Paper type: Essay Pages: 12 (2916 words)
ANATOMY OF THE RETINA:
The retina of the vertebrates is divided into 3 cellular types namely: the outer nuclear layer,inner nuclear layer and the ganglionic layer. Photoreceptors present on the outer nuclear layer can sense light and transduce signals and information to ganglionic cells through 3 different types of interneurons :bipolar cells,amacrine and horizontal cells.The axons of the ganglion cells runs beneath the ganglionic cell layer that constitutes the nerve fibre layer.The outer and inner plexiform layer is formed by synapses between photoreceptors and interneurons for the former,while the latter is formed by synapses between interneurons and ganglion cells.
Muller glial cells contributes to the formation of inner and outer limiting membrane. These muller glial cells in mammals can proliferate in response to neuro-toxin induced damage thus capable of generating new retinal cell neurons.
THE CAUSES AND TREATMENT METHODOLOGIES:
The various diseases/causes that leads to retinal ganglion cell death are glaucoma,diabetic retinopathy,macular degeneration and retinitis pigmentosa also from traumatic injuries.
Apart from this various mechanisms such as excitotoxicity,ischaemia, oxidative stress and abnormal protein trafficking also induces retinal ganglion cell death.Apoptosis and Necrosis are the important mechanisms that plays crucial role in this process.
2 : RETINAL DISORDERS IN HUMANS:
The diseases that leads to degeneration :
A normal retinal pigment epithelium is derived from the neuroepithelium of the anterior neural plate in a developing embryo, it is polarised monolayer in nature.
Its primary function is it helps in transport of nutrients,recycling of retinol, production of pigment and the phagocytosis of cones and rods.
Degenerated RPE is discontinuous, shows the loss of adherence (yellow bars) to Bruch’s membrane (BM, green) and the loss of tight junctions (red bars) between Retinal Pigment Epithelium cells. Photoreceptor loss also occurs due, in part, to the inability of the degenerated Retinal Pigment Epithelium to phagocytose the photoreceptor outer segments, as depicted above,by the lack of phagosomes (Ph) within the Retinal Pigment Epithelium cells.
Age related macular degeneration:
It is a complex degenerative disease with a polygenic hereditary component that arises as a result of chronic,low grade inflammation in the central outer retina that leads to the degeneration of retinal pigment epithelium and its basement membrane,bruchs membrane.Under normal conditions, each Retinal Pigment Epithelial cell is responsible for the phagocytosis of millions of PR OS discs. Over time, the incomplete digestion of each phagosome results in accumulation of the lysosomal protein lipofuscin, which is toxic to Retinal Pigment Epithelial cells. In the case of dry Age related Macular Degeneration, this gradual deterioration of Retinal Pigment Epithelium leads to subsequent PhotoReceptor loss at the macula. In wet AMD, fluid accumulation occurs as the result of unwanted neovascular membranes growing from the choroid through Bruch’s membrane and the RPE into the subretinal space and occasionally through the retina. This neo vascularisation is driven by the presence of excess Vascular Epithelial Growth Factor on the apical side of the RPE, which promotes the growth of fenestrated, leaky capillaries that allow the build up of fluid. Occasionally, these fragile vessels cause haemorrhage at the macula, which may result in scarring.
Stargardt’s disease is an inherited disease of the retina which is the most common cause of macular disease in children, with an incidence of 1:10,000 live births. The disease progressively leads to blindness in adults as the central vision fades.In the macular region that leads to abnormal accumulation of the waste pigment lipofuscin in the PhotoReceptors, and subsequently the Retinal Pigment Epithelium,after PhotoReceptor phagocytosis. The most common form of the disease is STGD1, which is autosomal recessive. It is caused by mutations in the ABCA4 gene, which encodes a transporter protein that is normally expressed on the PR OSs.
Retinitis pigmentosa refers to a group of inherited retinal degenerations that mostly affect the rod visual system. There are over 100 defined genetic mutations that may lead to Retinitis Pigmentosa, and it may be inherited in a dominant, recessive or X-linked fashion. In many cases, RP progresses to involve the central visual field. Of the 1 in 4000 people affected, the most common RP subtype occurs due to mutations in the gene encoding rhodopsin and accounts for 30% of autosomal dominant cases. Although many of RP mutations code for genes in PRs, many RP subtypes begin with primary failure of the RPE.
b) GLOBAL STATISTICS:
Comparison of a healthy and neurodegenerative retina.
People suffering from glaucoma,age related macular degeneration(AMD) the interaction by the outer and inner limiting membrane with the retinal pigment epithelium is affected by the formation of drusen that gradually leads to the retinal cells detachment and ultimately in apoptosis.
Also due to the increased intraocular pressure in patients with glaucoma might result in axon damage in the optic nerve head on the base of the retina.Due to this the ganglionic cells of the retina loses its synaptic connection and thus making it apoptotic .
According to the global statistics by the National Eye Institute ,patients suffering from AMD in the age group of 45-85 years is 8.7% of the global population.this is estimated to affect the 196 million people by 2020.
76 million people worldwide is affected by glaucoma.Hence preventive measures are being taken to prevent its further occurence .
3: RETINAL REGENERATION :
MATERIALS AND TECHNIQUES USED:
Immunohisto-chemistry: 5 whole eyes from donor adult animals (9-18 months old) was immersed in 9:1 ethanolic formaldehyde , overnight at 4 degree celsius.Eyes were cryopreserved and embedded in a freezing medium.Immunohistochemistry was carried out with primary antibodies like: mouse Zpr-1 antibody, rabbit anti-Rhodopsin antisera , rabbit anti-GFP, mouse anti-PCNA , rabbit anti-Blue opsin, rabbit anti-UV opsin, mouse anti-Glutamine Synthetase and mouse 4C4. The slides were pre-heated on a slide-warmer to 55 degree celsius, glass coplin jar was also pre-heated for 30 min. Sodium citrate buffer was poured into the coplin jar that was heated until boiling in a microwave . the slides were immersed in the buffer solution. The jar is then removed from the streamer after 30 mins and is allowed to cool. Slides are rinsed three times for 5 min with 1XPBS/.5% Triton X-100. Retinal sections are covered with a blocking solution of 1XPBS/.5% Triton X-100/20% sheep serum for 1 hour. Sections are kept for overnight incubation at 4 °C in primary antibody diluted into 1XPBS/.5% Triton X-100/2% sheep serum.The sections are rinsed three times for 10 min with PBS/.5% Triton X-100 at room temperature after overnight incubation. Sections are then incubated in the secondary antibody solution diluted in 1XPBS/.5% Triton X-100/2% sheep serum for 1 hour. After secondary antibody incubation, sections are rinsed in 1XPBS/.5% Triton X-100 3 times for 10 min. Slides are covered with a coverslip using ProLong Gold Secondary antibodies included AlexaFluor-conjugated 488, 594 and 647 goat anti-primary. Nuclei are stained using TO-PRO-3 or DAPI.
This can be performed with a Leica TCS SP8 confocal microscope. Z-stacked images can be taken within a 4 µM thickness with 0.5 µM between slices. Quantification of retinal cell number can be performed by manual count of a 300 micron linear distance on the central dorsal retina using five captured images in each group of biological replicates.
Qualitative real time PCR:
Retinal tissues isolated at 36 hpL, 72 hpL, and 28 days post light (dpL). Tissue can be collected into 1 mL of Trizol, manually homogenized, and frozen in Trizol at ?80 °C. RNA is isolated using Trizol Reagent. RNA is then quantified and assessed for purity using a nanodrop. 1 µg RNA can be used as the input for cDNA synthesis, which is performed using Superscript II polymerase. cDNA is diluted 1:25 in nuclease free H2O and 2 µL is used in the reaction.
Quantitative Real-Time PCR :
This Is carried out in technical and biological triplicate in a 10 µL reaction using SYBR green reagent on a CFX Connect Real-Time System. Cycling conditions is as follows: step 1. 95 °C hold for 10 min,step 2. 95 °C denature for 15 s, 6063 ° C anneal/extend for 1 min (cycle step 2. 39 times), step 3. 6595 °C melt-curve increasing by 0.5 °C every 5 s.
4: SOURCES OF STEM CELLS
EMBRYONIC STEM CELLS:
The endogenous tissue morphology and function can be replicated more accurately from the human embryonic stem cells derived from the retinal pigment epithelium.These cells display polarity with the correct acute orientation of Na+K+ATPase and expresses proteins with tight junction.similar to that of the RPE of the foetal cells these cells also express PAX6 in equal levels.In order to evaluate the safety,the hESC derived RPE is transferred into NIH -III mice that are immune deficient and does not contain mature B ,T and N.K cells.These mice are desirable for assessing tumor formation as they exhibit reduced ability to fight against cancer and infections.as these cells display every characteristics as that of the parent RPE such as the morphology,retinol cycling,phagocytosis etc, they are believed to rescue the visual parameters,there is no teratoma formation and hence no loss of vision.
b) ADULT STEM CELLS:
It has been observed that in mice, the autogenous human adult stem cells initiates the repair mechanism by migrating to the sub retinal space in the damaged retina where they multiply to express RPE65,thus indicating that the transplanted cells are reacting to their new niche.As these cells have the capacity to rejuvenate while functionally repopulating the cells of the blood and the lymph.we can thus see if the adult stem cells is sufficiently plastic so as to mimic as a functional hemangioblast in an adult animal with visual defects in the retina.
5: PRESENT CONDITION OF RETINAL REGENERATION:
RESPONSE OF THE CELLS IN THE PRESENCE OF GROWTH FACTORS:
The cell secreted mediators such as:the growth factors,neurotrophic factors, cytokines and chemokines are important for the cell,maintenance ,proliferation, angiogenesis,vascular permeability and inflammation.The retinal pigment epithelium is an important source of growth factor that is involved in homeostasis,inflammation & choroid neovascularization(CNV). Damage or destruction of RPE leads to the atrophy of choriocapillaris and photoreceptors.Growth factors are involved in the anatomical function of RPE with the retinal cells.The major function of RPE is to give trophic support to the retina by the production of neurotrophic growth factors like nerve growth factor (NGF),brain-derived growth factor (BDGF) pigment epithelium derived growth factor (PEDF). PEDF,a 50 kDa secreted glycoprotein ,produced by the reclined RPE is an important neurotrophic factor for retinal cells and is also a potent inhibitor of angiogenesis. Another angiogenic inhibitor is Thrombospondin-1(TSP-1) produced by the RPE cells where it tries to maintain anti-angiogenic state in the resting choroid. It has been observed that the integrity of the ocular wall can be restored in the presence of these growth factors in patients where there is significant collateral damage to visual function caused by CNV.
b) RETINAL REPAIR:
Various ways by which the retina can be regenerated differs in different organisms for eg the regeneration of retina in fish,birds,amphibians egg/larvae is by transdifferentiation of Retinal pigment epithelium. In this process of transdifferentiation the RPE loses its characteristics of genesis and re-enters the cell cycle to form the neuroepithelial cell layer that ultimately differentiates into all cell types of the retina. The regeneration in teleost fish occurs via the use of rod precursors,intrinsic stem cells in the inner nuclear layer and muller glial cells that proliferate and relocate to the outer nuclear layer.in this case the repair / replacement depends on the damage evoked and must have damage in the outer nuclear cell layer,else regeneration will not take place. Photoreceptor(PR) death in AMD occurs as a result of dysfunction of RPE. when the PR is lost it results in permanent visual impairment . In order to regain vision the degenerated PR cells can be replaced,where the ganglionic cells can survive in the retina even when the PR cells are lost. Foetal retina is the most suitable source of retinal precursors that has the capacity to reintegrate into degenerating retina. The retinal precursors isolated from the foetal retina when transplanted into the subretinal space of mice and human subject with PR loss,show improved visual behaviour. The RPE when replaced so as to mimic the nourishing environment it can be seen that the survival of PR progenitor cells was increased when co-cultured with hESC-derived RPE,thus indicating that dual replacement is a promising strategy.
Various sources for retinal repair:
Animal model Embryonic-larval stages Primary sources or potential sources in adults
Teleost fish CGZ=CMZ Rod precursors
Rod precursors in ONL Quiescent stem cells in INL
Transdifferentiation of RPE M?ller glial cells?
CMZ Transdifferentiation of RPE, i.e. newts
Transdifferentiation of RPE, i.e. newts and axolotls CMZ-partial only, i.e. axolotls
CMZ, i.e. Rana esculenta; Rana temporaria CMZ-partial only, i.e. Xenopus laevis
Transdifferentiation of RPE, i.e. Rana catesbiana Birds CMZ/ciliary epithelium M?ller glial cells
Transdifferentiation of RPE Mammals Transdifferentiation of RPE in vitro and in association with transplantation PCE in vitro
experiments Iris in vitro
Corneal limbal epithelium in vitro/transplantations
Choroid and sclera in vitro
CMZ, ciliary marginal zone; CGZ, circumferential germinal zone; PCE, pigmented ciliary epithelium;
SOLUTION TO THE PROBLEM:
The major goal in retinal cells regeneration is to apply regenerative strategies to eye diseases and injuries that causes blindness. The use of endogenous stem cells for repair and treatment is concerned with prosthetic devices and cell transplant. From the use of stem cells it has been observed that due to the hyper-reflectivity of the pigment the transplanted RPE can be observed on OCT while the other retinal neurons are indistinguishable as they are transparent. From this the functional and structural image of the retina is remarkable with high resolution fundus imaging, OCT and spectral domain optic coherence tomography (SD OCT), heidelberg retinal tomography (HRT) and adaptive optics scanning laser ophthalmoscopy (AO-SLO) when combined provides single cell resolution in all dimensions. Thus the problems like inflammation,ischemia or neovascularization is monitored safely. One of the initial concerns for subretinal delivery, is the persistent detachment of the retina from the RPE in the transplant area, can be resolved, as for hundreds of patients treated by now with cells or gene vectors, the initial detachment was observed to spontaneously resolve within 48 hours.
b) PERSPECTIVES OF FURTHER DEVELOPMENT:
Stem cell transplantation for retinal disease is currently transitioning from over a decade of preclinical research to phase I/II clinical trials. Further trials are expected to help determine whether an RPE suspension is adequate or whether an RPE monolayer is required to rescue retinal function. The transplantation of iPSC-derived RPE will be one of the first clinical applications of iPSCs and will help to define how immunogenic iPSCs are in comparison with hESCs. In addition, further preclinical research is under way to develop a source of PRs from stem cells that can be used for neural retinal replacement. The therapeutic application of stem cells for retinal disease has thus begun.REFERENCES:
1.Lund, R. D., Kwan, A. S., Keegan, D. J., Sauve, Y., Coffey, P. J. & Lawrence, J. M. (2001) Prog. Retin. Eye Res. 20, 415449.
2. Takahashi, M., Palmer, T. D., Takahashi, J. & Gage, F. H. (1998) Mol. Cell. Neurosci. 12, 340348.
3. Young, M. J., Ray, J., Whiteley, S. J., Klassen, H. & Gage, F. H. (2000) Mol. Cell. Neurosci. 16, 197205.
4. Nishida, A., Takahashi, M., Tanihara, H., Nakano, I., Takahashi, J. B., Mizoguchi, A., Ide, C. & Honda, Y. (2000) Invest. Ophthalmol. Vis. Sci. 41, 42684274.
5. Akita, J., Takahashi, M., Hojo, M., Nishida, A., Haruta, M. & Honda, Y. (2002) Brain Res. 954, 286293.
6. Temple, S. (2001) Nat. Rev. Neurosci. 2, 513520.
7. Gould, E. & Gross, C. G. (2002) J. Neurosci. 22, 619623.
8. Johansson, C. B., Momma, S., Clarke, D. L., Risling, M., Lendahl, U. & Frisen, J. (1999) Cell 96, 2534.
9. Magavi, S. S., Leavitt, B. R. & Macklis, J. D. (2000) Nature 405, 951955.
10. Nakatomi, H., Kuriu, T., Okabe, S., Yamamoto, S., Hatano, O., Kawahara, Tamura, A., Kirino, T. & Nakafuku, M. (2002) Cell 110, 429441. 11.Fausett, B. V. & Goldman, D. A role for ?1 tubulin expressing M?ller glia in regeneration of the injured zebrafish retina. J. Neurosci. 26, 63036313 (2006).
12. Qin, Z., Barthel, L. K. & Raymond, P. A. Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish. Proc. Natl Acad. Sci. USA 106, 93109315 (2009).
13. Nagashima, M., Barthel, L. K. & Raymond, P. A. A self-renewing division of zebrafish M?ller glial cells generates neuronal progenitors that require N-cadherin to regenerate retinal neurons. Development 140, 45104521 (2013).
14. Kassen, S. C. et al. Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish. Dev. Neurobiol. 67, 10091031 (2007).
15. Turner, D. L. & Cepko, C. L. A common progenitor for neurons and glia persists in rat retina late in development. Nature 328, 131136 (1987).
16.Carr, A.-J., Vugler, A. A., Hikita, S. T., Lawrence, J. M., Gias, C., Chen, L. L., Buchholz, D. E., Ahmado, A., Semo, M., Smart, M. J. K. et al. (2009). Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS ONE 4, e8152.
17.Carr, A.-J. F., Smart, M. J. K., Ramsden, C. M., Powner, M. B., da Cruz, L. and Coffey, P. J. (2013). Development of human embryonic stem cell therapies for age-related macular degeneration. Trends Neurosci. doi: 10.1016/ j.tins.2013.03.006.
18.Chen, F. K., Uppal, G. S., MacLaren, R. E., Coffey, P. J., Rubin, G. S., Tufail, A., Aylward, G. W. and Da Cruz, L. (2009). Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration. Clin. Experiment. Ophthalmol. 37, 275-285.
19.Churchill, A. J., Carter, J. G., Lovell, H. C., Ramsden, C., Turner, S. J., Yeung, A., Escardo, J. and Atan, D. (2006). VEGF polymorphisms are associated with neovascular age-related macular degeneration. Hum. Mol. Genet. 15, 2955- 2961.
20.DCruz, P. M., Yasumura, D., Weir, J., Matthes, M. T., Abderrahim, H., LaVail, M. M. and Vollrath, D. (2000). Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum. Mol. Genet. 9, 645-651.
Cite this page
retinal cell regen. (2019, Dec 17). Retrieved from https://studymoose.com/retinal-cell-regen-best-essay