Expression of cell cycle-related genes in dying cells in retinal ischemic injury.
OBJECTIVE
To investigate whether cell cycle-related genes play a role in neuronal cell death in retinal ischemia-reperfusion injury.


METHODS
Retinal ischemia-reperfusion injury was induced in rats by a ligation method and also by increasing the intraocular pressure. After 1 hour-of ischemia, cell death in the retina was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method, propidium iodide (PI) staining, DNA ladder formation, and ultrastructural studies. Immunohistochemical studies using antibodies against cell cycle-related genes were conducted. Changes in expression of cyclin D1 mRNA were quantitated using competitive quantitative polymerase chain reaction.


RESULTS
At 3 hours after reperfusion, cells in the ganglion cell layer were the first to die, followed by those in the inner nuclear layer (at 6 hours) and outer nuclear layer (at 9 hours). Ultrastructural studies revealed condensed nuclei and relatively preserved mitochondria; DNA ladder formation was also detected. Immunostaining was positive for the cell cycle-related gene products c-Jun, cyclin B1, and cyclin D1. The time course of TUNEL-positive cells and that of cells positive for c-Jun or cyclin D1 in the inner nuclear layer was similar. A double-labeling study, using PI or TUNEL, and immunohistochemical analysis revealed that dying cells expressed c-Jun and cyclin D1, whereas cyclin B1 expression was observed in Müller cells. Quantitation of cyclin D1 mRNA revealed an approximate 4-fold increase at 24 hours after reperfusion.


CONCLUSIONS
Aberrant expression of cell cycle-related genes may play an important role in the cell death that accompanies retinal ischemia-reperfusion injury.
Kuroiwa
S
S
Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
Katai
N
N
Shibuki
H
H
Kurokawa
T
T
Umihira
J
J
Nikaido
T
T
Kametani
K
K
Yoshimura
N
N
D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle / Q000235:genetics; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism*; D056744:Cyclin B1; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D007124:Immunoenzyme Techniques; D008297:Male; D009475:Neurons, Afferent / Q000378:metabolism* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D012160:Retina / Q000378:metabolism* / Q000648:ultrastructure; D012171:Retinal Vessels
C534701:Ccnb1 protein, rat; D019926:Cyclin B; D056744:Cyclin B1; D017931:DNA Primers; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D019938:Cyclin D1; D004247:DNA