPE survival in allogeneic grafts, autologous cell suspension grafts and autologous RPE-choroid patch grafts respectively.

2.3.7.1 Allogeneic RPE suspension and patch grafts

As discussed above, a short course of oral steroid (Algvere et al. 1994), or a 6-month course of cyclosporine and azathioprine with or without systemic steroid (Del Priore et al. 2001; Tezel et al. 2007) have been used for immunosuppression in patients receiving

allogeneic grafts. RPE allograft survival after delivery as cell suspension is difficult to evaluate. Algvere et al. (1994) have reported increased pigmentation in the macular region as a sign of cell adherence and growth. Although no specific features on autofluorescence imaging or angiography have suggested RPE suspension graft survival, improvement in retinal sensitivity on microperimetry may reflect rescued photoreceptor by viable donor RPE (Weisz et al. 1999). However, given the poor adhesion of uncultured adult, fetal and immortalised RPE cell lines to aged or damaged human BM (Zarbin 2003; Del Priore and Tezel 1998; Tezel et al. 2004), it is unlikely that cell suspension graft would have survived (see below). On the other hand, fetal and adult allogeneic RPE patch grafts appear to survive while the patient was immunosuppressed (Algvere et al. 1994; Tezel et al. 2007). There has been no OCT study of the structure allogeneic grafts.

Immune rejection is a significant barrier to long-term graft survival in allograft.

It was proposed that the loss of immune privilege in the subretinal space following membrane removal was the cause of patch allograft rejection in all 7 patients with neovascular AMD in the study by Algvere et al. (1999). Interestingly, these grafts maintained visual function during the first month. Between 1 and 3 months, macular oedema, fluorescein leakage and graft depigmentation occurred, followed by progression to fibrous encapsulation after 3 months (Algvere et al. 1994). In contrast, extrafoveal fetal RPE patch allografts in GA were not rejected after 30 months with the exception of one case which was rejected within 6 months. In this patient, the graft was placed adjacent to the retinotomy; a site of damaged blood retinal barrier. Subfoveal fetal RPE allograft as a cell suspension in atrophic AMD was rejected after 12 months.

Similarly, in 2 patients with RPE rip, the fetal RPE suspension allograft was also rejected within 6 months. Algvere et al. (1999) concluded that rejection is common but not inevitable and the risk factors for rejection include CNV removal, larger number of cells transplanted and proximity of transplant to fovea or retinotomy. As described previously, 2 of 12 patients from Tezel and colleagues’ series (2007) developed fibrosis and haemorrhage around the graft within 1 month of discontinuing immunosuppression.

The authors attributed this to graft rejection given the temporal relationship.

Other evidence of immune activation is the presence of autoreactive lymphocytes (against retinal antigens) in a patient who received RPE allograft.

However, it was not possible to determine if the surgical procedure or the transplantation was the cause (Weisz et al. 1999). Histological examination of the eye at 2 – 3 months after adult allogeneic RPE patch graft did not demonstrate excessive

inflammatory cell infiltrate (Del Priore et al. 2001). As the mechanism of RPE allograft rejection is unknown, current immunosuppressive regime will only be empirical until the mechanism of RPE rejection is understood.

2.3.7.2 Autologous RPE suspension and patch grafts

Survival of autologous RPE suspension on damaged BM after subretinal injection has been difficult to demonstrate. Binder and colleagues (Binder et al. 2004; Krebs et al.

2008) were unable to provide evidence of cell survival following autologous RPE suspension graft although they showed visual improvement in some patients. Although the authors suggested the use of an in vivo donor RPE marker to determine if these RPE cells survived, absence of donor RPE will not be a unexpected given previous ex vivo experiments have demonstrated poor adhesion of uncultured adult, fetal and immortalised RPE cell lines to aged or damaged human BM (Zarbin 2003; Del Priore and Tezel 1998; Tezel et al. 2004). Since cells are unlikely to have survived on damaged BM, the Long-term survival of submacular injection of peripheral RPE cell suspension becomes unlikely.

Early survival and function of RPE-choroid patch grafts depends on minimal surgical trauma to the RPE during harvesting and delivery and early perfusion of the graft. Factors that may impact on late equatorial RPE-choroid graft function include;

size of the patch, ability of equatorial RPE cell to maintain foveal cones, primary photoreceptor cell dysfunction and delayed graft ischaemia.

Surgical trauma to donor RPE may occur during harvesting of donor RPE, loading of the graft on instruments, insertion through retinotomy and release of the graft and withdrawal of instruments. Electron microscopic examination of the RPE-choroid donor patch demonstrated intact Bruch’s membrane but patchy loss of RPE or RPE cells with avulsed apical membrane (MacLaren et al. 2007). Placement of folded or even inverted graft significantly reduces the ability of the patch graft in supporting photoreceptor cells (Stanga et al. 2002; van Meurs et al. 2006; Joussen et al. 2006).

Further evidence of the importance of surgical trauma in relation to outcome was provided by Maaijwee and colleagues (Maaijwee et al. 2008c).

Graft perfusion has been detected as early as 1 week to as late as 4 months after patch graft using either early frames of FFA or high-speed ICG angiography (Joussen et al. 2006; Treumer et al. 2007a; Maaijwee et al. 2008d). It has also been shown that reconnection of vascular channels between the patch and the recipient choroidal bed can occur at the edge of the graft (see Figure 2.8). Joussen et al. (2006), on the other hand,

reported that the patch is perfused from the underlying choroid. Serial ICG angiography demonstrated initial reconnection of vascular channels and later remodelling of the RPE-choroid patch vasculature (Joussen et al. 2006). It is not known if there is ingrowth of underlying or adjacent choroidal vessels into the patch graft and recruitment of marrow-derived endothelial progenitor cells, similar to that found in skin graft integration (Capla et al. 2006). The process from initial connection to complete graft revascularisation may take up to 3 weeks (Joussen et al. 2006).

Failure of the graft to revascularise can be due to (1) separation of the graft from adjacent or underlying choroid by haemorrhage or (2) absence of choroid vessels around the graft. Absence of medium sized choroidal vessels may occur in areas affected by GA or following excision of CNV, previous repeated photodynamic therapy or possibly previous repeated anti-VEGF therapy. It is now generally accepted that a pedicle of choroid is not necessary for RPE-choroid patch survival as was suggested by Yepez and Arevalo (2003). With time, ischaemic RPE-choroid graft becomes encased in fibrous tissue. If the patch was only partially vascularised, the avascular portion of the graft may undergo fibrosis. The reported rates of failure to revascularise ranged from 6 to 11% (van Meurs 2005; Joussen et al. 2006; MacLaren et al. 2007; Treumer et al. 2007a;

Maaijwee et al. 2008d). In Joussen’s series (2006), 4 patients with failed graft vascularisation received a second RPE choroid patch, and 1 of these remained non-perfused. Revascularisation rate in patients with GA is similar to those in patients with neovascular AMD with only 2 of 12 patients demonstrated incomplete or absence of graft perfusion (Joussen et al. 2007). These 2 patients did not have intentional damage to BM prior to insertion of the grafts. Once perfused, the patch remained vascularised during the entire follow up period except for one case reported by Joussen et al. (2006) where patch perfusion had reduced at 6 months in a patient with GA. It is important to note that presence of vascularisation or autofluorescence signal do not always correlated with visual function (Joussen et al. 2006). Furthermore, a non-perfused graft (and presumably, dying RPE cells) may retain autofluorescence signal (derived from peripheral photoreceptors) for as long as 6 weeks after transplantation (Joussen et al.

2006; Treumer et al. 2007a). Conversely, a vascularised graft may also loose its autofluorescence signal years later due to possible delayed photoreceptor (MacLaren et al. 2005) or RPE cell death.

Long-term equatorial RPE-choroid graft survival has not been well characterised. Although there has been no study which correlated equatorial RPE cell function to outcome, it has been assumed that patients with peripheral degenerative

changes and those with full-field ERG abnormality may not benefit from autologous RPE-choroid patch graft. Following submacular RPE-choroid flap rotation or graft reposition, Stanga et al. (2002) noted development of fibrosis around the graft and lack of visual improvement after 10 months. Although they attributed these to primary graft failure from poor revascularization, long term (4-5 years) follow up on 4 of the 9 patients revealed that the grafts were in fact perfused. MacLaren et al. (2005) concluded that the failure of visual improvement and progressive visual loss may in fact be due to a primary progressive photoreceptor cell death rather than primary or secondary graft failure. It was proposed that the insult of surgery or continuation of natural history of the disease led to progressive photoreceptor cell loss. Joeres et al. (2008) used OCT To study the structure graft in 29 patients and found a trend that eyes with thinner grafts had better VA than those with thicker grafts at 6 months. Also, despite the significant undulation of the graft surface, most patient did not complain of metamorphopsia (Joeres et al. 2008).

Figure 2.8 Equatorial autologous RPE graft: angiography

Colour fundus photographs and indocyanine green angiographies (a, c) before and (b, d) after autologous RPE-choroid patch graft, showing intrinsic vascular filling pattern (white arrow) within the graft which is significantly different from the corresponding region (yellow outline) in the preoperative angiogram (yellow arrow), suggesting perfusion of the transplanted choroid.