The present results show that subretinal grafts of Schwann cell lines both preserve photoreceptors from degeneration and slow the deterioration of visual function in the RCS rat. The SCTM41-GDNF Schwann cell line produced more sustained preservation of photoreceptors and function than SCTM41-BDNF grafts. It is not known why BDNF and GDNF produced different degrees of rescue. It may simply be that the BDNF level achieved by the present number of cells was less than optimal. Alternatively, the two growth factors may activate different pathways. It is clear, however, that the cell lines engineered to secrete GDNF and BDNF showed better functional and anatomic rescue than the parent cell line, adding support to the concept that growth factor delivery is an important component. The parent cell line, by being homologous, provided an appropriate control not often available in experiments of this kind.
Both direct injection of GDNF and adenovirus delivery of GDNF to the eye have been shown to slow photoreceptor loss in various rodent models of retinal degeneration (e.g., Refs.
6 8 19 ), and GDNF receptors have been detected on photoreceptors
20 21 and on Müller glia.
21 In vitro studies have confirmed that GDNF can promote photoreceptor survival,
20 22 reduce apoptosis, and augment opsin expression,
22 suggesting a role as survival and differentiation factors. However, low concentrations of GDNF appear not to exert a rescue effect,
23 and endogenous levels of GDNF fail to prevent photoreceptor loss in the
rd mouse.
6 In accord with this, we found in pilot experiments preliminary to the present sequence of studies, that SCTM41-GDNF-secreting cells injected at the lower level of 2.2 × 10
5cells/100 μL failed to give better head-tracking than the parent SCTM41 line injected at the same concentration, suggesting a dose effect (Lawrence JM, unpublished results, 2001).
BDNF injected into the eye has also been shown to reduce photoreceptor loss after injection in vivo
2 7 ; however, in isolated cell culture
23 BDNF had no effect on rod outer segment survival. Because TrkB receptors (for BDNF) have not been identified on photoreceptors (except on red-green cones
24 ) but have been identified on RPE
25 26 and Müller cells,
25 the protective effect of most survival factors is likely to be mediated indirectly through these cells. In support of this, Wahlin et al.,
27 showed that BDNF, CNTF, and bFGF did not activate intracellular signaling pathways in photoreceptors but did so in Müller cells.
27 Because of the pigmentation in the RPE cells, they were unable to identify reactivity in RPE cells.
In transplantation studies, preservation of function is a necessary measure of success. Improved electroretinogram recordings have been observed after GDNF administration to the
rd mouse
6 and the TgN S344 ter-4 rat.
8 In this study, we used head-tracking behavior which, in previous studies, has been shown to be a good predictor of successful grafts.
28 The best-performing graft-recipient eyes tracked for far longer periods than those with sham surgery and GDNF-treated eyes showed better preservation of function than eyes treated in any other way. The pattern of head-tracking in the engineered rat Schwann cells is similar to that described for engineered human retinal pigment epithelial cells,
17 with a good response early on that deteriorates with time. Thus, at 9 to 10 weeks after surgery, dystrophic animals with engineered cell grafts (both RPE and Schwann) were able to track all gratings and performed significantly better than sham-surgery or nonsurgical dystrophic animals. By 17 to 20 weeks after surgery, sham-surgery animals in both experiments were unable to track any of the gratings. Animals with engineered cell (RPE and Schwann) grafts, however, performed significantly better at both the 0.25 and 0.125 gratings but showed little or no tracking with the 0.5 grating. Although dystrophic rats with GDNF- or BDNF-engineered cell grafts show a deterioration of head-tracking behavior with time, it not necessarily mean that all visual function has been lost, because photoreceptors remain
(Fig. 6) , and electrophysiological studies recording from the superior colliculus and visual cortex of animals whose eyes had received engineered RPE cells
17 or immortalized human RPE cell grafts
28 showed continued function for at least 6 to 8 months. It is not known why head-tracking behavior deteriorates over time when there is evidence of vision using other tests, but these studies have shown that good head-tracking behavior at earlier time points correlates with good performance in other visual tests.
28 It should be noted, however, that the donor cells are allografts and there may be a slow loss of cells because of some form of rejection.
29
Although levels of CyA were not determined in this study, Lund et al.,
17 found a mean blood level of 321.6 ± 21.9 μg/L in RCS rats where CyA from the same source was administered in an identical dose and manner. However, CyA is poorly absorbed,
30 and a sub-group of T cells have been shown to become resistant to immunosuppressive drugs (such as CyA, see Ref.
31 ). These cells may be important contributors to chronic graft rejection. Although there was no evidence of cell infiltration or vessel-cuffing in the initial weeks after grafting, it may be that as the vessels in the RCS rat become leaky (a feature of the vascular changes in this animal with time, e.g., Ref.
18 ) and the CyA treatment becomes less effective, some nonclassic form of rejection occurs, possibly reducing the area of the graft. This is the subject of further studies. Even when syngeneic donor cells are used in the RCS rat, there is a reduction in the number of rescued photoreceptors with time.
12 Possibly the extensive remodeling of the retina that occurs in the dystrophic RCS rat
18 affects graft survival.
This study shows that Schwann cell lines engineered to overexpress growth factors can prolong photoreceptor survival and function, supporting our original findings with syngeneic grafts of primary Schwann cells.
12 In accord with the findings of Wilby et al.,
13 the engineered cells performed better than the parent cell line, suggesting that it is the additional growth factor production that is affecting photoreceptor survival. It remains to be seen whether rescue cam be further improved by grafting a mixture of the two engineered cell lines to a single eye or adding yet other cells engineered to make additional factors.
The results add to recent work
32 that has indicated that ex vivo gene therapy coupled with transplantation may be a useful approach for preserving photoreceptors in retinal degenerative conditions. Transfected cells can be carefully monitored before introduction into the eye for factor production levels, for stability of factor production, and for any indication that the introduced factors might change the behavior of the cell in a detrimental way.
The authors thank Robin Howes for valuable assistance.