Abstract
BackgroundRetinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors. The preservation of cones would prevent blindness due to their essential role in human vision. Rod-derived Cone Viability Factor is a thioredoxin-like protein that is secreted by rods and is involved in cone survival. To validate the activity of Rod-derived Cone Viability Factors (RdCVFs) as therapeutic agents for treating retinitis Pigmentosa, we have developed e-conome, an automated cell counting platform for retinal flat mounts of rodent models of cone degeneration. This automated quantification method allows for faster data analysis thereby accelerating translational research.MethodsAn inverted fluorescent microscope, motorized and coupled to a CCD camera records images of cones labeled with fluorescent peanut agglutinin lectin on flat-mounted retinas. In an average of 300 fields per retina, nine Z-planes at magnification X40 are acquired after two-stage autofocus individually for each field. The projection of the stack of 9 images is subject to a threshold, filtered to exclude aberrant images based on preset variables. The cones are identified by treating the resulting image using 13 variables empirically determined. The cone density is calculated over the 300 fields.ResultsThe method was validated by comparison to the conventional stereological counting. The decrease in cone density in rd1 mouse was found to be equivalent to the decrease determined by stereological counting. We also studied the spatiotemporal pattern of the degeneration of cones in the rd1 mouse and show that while the reduction in cone density starts in the central part of the retina, cone degeneration progresses at the same speed over the whole retinal surface. We finally show that for mice with an inactivation of the Nucleoredoxin-like genes Nxnl1 or Nxnl2 encoding RdCVFs, the loss of cones is more pronounced in the ventral retina.ConclusionThe automated platform ℮-conome used here for retinal disease is a tool that can broadly accelerate translational research for neurodegenerative diseases.
Highlights
Retinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors
Retinitis pigmentosa (RP) is characterized clinically by an initial loss of night vision resulting from the degeneration of rod photoreceptors directly due to a genetic deficit, followed irreparably over a period of several years by the loss of central vision that results from the non-cell autonomous death of cone photoreceptors [1]
Rod-derived Cone Viability Factors (RdCVFs) is encoded by the Nucleoredoxin-like gene, Nxnl1 that belongs to the family of thioredoxin proteins, reducing oxidative stress, a condition encountered broadly in neurodegenerative diseases [8]
Summary
Retinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors. To validate the activity of Rod-derived Cone Viability Factors (RdCVFs) as therapeutic agents for treating retinitis Pigmentosa, we have developed e-conome, an automated cell counting platform for retinal flat mounts of rodent models of cone degeneration. This automated quantification method allows for faster data analysis thereby accelerating translational research. RdCVF is encoded by the Nucleoredoxin-like gene, Nxnl that belongs to the family of thioredoxin proteins, reducing oxidative stress, a condition encountered broadly in neurodegenerative diseases [8] This novel trophic signaling is part of an endogenous defense response since cone photoreceptors degenerate during aging in the Nxnl1-/- mouse and at an accelerated rate in the presence of high levels of oxygen [9]. The administration of RdCVF in patients suffering from RP at early stage of the disease could reduce secondary cone degeneration and prolong central vision
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