Animal Studies

a. RPE Transplant. An animal model of hereditary retinal degeneration, often used to study the effects of transplants in the eyes, is the Royal College of Surgeons (RCS) rats. These animals suffer from a well-characterized, early-onset form of photoreceptor cell degeneration. One of the earliest indications that grafts of human fetal RPE cells are capable of rescuing photoreceptor degeneration in RCS rats was found in a report by Little and coworkers (26). In the study, sheets of healthy fetal human RPE cells were transplanted into the subretinal space of dystrophic RCS rats that have been immunosuppressed with cyclosporine. The thickness of the outer nuclear layer (ONL), which contains the photoreceptor cells, was measured 4 weeks after transplantation. A fourfold increase in thickness of the ONL, in the area where the grafts were placed, was observed after the transplant period. Furthermore, the protected cells in the ONL were morphologically similar to their normal counterparts in healthy retinas. ONL thickness remained unchanged in areas of the retina distant from the site of transplant or in the retina of sham injected controls. This preliminary study indicates that transplanted RPE cells are capable of rescuing a significant percentage of photoreceptors from degenerating.

One question raised by many researchers in the ophthalmic field is: Are the rescued photoreceptor cells functional? Results from a follow-up study suggested that morphological as well as functional rescue of photo-receptor cells can be achieved with RPE transplants (27). Data from the study showed that a progressive central to peripheral loss of visual responsiveness occurs in pigmented dystrophic RCS rats. Recordings of single and multiunit receptive fields across the surface of the superior colliculus were used to determine the changes in visual responsiveness in the rat retinas. The potential of RPE transplants to slow down progressive loss of vision was subsequently tested in these animals by subretinal injections of healthy RPE cells into the eyes of the dystrophic RCS rats. Recordings taken 85-108 days after the transplantation procedure indicated that photoreceptor rescue occurred in the region of the graft and that the progressive central to peripheral loss of visual responsiveness in the animals was delayed by the healthy donor RPE cells. Many RPE transplant studies have now been carried out in a number of animal models, and most have confirmed the clinical potential of RPE transplants to integrate into the retina, to rescue photoreceptor cells, and to improve vision.

b. Cograft Transplant: RPE-Retina Complex. For transplant procedures to become clinically feasible, it is important that the transplanted tissue provide long-term benefits to blind individuals. In a study that examined the long-term survival of retinal transplants, Sharma and coworkers observed that neuronal components of retinal grafts did not survive as well as glial cells after transplant (28). They proposed that photoreceptors require contact and trophic support of the adjacent RPE cells to survive over an extended period of time. Cotransplantation of these two tissues, therefore, may be clinically more advantageous in promoting tissue survival and retina repair for diseases in which both the RPE and photoreceptor cells have degenerated. This hypothesis was supported by a recent study, which showed healthy morphology and normal cell function of RPE-retina cografts after successful transplant into the subretinal space of albino RCS rats (29). Morphological and biochemical evaluations of the transplanted tissue suggest that the photoreceptor cells in the cografts were well differentiated and capable of normal visual function (Fig. 1).

In support of the cograft study, another group demonstrated the beneficial effects of RPE-retina cografts on photoreceptor cell morphology and function in a study using a large number of dystrophic RCS rats (30). Recordings in the superior colliculus, from areas of the retina restricted to the site of transplant, indicated that visually evoked responses were restored in the brain after cograft integration into the retina. These preliminary results are encouraging and could have only been possible if normal retinal circuitry was established by functional photoreceptor connections within the cografts.

c. IPE Transplant. The need for readily available tissue alternatives that can promote photoreceptor survival has led many laboratories to examine the transplant effects of the IPE in the retina. Several clinically appealing features are associated with the use of IPE transplants for retinal degenerations. These features include: (a) the IPE contains pigmented cells of the same embryological origin as the RPE cells; (b) IPE cells have a high transdifferentiation potential; (c) they can be obtained with relative ease by peripheral iridectomy; and, perhaps the most advantageous aspect of this approach, (d) the promise of functionally autologous IPE transplants in patients with retinal degeneration.

A few studies have now demonstrated the clinical potential of IPE transplants for retinal diseases. In one of the initial experiments, IPE cells, obtained by iridectomy from Long-Evans rats, were transplanted into the choroid and subretinal space of RCS rats. The beneficial effects of the allografts were evaluated 6 months after the procedure using computerassisted morphometric and microscopic analyses. In the transplanted rat retinas, a remarkable effect on photoreceptor survival was observed even though a high percentage of the grafted IPE cells were located in the choroid. The rescue appeared to be specific as photoreceptors were absent in the nontransplanted group (31,32). These results confirmed earlier reports suggesting that the IPE cells are capable of delaying photoreceptor degeneration (22,35), and this may be a useful alternative approach to retinal transplants (Fig. 2).

d. Autologous IPE Transplant. Recently, the successful transplantation of autologous IPE cells in animals has made the use of IPE cells for retinal degeneration even more clinically appealing. In a study carried out with a group of 25 rabbits, autologous IPE cells were harvested by iridectomy from one eye of each rabbit and transplanted into the subretinal space of the contralateral eye. After a transplant period of 5 months, the presence of healthy IPE was observed in the rabbit's retina by light and electron microscopic examination. The IPE grafts formed a polarized monolayer above the retinal pigment epithelium and projected microvillous processes towards the photoreceptor cells. Furthermore, they appeared to be involved in the process of phagocytosis of shed photoreceptor outer segments, a biological activity associated with the RPE and one that is important to outersegment renewal. The lack of immunological side effects with the IPE transplants is of significant clinical importance (33). In addition to the experiments with rabbits, successful transplant of autologous IPE was also shown in monkey retinas. Monkey IPE cells, obtained by peripheral iridectomy, were first cultured with autologous serum and labeled with DIL prior to the experimental procedure. Labeled autologous IPE cells were subsequently transplanted into the monkey's submacular region, and the eyes were observed regularly by fluorescein angiography and fundus examination. Analysis of histological preparations, indicated that autologous IPE was present in the submacular region and that these appeared to interact with the RPE cells. Their presence was determined by the intense fluorescence of Dil-labeled IPE cells in the region. The transplanted cells stayed in the area for at least 6 months after the experiment and did not mount an immunological response in the animals (35) (Fig. 3). Autologous IPE transplants may, therefore, prove to be of significant therapeutic benefit in the treatment of retinal diseases because they are easy to obtain, are capable of RPE function, and do not promote host-graft rejection.

Figure 1 Albino RCS rat retinal degeneration model. This rat has a genetic defect in the RPE and needs both photoreceptors and RPE replaced. The transplants presented in B and C were achieved by transplanting sheets of fetal neural retina together with its RPE. (A) Recipient RCS rat retina, close to the transplant, age 3 months. The photoreceptor layer is almost completely degenerated, and the inner nuclear layer (IN) is immediately adjacent to the defective RPE. (Hematoxylin-Eosin staining.) (B) Reconstructed area of RCS host retina (H) by a transplant (T) of neural retina and RPE. Note the good integration between host and retinal transplant. (Toluidine blue staining.) Donor age E20, host age at time of transplantation, 2.2 months; age at time of death, 3.9 months. (From Ref. 29.)

Figure 1 Albino RCS rat retinal degeneration model. This rat has a genetic defect in the RPE and needs both photoreceptors and RPE replaced. The transplants presented in B and C were achieved by transplanting sheets of fetal neural retina together with its RPE. (A) Recipient RCS rat retina, close to the transplant, age 3 months. The photoreceptor layer is almost completely degenerated, and the inner nuclear layer (IN) is immediately adjacent to the defective RPE. (Hematoxylin-Eosin staining.) (B) Reconstructed area of RCS host retina (H) by a transplant (T) of neural retina and RPE. Note the good integration between host and retinal transplant. (Toluidine blue staining.) Donor age E20, host age at time of transplantation, 2.2 months; age at time of death, 3.9 months. (From Ref. 29.)

Figure 1 (C) Overview of a co-transplant. The transplanted pigmented RPE sheet can easily be seen because of the albino host. The parallel photoreceptor layer of the transplant in contact with the supporting RPE sheet shows strong immunoreactivity for the phototransduction protein rod a-transducin. Note that the photoreceptors in rosettes (arrows) at the edges of the transplant show only weak staining. (No stain in the host retina.) Host age at time of transplantation, 1.8 months; age at time of death, 3.2 months. The spaces in the section are tissue processing artifacts. (From Ref. 29.)

Figure 1 (C) Overview of a co-transplant. The transplanted pigmented RPE sheet can easily be seen because of the albino host. The parallel photoreceptor layer of the transplant in contact with the supporting RPE sheet shows strong immunoreactivity for the phototransduction protein rod a-transducin. Note that the photoreceptors in rosettes (arrows) at the edges of the transplant show only weak staining. (No stain in the host retina.) Host age at time of transplantation, 1.8 months; age at time of death, 3.2 months. The spaces in the section are tissue processing artifacts. (From Ref. 29.)

e. Genetically Modified Transplant. Trophic factors, released in a sustained manner by transplanted RPE cells, are known to influence the progression of hereditary retinal degeneration in the RCS rats. Examples of these are: basic fibroblast growth factor (bFGF), glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF), all of which have been reported to promoted photoreceptor survival and delay the progression of retinal degenerations (34).

An interesting variation in autologous IPE transplants involves the genetic engineering of IPE cells to secrete high levels of specific cytokines or growth factors, above their endogeneous levels, before transplanting them into the retina. In a study designed to test whether genetically modified cells were more potent in promoting photoreceptor survival, IPE cells were transfected with the bFGF cDNA, which was cloned into the high-expression vector pCXN2 and subsequently transplanted into the subretinal space of dystrophic RCS rats. An increase in the expression of bFGF in the retina was observed as expected, and this level correlated with prolonged photo-receptor survival as compared to the control experiments (35). The increased beneficial effects on photoreceptor cells were attributed to the soluble trophic factor secreted by the IPE cells. Genetically engineered transplants may have an advantage in prolonging cell survival by transferring therapeutic genes that have the potential to augment the intrinsic pharmacological efficacy of the transplanted cells. Gene transfer approaches for retinal degenerative diseases are discussed in more detail in Sec. III.D.

Figure 2 (A) IPE or bFGF transfected IPE cells were transplanted into the sub-retinal space of 21-day-old RCS rat eyes. Eyes were enucleated at 30 days after transplantation. (A) RCS rat retina; (B) IPE-transplanted retina; (C) bFGF-IPE transplanted retina. Asterisk and arrows indicate debris and transplanted bFGF-IPE, respectively. Bar = 50 ^.m. (D) Thickness of outer nuclear layer in bFGF-IPE or IPE transplanted retina. (Courtesy of Dr. Toshiaki Aloe, Tohoku University, Sendai, Japan.)

Figure 2 (A) IPE or bFGF transfected IPE cells were transplanted into the sub-retinal space of 21-day-old RCS rat eyes. Eyes were enucleated at 30 days after transplantation. (A) RCS rat retina; (B) IPE-transplanted retina; (C) bFGF-IPE transplanted retina. Asterisk and arrows indicate debris and transplanted bFGF-IPE, respectively. Bar = 50 ^.m. (D) Thickness of outer nuclear layer in bFGF-IPE or IPE transplanted retina. (Courtesy of Dr. Toshiaki Aloe, Tohoku University, Sendai, Japan.)

Was this article helpful?

0 0

Post a comment