Structural and functional protection of photoreceptors from MNU-induced retinal degeneration by the X-linked inhibitor of apoptosis.
OBJECTIVE
To evaluate the neuroprotective effects of adenoassociated virus delivery of XIAP in N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats.


METHODS
Sprague-Dawley rats were injected subretinally with recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control). Six weeks after injection, the animals received an intraperitoneal injection of MNU, a DNA methylating agent, at a dose of 60 mg/kg. Electroretinograms (ERGs) were recorded at 0, 24, 48 and 72 hours and 1 week after MNU. The rats were killed after the ERG was performed and were perfused with 4% paraformaldehyde. Eyes were then enucleated and embedded for cryosectioning. Eye sections were analyzed by TUNEL and histologic techniques. Real-time PCR and Western analysis were performed to confirm the overexpression of XIAP in injected eyes.


RESULTS
Real-time PCR and Western analysis confirmed the overexpression of XIAP in virus-injected eyes in comparison to uninjected control eyes. At 24 hours after MNU injection, fewer cells had undergone apoptosis in the XIAP-treated eyes in comparison with GFP-injected or uninjected eyes. Hematoxylin and eosin staining revealed that the uninjected and GFP-injected photoreceptors were destroyed by 72 hours after injection of MNU, whereas the AAV-XIAP-injected eyes showed structural protection of the photoreceptors at all time points throughout the 1-week sampling period. ERGs showed functional protection up to 1 week after MNU injection in the AAV-XIAP-injected eye, whereas no response was observed in the control eye.


CONCLUSIONS
The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeutic agent in gene therapy approaches to treating retinitis pigmentosa.
Petrin
Dino
D
Children's Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada.
Baker
Adam
A
Coupland
Stuart G
SG
Liston
Peter
P
Narang
Monica
M
Damji
Karim
K
Leonard
Brian
B
Chiodo
Vince A
VA
Timmers
Adrian
A
Hauswirth
William
W
Korneluk
Robert G
RG
Tsilfidis
Catherine
C
D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D019610:Cytoprotection; D000229:Dependovirus / Q000235:genetics; D004596:Electroretinography; D004791:Enzyme Inhibitors; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D020287:In Situ Nick-End Labeling; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008770:Methylnitrosourea / Q000633:toxicity; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000502:physiology*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051636:X-Linked Inhibitor of Apoptosis Protein
D000477:Alkylating Agents; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D011506:Proteins; D012333:RNA, Messenger; D051636:X-Linked Inhibitor of Apoptosis Protein; D049452:Green Fluorescent Proteins; D008770:Methylnitrosourea