J Korean Ophthalmol Soc.  2013 Mar;54(3):479-489. 10.3341/jkos.2013.54.3.479.

Effect of ROCK Inhibitor on the Expansion and Wound Healing of Human Corneal Endothelial Cell

• Kim KH
• Chung JK
• Ryu JS
• Koh AY
• Kim MK
• Wee WR

• KMID: 2216770
• DOI: http://doi.org/10.3341/jkos.2013.54.3.479

Abstract

PURPOSE
To investigate the effect of ROCK inhibitor Y27632 on the human corneal endothelial cell proliferation in vitro and in vivo.
METHODS
Using corneal endothelial cells isolated and cultured from human donor cornea, we compared the effect of Y27632 (10 microM) on the proliferation in vitro by flow cytometry analysis. For the evaluation of the effect of Y27632 (10 mM) in vivo, corneal thickness and wound area were analyzed for the corneal endothelial wound rabbit model induced by transcorneal freezing.
RESULTS
Ki67 positive cells were increased in the Y27632 group (9.1 +/- 4.1%) than the control group (8.0 +/- 5.9%), whereas annexin V positive cells in the Y27632 group (2.9 +/- 1.0%) were decreased compared to the control group (4.2 +/- 2.2%). However these were not statistically significant. Wound area after Y27632 application in animal model is concerned, the control group showed significant smaller area (45.6 +/- 0.6 mm2) compared to the Y27632 group (49.3 +/- 0.8 mm2; p = 0.029, Mann-Whitney U test), however, these were not significantly different from the baseline. Corneal thickness was not different between the two groups.
CONCLUSIONS
Different from other reports for the effect of Y27632, no significant effect on the proliferation in vitro and wound healing in vivo, regarding human corneal endothelial cell, were found in this study.

Keyword

Human corneal endothelium; Rho-associated kinase inhibitor; ROCK inhibitor; Wound healing; Y27632

MeSH Terms

Amides
Annexin A5
Cornea
Endothelial Cells
Flow Cytometry
Humans
Models, Animal
Pyridines
Tissue Donors
Wound Healing
Amides
Annexin A5
Pyridines

Figure

• 

  Figure 1. (A) Cylindrical dry-ice block was prepared using 7.75 mm sized corneal trephine. (B) Dry-ice block was applied on the corneal surface of the animal for 25 seconds (C, D) for the induction of transcorneal freezing injury.

• 

  Figure 2. (A) Alizarin red staining was performed using the cornea isolated 48 hours after corneal endothelial wound induction. Gross appearance and (B) microscopic appearance shows the area of the induced corneal endothelial wound. (C) There was no significant difference between Y-27632 group and control group for the wound area change from the baseline (*M ann-W hitney U test).

• 

  Figure 3. (A) M orphological analysis of cultured corneal endothelial cells showed relatively uniform and similar sized appearance. Until the second passage cells were kept hexagonal confluent single cell layers and showed less phenotypical change due to the replicative scenecence. (B, C) Cells during primary culture and subcultures were stained positively by both the COL8A2 and Na/K ATPase. Different from intracellular homogenous distribution of COL8A2, Na/K ATPase was distinctively found around the cellular membrane.

• 

  Figure 4. (A-C) The number of Ki67 positive cells were analyzed using flow cytometry. (A: Control group, B: Y-27632 group, C: FGF-2 group) (D) Ki67 positive cells were increased in the Y-27632 group compared to the control group, however there was no statistical significance (*M ann-W hitney U test).

• 

  Figure 5. (A-C) The number of annexin V positive cells were analyzed using flow cytometry. (A: Control group, B: Y-27632 group, C: FGF-2 group) (D) Annexin V positive and propidium iodide (PI) negative cells were decreased in the Y-27632 group compared the the control group with no statistical significance (*M ann-W hitney U test).

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  Figure 6. Baseline endothelial cell profile analyzed with specular microscope was not different between the Y-27632 group (A) and the control group (B).

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  Figure 7. (A, B) As far as corneal transparency and edema are concerned, there was no significant difference between the Y-27632 group and the control group 48 after corneal endothelial wound induction. However, Y-27632 group showed relatively more surface irregularity compared to the control group.(A: Y-27632 group, B: Control group) (C) Corneal thickness confirmed by ultrasound pachymetry 48 after corneal endothelial wound induction revealed no significant difference between the two groups (*Mann-W hitney U test).

Reference

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