Activation of microglia and chemokines in light-induced retinal degeneration.
OBJECTIVE
Microglial cells, which are activated and recruited by chemokines, have been shown to play crucial roles in neuronal degenerations of the central nervous system (CNS). This study investigated the activation and migration of retinal microglial cells and expression of chemokines in retinas in light-induced photoreceptor degeneration in mice.


METHODS
Ninety-five Balb/cJ mice were kept in cyclic light for 1 week followed by dark adaptation for 48 h prior to light exposure of 3 h at 3.5 Klux. Animals were enthuanized at various times after light exposure. Terminal deoxynucleotidyl transferase-mediated dUTP nick end label (TUNEL) assay, rat-anti-mouse CD11b and 5D4 antibodies, isolectin-B4, and a chemokine-specific gene array were used to detect DNA fragmentation during retinal degeneration, to label retinal microglial cells, and to determine the expression of retinal chemokines and chemokine receptors, respectively. Reverse-transcriptase coupled polymerase chain reactions (RT-PCRs) were conducted on selected chemokine mRNAs to confirm the gene array findings.


RESULTS
After intense light exposure, TUNEL-positive cells were noted in the outer nuclear layer (ONL) of the retina at 3 h, and their presence were noticeably increased at 1 day but declined at 3 days and 7 days after light exposure. In contrast, CD11b- or isolectin-B4-positive cells were seen in the ONL as early as 6 h and their presence increased significantly at 1 day and 3 days after light exposure. These cells displayed a round or ovoid morphology at 6 h and 1 day but assumed a more ameboid configuration at 3 days. By 7 day, the number of the microglial cells declined in the ONL and they became ramified, and were present mostly in the subretinal space. 5D4-positive cells with large cell bodies were only noted at 3 day and 7 day but not earlier. With chemokine-specific gene array analysis, we identified four chemokines and two chemokine receptors showing significant increases in their gene expressions. Among them, monocyte chemoattractant protein-3 (MCP-3), showed a remarkable 4.4 fold increase in its gene expression. RT-PCR confirmed a marked increase of MCP-3 expression in retinas at 3 h to 1 day, and a return to normal at 3 days following light injury.


CONCLUSIONS
Retinal chemokines such as MCP-3 and their receptors are involved in the activation and migration of retinal microglia in light-induced retinal degeneration, which in turn modulate the apoptotic loss of photoreceptor cells in the outer retina.
Zhang
Cheng
C
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. czhang1@jhmi.edu
Shen
Ji-Kui
JK
Lam
Tim T
TT
Zeng
Hui-Yang
HY
Chiang
Samuel K
SK
Yang
Fang
F
Tso
Mark O M
MO
D000818:Animals; D039481:CD11b Antigen / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D008027:Light; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017628:Microglia / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011833:Radiation Injuries, Experimental / Q000378:metabolism*; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000528:radiation effects*; D012162:Retinal Degeneration / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D039481:CD11b Antigen; D018925:Chemokines; D006023:Glycoproteins; D012333:RNA, Messenger; D019707:Receptors, Chemokine; C473590:isolectin B4-binding glycoprotein, mouse