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The Cdk5 inhibitor olomoucine promotes corneal debridement wound closure in vivo.

Tripathi BK, Stepp MA, Gao CY, Zelenka PS - Mol. Vis. (2008)

Bottom Line: Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro.Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05).The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT

Purpose: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo.

Methods: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting.

Results: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization.

Conclusions: Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization.

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Related in: MedlinePlus

Olomoucine treatment decreased localization of MMP-2 protein at the wound edge. A: MMP-2 immunofluorescence is elevated at the wound edge in untreated control corneas. B: The immunostaining of MMP-2 at the wound edge (arrow) is reduced in olomoucine-treated corneas. C: Whole mounted corneas without primary antibody showed no immunofluorescence. D: Paraffin sections of wounded control corneas showed MMP-2 immunofluorescence in all layers of the cornea at wound edge (arrow) and in basal epithelial cells distal to the wound (arrowhead). E: The paraffin section of olomoucine-treated cornea showed reduced immunofluorescence of MMP-2 at the wound edge (arrow). MMP-2 immunofluorescence persisted in the basal layers of epithelium (arrowhead). F: No immunostaining was detected in the paraffin section of the cornea when primary MMP-2 antibody was omitted. Scale bar=100 μM.
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f6: Olomoucine treatment decreased localization of MMP-2 protein at the wound edge. A: MMP-2 immunofluorescence is elevated at the wound edge in untreated control corneas. B: The immunostaining of MMP-2 at the wound edge (arrow) is reduced in olomoucine-treated corneas. C: Whole mounted corneas without primary antibody showed no immunofluorescence. D: Paraffin sections of wounded control corneas showed MMP-2 immunofluorescence in all layers of the cornea at wound edge (arrow) and in basal epithelial cells distal to the wound (arrowhead). E: The paraffin section of olomoucine-treated cornea showed reduced immunofluorescence of MMP-2 at the wound edge (arrow). MMP-2 immunofluorescence persisted in the basal layers of epithelium (arrowhead). F: No immunostaining was detected in the paraffin section of the cornea when primary MMP-2 antibody was omitted. Scale bar=100 μM.

Mentions: It is known that MMP-2 (gelatinase A) participates in epithelial repair and stromal remodeling [12]. To determine whether olomoucine affects the expression of MMP-2 in the wounded corneal epithelium, whole mounted corneas and paraffin sections were immunostained for MMP-2. In contrast to the results seen for MMP-9, immunofluorescence of this matrix metalloproteinase was significantly less in corneal epithelium upon treatment with olomoucine in both the whole mounted corneas and the paraffin sections (Figure 6).


The Cdk5 inhibitor olomoucine promotes corneal debridement wound closure in vivo.

Tripathi BK, Stepp MA, Gao CY, Zelenka PS - Mol. Vis. (2008)

Olomoucine treatment decreased localization of MMP-2 protein at the wound edge. A: MMP-2 immunofluorescence is elevated at the wound edge in untreated control corneas. B: The immunostaining of MMP-2 at the wound edge (arrow) is reduced in olomoucine-treated corneas. C: Whole mounted corneas without primary antibody showed no immunofluorescence. D: Paraffin sections of wounded control corneas showed MMP-2 immunofluorescence in all layers of the cornea at wound edge (arrow) and in basal epithelial cells distal to the wound (arrowhead). E: The paraffin section of olomoucine-treated cornea showed reduced immunofluorescence of MMP-2 at the wound edge (arrow). MMP-2 immunofluorescence persisted in the basal layers of epithelium (arrowhead). F: No immunostaining was detected in the paraffin section of the cornea when primary MMP-2 antibody was omitted. Scale bar=100 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2274924&req=5

f6: Olomoucine treatment decreased localization of MMP-2 protein at the wound edge. A: MMP-2 immunofluorescence is elevated at the wound edge in untreated control corneas. B: The immunostaining of MMP-2 at the wound edge (arrow) is reduced in olomoucine-treated corneas. C: Whole mounted corneas without primary antibody showed no immunofluorescence. D: Paraffin sections of wounded control corneas showed MMP-2 immunofluorescence in all layers of the cornea at wound edge (arrow) and in basal epithelial cells distal to the wound (arrowhead). E: The paraffin section of olomoucine-treated cornea showed reduced immunofluorescence of MMP-2 at the wound edge (arrow). MMP-2 immunofluorescence persisted in the basal layers of epithelium (arrowhead). F: No immunostaining was detected in the paraffin section of the cornea when primary MMP-2 antibody was omitted. Scale bar=100 μM.
Mentions: It is known that MMP-2 (gelatinase A) participates in epithelial repair and stromal remodeling [12]. To determine whether olomoucine affects the expression of MMP-2 in the wounded corneal epithelium, whole mounted corneas and paraffin sections were immunostained for MMP-2. In contrast to the results seen for MMP-9, immunofluorescence of this matrix metalloproteinase was significantly less in corneal epithelium upon treatment with olomoucine in both the whole mounted corneas and the paraffin sections (Figure 6).

Bottom Line: Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro.Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05).The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT

Purpose: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo.

Methods: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting.

Results: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization.

Conclusions: Topical application of olomoucine in 1% DMSO significantly enhances closure of small epithelial debridement wounds without increasing inflammation or impairing reepithelialization.

Show MeSH
Related in: MedlinePlus