1 Introduction Retinal degeneration is the leading cause of untreatable blindness in the developed world. Current clinical treatments are limited, at best only slowing disease progression and very rarely restoring visual function. Cell transplantation offers a novel therapeutic approach, enabling the replacement of photoreceptor cells lost in the degenerative process. Photoreceptor transplantation may be more feasible than other types of neuronal transplantation, because photoreceptors are stimulated by light and their function is not, therefore, dependent on the reformation of complex afferent connections. Nevertheless, an efferent connection to host second order sensory neurons in the retina is essential for visual function and this is arguably best achieved if the transplanted photoreceptor is fully integrated into the host outer nuclear layer (ONL). Ghosh and Ehinger, 2000; Royo and Quay, 1959; Seiler et al., 1990; Zhang et al., 2003b Takahashi et al., 1998; Young et al., 2000 Ahmad et al., 2000; Chacko et al., 2000; Coles et al., 2004; Klassen et al., 2004; Qiu et al., 2005; Yang et al., 2002 Sakaguchi et al., 2003, 2004 MacLaren, 1996 MacLaren et al., 2006 Komuro and Rakic, 1998a,b; Nadarajah and Parnavelas, 2002; Parnavelas et al., 2002; Pearson et al., 2005 Fig. 1 Uga and Smelser, 1973; Woodford and Blanks, 1989 Fig. 1 Karlsen et al., 1982; Pedersen and Karlsen, 1979 Ishikawa and Mine, 1983 2 Materials and methods 2.1 Animals +/+ Akimoto et al., 2006 +/+ 2.2 α-Aminoadipic acid formulation and administration dl For histological assessment, mice were sacrificed at various time points (3–4 mice per time-point) and the eyes were fixed in buffered formalin for 48 h at 4 °C. Retinal sections were prepared by overnight dehydration and paraffin embedding (Histocentre). Sections (5 μm thick) were cut and affixed to glass slides and stained using standard haematoxylin and eosin protocols. 2.3 Dissociation of retinal cells and transplantation +/+ MacLaren et al., 2006 5 MacLaren et al., 2006 2.4 Histology and immunohistochemistry in situ 2.5 Electron microscopy Tschernutter et al., 2005 2.6 Confocal microscopy MacLaren et al., 2006 2.7 Integrated cell counts MacLaren et al., 2006 Ishikawa and Mine, 1983 3 P N 2.8 Apoptotic cell counts The number of apoptotic cells was determined by counting all TUNEL-positive profiles in each layer of the retina in alternate serial sections. Only sections that encompassed the site of intravitreal injection were used and are thus not representative of apoptosis in the whole eye. 2.9 Statistics t P N n 3 Results 3.1 Dosage and route of AAA administration N N N Ishikawa and Mine, 1983; Pedersen and Karlsen, 1979; Rich et al., 1995 N N N Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Fig. 2 Pedersen and Karlsen, 1979; Rich et al., 1995 3.2 Window of OLM disruption N +/+ Akimoto et al., 2006; Mears et al., 2001; Swain et al., 2001 Fig. 3 Fig. 1 Fig. 3 Karlsen et al., 1982; Pedersen and Karlsen, 1979 Fig. 3 n n n d l Tsai et al., 1996 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Together, these findings demonstrate that intravitreal administration of 100 μg AAA in the mouse causes a transient, reversible disruption of the OLM approximately 72 h post injection. 3.3 Cell integration with OLM disruption +/+ Nrl.gfp Akimoto et al., 2006; Mears et al., 2001; Swain et al., 2001 +/− MacLaren et al., 2006 N P t Fig. 5 N N Fig. 5 Fig. 5 N P t Fig. 5 Ishikawa and Mine, 1983; Khurgel et al., 1996 Rich et al. (1995) Supplementary Fig. 1 MacLaren et al., 2006 Fig. 5 4 Discussion Here we demonstrate that the OLM in the adult mouse retina can be transiently disrupted by the intravitreal administration of AAA. OLM disruption is maximal approximately 72 h post administration. When combined with precursor cell transplantation, this time point correlates with a significantly enhanced level of transplanted photoreceptor cell integration into the recipient ONL, compared with sham-injected controls. These findings suggest that the OLM represents a natural barrier to the successful integration of photoreceptor precursor cells transplanted into the subretinal space. Consideration of the OLM may therefore be important in any future clinical photoreceptor transplantation strategies directed towards retinal repair. 4.1 Effect of AAA on Müller cells and the OLM Karlsen et al., 1982; Olney, 1982; Pedersen and Karlsen, 1979 Sugawara et al., 1990 Ishikawa and Mine, 1983; Khurgel et al., 1996 Pedersen and Karlsen, 1979 Tsai et al., 1996 Kato et al., 1993 Chang et al., 1997; McBean, 1994 Tsai et al., 1996 4.2 OLM disruption and photoreceptor integration MacLaren et al., 2006 Hagg, 2005; Komuro and Rakic, 1998a; Nadarajah and Parnavelas, 2002; Parnavelas et al., 2002; Pearson et al., 2005 Zhang et al., 2003a Zhang et al., 2004 Ghosh et al., 1999; Gouras et al., 1994; Zhang et al., 1999 Zhang et al., 2003a Nishida et al., 2000 Paffenholz et al., 1999; Williams et al., 1990 Rich et al., 1995 Kljavin and Reh, 1991 MacLaren et al., 2006 Rich et al., 1995 Bignami and Dahl, 1979; Bjorklund et al., 1985 Kinouchi et al. (2003) Kinouchi et al., 2003 4.3 Therapeutic implications l Kato et al., 1990; Pedersen and Karlsen, 1979; Sugawara et al., 1990 Yanoff et al., 1984 Appendix Supplementary information Figure 1 a b a, b,  Appendix A Supplementary data 10.1016/j.exer.2008.01.004