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

7. CONCLUSION

Our work has capitalised on two models of optic nerve regeneration to examine factors that are involved in the restoration of topographic maps after injury to the adult brain. In goldfish, guidance molecules instrumental in establishing topography during development are involved functionally in restoring topography during optic nerve regeneration in adults. Furthermore, in lizard, a model in which RGC axon regeneration is robust but topography is lacking, specific visual training results in the restoration of topography and useful vision. Taken together, the work suggests that restoration of vision in mammals will require the recapitulation of developmental guidance cues during the restoration of a coarse topographic map and appropriate levels of relevant neural activity to ensure the return useful vision.

8. ACKNOWLEDGEMENTS

We acknowledge the financial support provided by the National Health and Medical Research Council (Australia), the Neurotrauma Research Program (Road Safety Council, Western Australia) and Woodside Energy Limited.

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are characteristic of neurons in chronic neurodegenerative diseases. Work in the primate model of experimental glaucoma has revealed remodelling at the level of the cell soma and the dendritic tree (Weber et al., 1997). Furthermore, other studies have failed to find evidence supporting the selective loss of magnocellular retinal ganglion cells in experimental glaucoma (Morgan et al., 2000). It is important to note that these observations have only been made in the primate glaucoma model. As such, they remain contentious and further work is needed to establish whether changes in RGC morphology are a general feature of other glaucoma models and of human disease. Although the primate has been a popular model for studying these changes in the past, because of its great cost represent it is not a feasible model for the study of the early pathophysiology of RGC death.

Therefore, we have adopted the rodent glaucoma model in which moderate increases in intraocular pressure can be produced by the injection of hypertonic saline into the episcleral venous system (Morrison et al., 1997). The principal aims of our work are to establish whether morphological changes are occurring in the retinal ganglion cell population prior to the onset of cell death and to develop techniques that will allow us to evaluate strategies for reversing any changes that occur. In this paper, we outline techniques for the analysis of RGC morphology in the rat model and report our preliminary findings.

2. METHODS

Unilateral ocular hypertension was induced in adult Norwegian Brown rats (retired male breeders, weight range: 335 to 465 gm). All experiments were conducted in accordance with Home Office (UK) regulations. Animals were maintained in a constant low light environment (40-60 lux) to minimise diurnal fluctuations in IOP. IOPs were measured at least every other day using a factory-calibrated Tonopen XL (Mentor) with the IOP taken as the mean of 10 readings. All measurements were made in awake animals in which the cornea was anaesthetised using topical benoxinate drops (0.5%).

Ocular hypertension was induced in left eyes with the right eye acting as an unoperated control. In each case, a single episcleral vein was exposed by conjunctival dissection and injected using a glass microcannula (outside diameter 15-35 mm) with sufficient hypertonic (1.75 M) saline (approximately 50-75 ml) to blanch the vessel and clear blood from the episcleral venous system. We obtained unilateral elevation of IOP within 24-48 hours in over 90% of animals following a single injection. A repeat injection was given to those animals that failed to show an increase after 7 days.

Following defined periods of sustained elevation of IOP, animals were sacrificed and the eyes dissected out rapidly for culture in oxygenated Ames medium. Retinal ganglion cells were labelled using carbocyanine dyes delivered ballistically using a Gene Gun (BioRad) in which tungsten microparticles (1.7 mm diameter) were coated in dye and injected under high pressure directly into retinal ganglion cells (Sun et al., 2002). When injected in viable tissue, the dyes are rapidly transported within the cell membrane and reveal neuronal structure. Labelled cells can be viewed by fluorescence microscopy within minutes of injection to determine the extent of labelling and further injections administered as required. Images were captured at high resolution at a series of focal planes through the dendritic tree and then compressed in the z-axis for the analysis of dendritic structure in a 2 dimensional view. Two methods were used to determine changes in dendritic structure. Firstly, the number of dendritic branches was estimated using a modified Sholl analysis (Sholl, 1953) in which

a series of concentric rings centred on the cell soma are drawn at regular intervals to the outermost boundary of the dendritic field. The number of dendritic branches intersecting with each ring was then determined to provide an index of dendritic complexity. Secondly, the maximum diameter of the dendritic tree was measured based on a perimeter that connected the tips of the terminal dendrites.

The determination of retinal ganglion cell loss in this paradigm can be problematic. Conventionally, retinal ganglion cells are labelled prior to the induction of ocular hypertension by retrograde labelling from injections made in the superior colliculus (Laquis et al., 1996). While this method can provide robust estimates of the population of surviving RGCs, it has the potential limitation that injections into the superior colliculus can compromise retinal ganglion cell function as a result of direct damage to retinal ganglion cell axons (Leahy et al., 2004). We therefore adopted an alternative strategy in which retinal ganglion cells were identified immunohistochemically using an antibody (TUJ1) against a neuron-specific b-3 tubulin (Covariance, UK) which can be used to distinguish RGC’s and amacrine cells within the retinal ganglion cell layer (Cui et al., 2003). Use of immunohistochemical stains in the retina in which cells have been labelled intracellularly with a suitable fluorescent dye can be complicated by dispersion of dye following permeabilisation of cells during immunohistochemistry. We overcame this technical difficulty by using DiI which has been modified by the inclusion of a thiol reactive chloromethyl group (CMDiI, Molecular Probes, OR) to increase binding to the plasma membrane and to diminish the dispersion of fluorophore within the retina following permeabilisation. Once labelled RGCs had been analysed in detail, the surrounding retinal ganglion cell population was labelled immunohistochemically to determine the degree of local RGC loss.

3. RESULTS

Consistent and moderate increases in intraocular pressure were obtained using the episcleral vein injection model. Typical intraocular pressure elevation profiles are shown in Figure 56.1.

Days post-microinjection

Figure 56.1. Plot showing the change in intraocular pressure following injection of hypertonic saline into an episcleral vein.

Ring Number

Figure 56.2. DiI labelled RGCs. (a) a normal eye. (b) glaucomatous eye. Scale bar 100 microns. (c) Sholl rings overlying a labelled retinal ganglion cell. (d) Plot showing reduction in the Sholl count for 8 cells from 4 glaucomatous eyes. The number at each ring indicates the number of dendrites crossing that ring. SEMs are indicated.

The Gene Gun method provided extensive labelling of retinal ganglion cells. However, excessive labelling can reduce the numbers of cells in which the dendritic tree can be isolated and accurately reconstructed. We therefore focused on cells in areas of retina with sparser labelling in which the dendritic tree could clearly be distinguished from those of adjacent cells. In Figure 56.2, sample cells are shown from normal and glaucomatous flat-mounted retinae that indicate pruning of the dendritic tree with reduction in overall dendritic area and in the complexity of the dendritic tree. This shrinkage and remodelling has been a consistent feature of RGCs labelled in glaucomatous retinae. The mean reduction in dendritic tree diameter for RGCs from the glaucomatous retinae was 25.7% compared with control eyes (P < 0.001, ANOVA).

Immunohistochemical labelling with TUJ1 allowed us to determine the degree of RGC loss around cells labelled with DiI. RGC identification could be confirmed by the presence of axonal labelling. An example of RGC labelling with this technique is shown in Figure 56.3.

4. DISCUSSION

Analysis of intracellularly labelled cells in this rodent supports the hypothesis that changes in dendritic morphology precede the onset of retinal ganglion cell death in exper-

Figure 56.3. RGC’s labelled immunohistochemically with TUJ1. (a) Small arrow: RGC labelled with TUJ1 alone. Large arrow: RGC labelled with DiI and TUJ1. (b). TUJ1 labelled RGCs in a glaucomatous retina (large arrow). Note the reduction in number of labelled cells in (b).

imental glaucoma. Our observations are important because they suggest that widespread remodelling occurs in the retinal ganglion cell population prior cell loss. In the primate, these changes occur in step with reduction in cell soma size (Weber et al., 1998) and similar changes appear to occur in the rodent model. An important implication of these findings is that the apparent selective loss of larger retinal ganglion cells in glaucoma could also be explained by a reduction in retinal ganglion cell soma area and dendritic tree area rather than the selective loss of one particular class of retinal ganglion cells (Morgan, 2002). These observations are consistent with clinical physical studies which have failed to demonstrate selective damage to magnocellular or parvocellular retinogeniculate pathways (Graham et al., 1996, Ansari et al., 2002) in early glaucoma.

Perhaps of greater significance for our understanding of the early changes in retinal ganglion cells in glaucoma is that our data indicate that subtle changes occur in the retinal ganglion cell population prior to the onset of retinal ganglion cell death. We hypothesise a model in which large populations of RGCs are diffusely affected by elevated IOP which predisposes to programmed cell death. It is interesting to note that in the rodent glaucoma model changes in neurofilament mRNA levels are seen early in the disease process (Johnson et al., 2000). Neurofilaments are important for neuronal structural integrity and reduced expression of NFL (the light neurofilament subtype) can correlate with reductions in dendritic complexity (Zhang et al., 2002). Changes in neurofilament expression would provide a plausible link between ocular hypertension and changes in RGC structure.

In conclusion, our data support the hypothesis that retinal ganglion cells undergo structural changes prior to the onset of cell death in experimental glaucoma. These observations have far reaching implications in terms of our understanding of the processes that precede cell death in this disease.

5. ACKNOWLEDGEMENTS

Support: National Eye Research Centre (UK), Allergan Inc.

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PART VII

INDUCED RETINAL DEGENERATIONS