Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina
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Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina. / Tura, Aysegül; Schuettauf, Frank; Monnier, Philippe P; Bartz-Schmidt, Karl U; Henke-Fahle, Sigrid.
In: INVEST OPHTH VIS SCI, Vol. 50, No. 1, 01.2009, p. 452-61.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina
AU - Tura, Aysegül
AU - Schuettauf, Frank
AU - Monnier, Philippe P
AU - Bartz-Schmidt, Karl U
AU - Henke-Fahle, Sigrid
PY - 2009/1
Y1 - 2009/1
N2 - PURPOSE: To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions.METHODS: Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days.RESULTS: Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC.CONCLUSIONS: These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.
AB - PURPOSE: To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions.METHODS: Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days.RESULTS: Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC.CONCLUSIONS: These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.
KW - 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives
KW - Animals
KW - Apoptosis/physiology
KW - Astrocytes/metabolism
KW - Blotting, Western
KW - Calmodulin-Binding Proteins/metabolism
KW - Cell Survival/drug effects
KW - Cytokines/metabolism
KW - Enzyme Inhibitors/pharmacology
KW - Female
KW - Fluorescent Antibody Technique, Indirect
KW - Glial Fibrillary Acidic Protein/metabolism
KW - Gliosis/metabolism
KW - Male
KW - Mice
KW - Neuroprotective Agents/pharmacology
KW - Optic Nerve Injuries/metabolism
KW - Organ Culture Techniques
KW - Phosphorylation
KW - Rats
KW - Rats, Inbred BN
KW - Retinal Diseases/metabolism
KW - Retinal Ganglion Cells/metabolism
KW - rho-Associated Kinases/antagonists & inhibitors
U2 - 10.1167/iovs.08-1973
DO - 10.1167/iovs.08-1973
M3 - SCORING: Journal article
C2 - 18757509
VL - 50
SP - 452
EP - 461
JO - INVEST OPHTH VIS SCI
JF - INVEST OPHTH VIS SCI
SN - 0146-0404
IS - 1
ER -