Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

3.2.2. Brain-Derived Stem Cells

Most of the NSC transplant studies in the retina have focused on the survival, integration, and differentiation of the transplanted cells. Again, a lack of integration and differentiation was noted when NSC were transplanted into normal rat retinas (Kurimoto et al., 2001). Intravitreal injection of adult rat NSC into mechanically injured rat retinae differentiated into cells expressing markers of astrocytes and neurons together with some evidence of synapse-like formations between graft and host cells but did not express markers for photoreceptors (Nishida et al., 2000). Similar results were obtained by Young et al. (2000), subsequent to intravitreal injection of NSC into the dystrophic RCS rat retina. Pressmar et al. (2001) transplanted NSC into the retinae of b2/b1 knock-in mice that serve as a model of photoreceptor apoptosis and into normal mice. Transplanted cells were shown to differentiate into astrocytes and oligodendrocytes and the mutant mice were seen to contain more grafted cells than the wild type mice. Transplantation of mouse NSC into Brazilian opossum retinae (Van Hoffelen et al., 2003) also showed that the age of the host determined the fate of the differentiating cells in vivo.

3.2.3. Bone Marrow-Derived Stem Cells

Intravitreal injection of MSC into injured adult Brown Norway rats showed incorporation, mostly in the ONL in the vicinity of the site of injury, and differentiation into retinal neural cells (Tomita et al., 2002). Otani et al. (2002) demonstrated that MSC transplant into the eye rescued the retinal vasculature from degeneration. The rescued vasculature was seen to be responsible for the preservation of retinal nuclear layer thickness and improved ERG recordings following intravitreal injection of MSC into mouse models of retinal degeneration, rd1 and rd10 (Otani et al., 2004). The rescued retina was found to consist of mostly cones as opposed to the normal retina which is composed of mostly rods, and microarray analysis of the rescued retinae found up-regulation of many anti-apoptotic genes. Additional substantiation of MSC suitability for treatment of neural degenerative disease is provided by the fact that Priller et al. (2001) found bone marrow-derived fully differentiated Purkinje neurons in the brains of lethal irradiated C57BL/6J mice that had received tail vein injections of MSC.

4. ADVANTAGES AND DISADVANTAGES OF MSC FOR THE TREATMENT OF RETINAL DEGENERATIONS

The pool of MSC is significantly larger and more accessible than the pools of SC in the eye or brain hence our laboratory chose to analyze MSC in more depth. As outlined in section two, there exists both benefits and disadvantages for the use of ESC and ASC. The less controversial nature of using adult-derived SC, the potential for autologous grafts, and the lack of evidence for tumor formation after transplant into the eye made the study of adult-derived MSC more attractive for initial investigations. It is possible that ESC may be more malleable than ASC in their ability to respond to extrinsic cues due to the evidence of various levels of competence noted during development (Cepko et al., 1996; Belliveau and Cepko, 1999) and therefore it is also important to investigate ESC in this setting.

To assess the feasibility of MSC for cell replacement in retinal degenerative disease, our laboratory performed both in vitro and in vivo analyses. The in vitro analyses of rat CD90+ MSC have involved the use of activin A and taurine to induce differentiation and resulted in the expression of rhodopsin, opsin and recoverin (Kicic et al., 2003a). The rat MSC were transduced with rAAV.GFP prior to transplantation into the subretinal space of 4-5 week old normal RCS rats in order to demonstrate the viability of combining gene therapy with cell replacement (Kicic et al., 2003a). This was performed because autologous transplantation may necessitate the correction of the genes responsible for the retinal degenerative condition in order to prevent the transplanted cells from degenerating. The transplanted cells showed no morphological differentiation into photoreceptors and thus we suspect that the rhodopsin positive transplanted cells are immature photoreceptors. Despite little morphological resemblance, transplanted MSC were able to attract synapses, which provides evidence for some level of functional restoration.

Having established the safety of grafting MSC into the subretinal space and demonstrating that some signals inducing photoreceptor differentiation did exist in the RCS retina we next questioned whether the characteristics of MSC differed between species. The majority of studies have been performed in both mouse and rat models of normal and degenerate retinae and therefore it seemed natural to question whether results obtained with one species could be extrapolated to the other. Investigations of stem cell differences are also important when assessing the clinical application of cell replacement therapy in humans. The population profiles of CD45, CD11b and CD90 expression for MSC were compared between the rat, (RCS rdy+p+) and the mouse, (C57BL/6J) (Kicic et al., 2003b). The rat MSC population appeared to sustain a high level of sole CD90 expression, a marker of undifferentiated cells (Woodbury et al., 2000) through passages 1-8, however, the mouse MSC population did not show significant levels of sole CD90+ cells until passage 8. These results were comparable to those seen by Woodbury et al. (2000) in the rat and by Phinney et al. (1999) in the mouse. Differences between these two species were also observed with respect to their ability to differentiate into photoreceptors following activation with 100 ng/mL activin A with a larger proportion of rat MSC expressing rhodopsin than mouse MSC. However MSC from mice and rats do show some similarities in that they can be transduced with comparable efficiencies by both adenovirus and adeno-associated virus vehicles encoding GFP (Kicic et al., 2003b).

Having revealed differences in the characteristics of MSC between species it was then necessary to investigate if differences exist between the MSC of the healthy animal model compared with the MSC from a model with retinal degenerative disease (Kicic et al., In Press). Again, the population profiles of MSC from C57BL/6J normal mice and C3H/HeJ retinal degenerative mouse model were compared and both were shown to have similar profiles at early passage as reported for the mouse model above. In contrast to the normal model, the MSC derived from the retinal degenerate model showed a more rapid increase in the proportion of sole CD90+ cells with increasing time in culture. Marrow stromal cells from the healthy and disease origins were both found to respond to inductive cues for adipocytes and photoreceptor differentiation as determined by expression of cell-specific genes with RT-PCR and their associated protein products with Western blot analysis. There were no significant differences observed in the efficiency of adenovirus and adeno-associated virus to transduce MSC from disease and healthy origins. Collectively these results demonstrate that there do not appear to be limitations to the retinal transplant of autologous MSC in retinal degenerative animal models.

These studies have proven that MSC may be ideal candidates for cell replacement therapy due to their stem cell characteristics, their ease of isolation, their accessibility and the large pool of cells available requiring less time in culture. The safety of MSC transplant in the retina and the ability of MSC to differentiate into cells of retinal lineage have both been demonstrated. Variations exist between MSC from different species and further investigations are required to examine whether differences exist between SC from human and animal models as currently thought (Ginis et al., 2004; Rao et al., 2004). There do appear to be intrinsic differences in the MSC from disease and healthy models which affect their ability to respond to extrinsic cues however, we have shown that the MSC from retinal degenerative models do still have the potential for photoreceptor differentiation. Therefore it is feasible that autologous MSC may be used in the future for cell replacement therapy in the treatment of retinal degenerative disease.

5. CONCLUSION

The prevalence of retinal degenerative disease and the lack of available treatments have prompted investigation into the feasibility of cell replacement therapy. Studies using allogeneic and xenogeneic transplants have supported the notion that the photoreceptor layer may be repaired or regenerated if suitable signals and support cells are present in the eye. The lack of suitably matched tissue available for transplant severely limits the clinical application of such methods. Stem cells provide a source of cells that could be used to enhance sight in retinal degenerative patients. The potential of stem cells for the treatment of retinal disease is substantiated by in vitro studies indicating the capacity of both adult and embryonic stem cells to differentiate into photoreceptors and in vivo studies where transplantation of stem cells into injured or diseased retinae results in the survival, integration, and a level of differentiation. Many questions still exist regarding the behavior of stem cells after transplantation in the retina and these factors are important for translating current knowledge into a safe and effective treatment for retinal degenerative diseases.

6. ACKNOWLEDGEMENTS

We thank the National Eye Institute for supporting the participation of Christine Hall at the XIth International Symposium on Retinal Degenerations.

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2. SUCCESSFUL OPTIC NERVE REGENERATION IN FISH AND AMPHIBIA

Comparative studies of optic nerve regeneration across the vertebrate classes have provided powerful insights whereby the pre-requisites for neural repair are fulfilled (reviewed: Beazley, 2000; Beazley and Dunlop, 2000). In fish and amphibia, RGC survival is robust; almost 100% of RGCs survive in goldfish and up to 50-70% in amphibia with RGC axons regaining the major visual centre, the optic tectum, by approximately 2 weeks (Beazley and Humphrey, 1985; Murray and Edwards, 1982). The restoration of topography appears to be biphasic requiring an initial activity-independent phase followed by a phase that is critically dependent on normal patterns of neural activity (Fig. 55.1).

During the initial phase, a coarse topographic map is restored in which receptive fields map approximately correctly within the optic tectum but are somewhat larger than normal (Schmidt and Edwards, 1983). The anatomical correlate is that regenerating RGC axonal arbors are more widespread than normal and are located within both topographically appropriate and inappropriate tectal regions (Schmidt et al., 1988). The initial phase appears to be activity-independent since it occurs if animals are raised in the dark, a condition permitting only spontaneous activity, or in a stroboscopic environment which synchronises all activity and prevents normal patterned form vision (Schmidt and Edwards, 1983; Schmidt et al., 1983). Similarly, a coarse map forms if sodium channel-mediated activity is blocked with tetrodotoxin or if glutamatergic NMDA receptor-mediated activity is prevented with

Making a map – a two step process

1. Coarse map: activity-independent mechanisms molecular guidance cues

2. Refinement: activity-dependent mechanisms

Figure 55.1. Diagram of two overlapping phases involved in restoration of retinotectal topography. During the activity-independent phase (left panel), RGC axons (dots in retinal outline) project axons with wide-spread terminal arbors in the optic tectum (jagged shapes in tectal outline). In the second phase, activity-dependent mechanisms focus terminal arbors and produce precise topography. See also color insert.

the antagonist APV (Schmidt, 1990). In the second phase, the map is refined via activitydependent mechanisms, whereby incorrectly located terminal arbors, or parts thereof, are pruned or silenced by inhibition or are inactive (Lin et al., 1998); absence of appropriate activity results in failure to refine.

Here we describe some of our experiments in different models of optic nerve regeneration which throw new light on mechanisms underpinning both phases. We summarise evidence which implicates a role for molecular guidance cues in the first, activity-independent phase. We also highlight the importance, during the second phase, of normal patterns of neural activity, which can be induced by training on a specific visual task, to restore topography and therefore useful vision.

3. MOLECULAR PRE-REQUISITES FOR RESTORATION OF TOPOGRAPHY

In his “chemoaffinity hypothesis”, the Nobel Laureate Roger Sperry predicted that matching gradients of guidance molecules would be involved in conferring positional identity to both RGCs in the eye and their postsynaptic target cells within the optic tectum, allowing the establishment of topographic connections between retinal and tectal cells with matched identity (Sperry, 1963). The hypothesis received widespread experimental support although the molecular basis remained elusive until the identification of an orphan tyrosine kinase Eph receptor with a graded expression pattern in the developing visual system (Cheng et al., 1995). Subsequent studies identified classes of Eph receptors and their ligands, the ephrins, which were involved in the development of retino-tectal topography (Flanagan and Vanderhaeghen, 1998).

The “A” family of Eph/ephrins is involved in the establishment of the retinal temporonasal to the tectal rostro-caudal axis. EphA receptors are expressed as temporalhigh to nasallow gradients on RGCs and their axons. Within the tectum, ephrin-As are expressed in a rostrallow to caudalhigh gradients. EphA/ephrin-A binding is primarily repulsive. Developing temporal axons, with high EphA receptor expression, are repulsed by high ephrin-A expression in caudal tectum and map rostrally. Conversely, nasal axons with low EphA receptor expression are less sensitive to high ephrin-A expression and map caudally. At maturity, such gradients are down-regulated. Functional involvement of EphA/ephrin-As is evident from ephrin-A2/-A5 double knock-out mice and EphA3 knock-in mice in which topography is disrupted (Feldheim et al., 2000; Brown et al., 2000). In vitro experiments demonstrate that temporal and nasal growth cones display opposite behaviours when presented with high ephrin-expressing tectal cells; temporal axons are repelled whilst nasal ones are not.

Given the well-established role for EphA/ephrin-As in the development of retinotectal topography, we investigated whether such developmental guidance molecules were involved in the restoration of topography after injury to the adult visual system. In normal goldfish, EphA3 and EphA5 have a uniform profile across the temporo-nasal retinal axis (King et al., 2003). However, during the time that a coarse map is restored, EphA3 and EphA5 are upregulated as a temporalhigh to nasallow gradient. Upregulation is transient, returning to normal levels once topography is refined. Similarly, within the optic tectum, ephrin-A2 is transiently up-regulated as an ascending rostrallow to caudalhigh gradient as the coarse map is restored (Rodger et al., 2000). We have also demonstrated a functional role for EphA/ephrin-As during optic nerve regeneration (Rodger et al., 2003). Blocking EphA/ephrin-A interactions

in vivo by intracranial injection of recombinant EphA3 (EphA3-AP) resulted in a degradation of retinotectal topography as assessed electrophysiologically. Furthermore, in vitro, goldfish temporal RGC axons were repulsed by ephrin-A5 whereas nasal ones were not.

4. RESTORING TOPOGRAPHY – THE NEED FOR A NOVEL MODEL

Although spontaneous optic nerve regeneration in mammals is largely abortive (Zeng et al., 1995), RGC axon regeneration can be assisted by, for example, grafting a piece of peripheral nerve (PN) between the back of the eye and the superior colliculus (SC; mammalian homologue of the optic tecum in lower vertebrates). After PN grafting, approximately 10% of RGCs survive but only a small proportion regenerate their axons along the graft, less than 1% enter the SC and precise topography is absent (Sauvé et al., 2001). However, the number of RGC axons entering the SC falls well below the 20% minimum required to restore topography, suggesting that once sufficient neuroprotection and neuroregeneration can be stimulated in mammals, further steps may be required to restore topography and therefore useful vision.

In searching for models lacking topography in visual projections, we turned our attention to reptiles, the class of vertebrate phylogenetically intermediate between the fish and amphibia and the birds and mammals. Although the response to axotomy is variable (reviewed: Dunlop et al., 2004), we identified a lizard, Ctenophorus ornatus, which was similar to fish and amphibia with large numbers of RGC axons regaining the tectum. However, the outcome of optic nerve regeneration mimicked peripheral nerve-assisted mammalian optic nerve regeneration in that topography was lacking (Beazley et al., 1997; Stirling et al., 1999; Dunlop et al., 2000).

5. NEURAL ACTIVITY AND VISUAL TRAINING TO RESTORE TOPOGRAPHY AND USEFUL VISION

In lizard, anatomical tracing throughout optic nerve regeneration revealed that RGC axons failed to restore topography (Beazley et al., 1997; Dunlop et al., 2000). Nevertheless, electrophysiological recording indicated the presence of a coarse topographic map at an intermediate stage. However, the map was transient, breaking down in the long term with blindness persisting throughout. Furthermore, whereas normal animals displayed purely AMPA-mediated glutamatergic fast secure excitatory synaptic neurotransmission accompanied by low levels of GABA-ergic inhibition, during optic nerve regeneration, responses were weak and habituated readily, were both NMDAand AMPA-mediated and displayed high levels of inhibition (Dunlop et al., 2003). Taken together, the data indicated that low levels of visually elicited activity from widely spread RGC arbors resulted in non-correlated firing of postsynaptic partner cells and therefore weak NMDA receptor activation. As a consequence, AMPA-mediated activity presumably decreased while GABA-ergic inhibition increased, thus preventing the maturation of fast, secure synaptic transmission (Shi et al., 1997) and the restoration of topography.

The corollary that low levels of activity will delay synaptic maturation is that high levels will accelerate it. Extensive evidence suggests that neural activity, either spontaneous or elicited, influences synaptic circuitry not only during development but also in both the

Figure 55.2. Visual training restores topography. Top panel: retinotectal maps assessed electrophysiologically. Numbers in tectal outlines (top) indicate electrode positions and those in retinal outlines (bottom) indicate the location of receptive fields. In normal and trained animals (left and centre) the map is topographic (rows). In untrained animals (right), topography is lacking. Bold numbers represent robust responses, non-bold weak responses. Bottom panel: retinotectal topography as assessed anatomically. Dorsal view of the optic tectum after placements of the carbocyanine dyes DiI (square dotted lines) and DiAsp (round dotted lines) respectively in dorsal or ventral retina. A retinotopic map is observed in normal and trained, but not untrained (complete overlap of dye labelling), animals. (Beazley et al., Journal of Neurotrauma, 2003, Vol 20, No. 11, 1263–1270, by the permission of Mary Ann Liebert, Inc. Publishers). See also color insert.

normal and damaged adult brain (reviewed, Dunlop and Steeves, 2003). Enhanced neural activity in the form of specific training improves functional outcome in a wide range of situations including peripheral nerve injury, stroke, spinal cord and head injury. We therefore assessed the influence of specific visual training on the outcome of optic nerve regeneration in lizard (Beazley et al., 2003). Food items were presented to the monocular field of the experimental eye in trained animals and to the unoperated eye in untrained animals twice weekly throughout the course of optic nerve regeneration. A topographic map was restored (Fig. 55.2) as well as glutamatergic excitation that was predominantly AMPA-mediated together with low levels of GABA-ergic inhibition (Beazley et al., 2003; Dunlop et al., 2003). Crucially, animals responded to and fed on prey items presented to the experimental eye indicating a return of useful vision.