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M. Hodges, F. Chang, K. Gregory-Evans; Prolonged Neuroprotection of Degenerating Photoreceptors in the S334ter4 rat Retina via Single Intravitreal Injection of Mouse Embryonic Stem Cells Expressing a GDNF Transgene. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5782.
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We have investigated the feasibility of long-term neuroprotectant delivery using embryonic stem (ES) cells because of their inherent ability to survive long-term in foreign tissue. We chose to use the rat Rhodopsin S334-ter4 transgenic model of retinal degeneration. This animal shows slow retinal degeneration allowing time for us to quantify changes in cell death rates with and without treatment. We chose the glial cell line-derived neurotrophic factor (GDNF) since this neuroprotectant has already been shown to slow degeneration in the S334ter4 rat in the short-term.
Mouse ES cells, derived from the mouse pluripotent embryonic stem cell E14TG2a, were genetically modified to include a transgene expressing GDNF, and green fluorescent protein (GFP) to highlight ES cells in histological sections. A 4µl injection containing approximately 200,000 ES cells and expressing approximately 35ng/106cells/24 hours GDNF was delivered into the vitreous cavity of heterozygous rat eyes at postnatal day 21 (P21) without immunosuppression. Control animals received mock injections of PBS alone or injections of ES cells not expressing GDNF. We determined a novel methodology to count photoreceptor nuclei in histological sections from superior and inferior retina and compared the number of photoreceptor nuclei in GDNF treated heterozygous animals versus untreated.
We observed GFP-expressing cells on the inner limiting membrane of the retina in retinal flatmounts up to P90. In each case most GFP-expressing cells were limited to a quadrant of the inferior retina. We were also able to see some integration of GFP-expressing cells into the retina in cryostat sections from P45 animals. Total photoreceptor cell counts were significantly higher in animals treated with GDNF secreting ES cells than in other control groups.
Photoreceptor cell counts from the inferior retina were significantly higher in GDNF treated animals than in other control groups, particularly at the periphery. Superior counts showed no difference to the control groups. The discrepancy in neuroprotection between the inferior and superior retina may be through an increased number of ES cells in the lower portion of the eye, through initial pooling of the injected cells in this region. This proof of principle methodology shows that a simple neuroprotection regime, involving a single injection into the eye, can give long term benefits.
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