Characterization of cytokine responses to retinal detachment in rats.
OBJECTIVE
Photoreceptor apoptosis is associated with retinal detachment (RD) induced photoreceptor degeneration. Previously, we demonstrated the importance of caspase activation for RD-induced photoreceptor death in a rat model of RD. However, extracellular signals that precede the activation of caspases and photoreceptor degeneration remain unclear. The aim of this study is to characterize the molecular and cellular responses that occur after RD. The expression of cytokines, chemokines, and growth factors were examined in a rat model of RD.


METHODS
RD was induced in adult rats by subretinal injection of sodium hyaluronate. Retinal tissues were collected at various times (1, 3, 6, 24, and 72 h) after the induction of detachment. To screen for expressional changes in response to RD, major candidates for cytokines, chemokines, and growth factors were broadly examined by quantitative real time polymerase chain reaction (QPCR). To identify the cellular sources of the expressed genes, cells from various layers of the retina were obtained using laser capture microdissection (LCM), and their mRNAs were isolated. Protein expression was quantified by immunohistochemistry and Enzyme Linked-Immuno-Sorbent Assay (ELISA). To assess the potential of early response genes after RD to induce photoreceptor degeneration, exogenous recombinant proteins were subretinally injected and the photoreceptor cell death was assessed using a TdT-dUTP terminal nick-end labeling (TUNEL) assay at 24 h after RD.


RESULTS
At 72 h after RD a significant increase in mRNA levels for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), monocyte chemotactic protein-1 (MCP-1), and basic fibroblast growth factor (bFGF) were detected in the neural retina. LCM revealed increased expression of mRNA for bFGF and MCP-1 in all retinal layers, though bFGF was especially evident in the outer nuclear layer (ONL) and MCP-1 in the inner nuclear layer (INL). TNF-alpha was increased in the ONL and the INL, and IL-1beta was increased in the ganglion cell layer. Time course experiments showed that TNF-alpha, IL-1beta and MCP-1 increased within 1 h after RD, while bFGF was increased by 24 h. Increased protein expression for TNF-alpha, IL-1beta, and MCP-1 was demonstrated by ELISA at 6 h after RD. Immunohistochemistry showed TNF-alpha and bFGF expression in the whole retina, with IL-1beta specifically expressed in astrocytes and MCP-1 in Müller cells. Subretinal administration of MCP-1 significantly increased TUNEL-positive cells in the ONL 24 h after RD, while injection of vehicle control, TNF-alpha, or IL-1beta showed no effect.


CONCLUSIONS
Retinal glial cells, including astrocytes and Müller cells, are a major source of cytokine induction after RD. The increased expression and release of MCP-1 may be an important cause of photoreceptor degeneration associated with RD. This study helps to understand the mechanisms of RD-induced photoreceptor degeneration. Our results may provide new therapeutic targets to prevent photoreceptor degeneration following RD.
Nakazawa
Toru
T
Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Matsubara
Akihisa
A
Noda
Kousuke
K
Hisatomi
Toshio
T
She
Haicheng
H
Skondra
Dimitra
D
Miyahara
Shinsuke
S
Sobrin
Lucia
L
Thomas
Kennard L
KL
Chen
Dong F
DF
Grosskreutz
Cynthia L
CL
Hafezi-Moghadam
Ali
A
Miller
Joan W
JW
D000818:Animals; D017209:Apoptosis; D001253:Astrocytes / Q000378:metabolism; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009457:Neuroglia / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011914:Rats, Inbred BN; D012160:Retina / Q000378:metabolism / Q000473:pathology; D012163:Retinal Detachment / Q000378:metabolism* / Q000473:pathology; D012165:Retinal Ganglion Cells / Q000378:metabolism; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016222:Fibroblast Growth Factor 2