Efficacy of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina

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 -