Jae Hwan Lee; Ei Tae Kim; Jung Hyub Oh

doi:10.3341/jkos.2009.50.3.450

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Lee, Kim, and Oh: The Inhibitory Effect of TGF-βInhibitor on the Corneal Opacity After Corneal Laceration

Original Article

J Korean Ophthalmol Soc 2009;50(3):450-461.

Published online: 31 August 2009

DOI: https://doi.org/10.3341/jkos.2009.50.3.450

The Inhibitory Effect of TGF-βInhibitor on the Corneal Opacity After Corneal Laceration

Jae Hwan Lee, MD, PhD, Ei Tae Kim, MD, Jung Hyub Oh, MD, PhD

Department of Ophthalmology, Inha University School of Medicine, Incheon, Korea

Address reprint requests to Jung Hyub Oh, MD, PhD, Department of Ophthalmology, Inha University School of Medicine #7-206 Shinheung-dong 3-ga, Jung-gu, Incheon 400-103, Korea Tel: 82-32-890-2400, Fax: 82-32-890-2403, E-mail: jhoh9707@hanmail.net

Received 19 March 2008 Accepted 30 September 2008

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

To evaluate the effects of TGF-β inhibitor on the wound healing process after corneal laceration, and its inhibitory effect on corneal scar formation.

Methods

Forty Lewis rats were randomly divided into one control and three experimental groups (groups I, II, and III). After partial-thickness vertical linear corneal incision, a diluted solution with 10, 25, and 50 μ g of TGF-β inhibitor was instilled into each eye of groups I, II, and III respectively. Corneal haze was measured by using slit-lamp biomicroscopic examination. Using histopathologic examination, we compared the number of stromal keratocytes and the arrangement of regenerated collagen fibers. We also performed immunohistochemistry to confirm the differential expression of fibronectin and α-smooth muscle actin in each group.

Results

Group III showed less corneal haze and more regular arrangement of regenerated collagen fibers than the other groups. The number of stromal keratocytes and immunoreactivity to fibronectin and α-smooth muscle actin decreased as the dose of TGF-β inhibitor increased.

Conclusions

TGF-β inhibitor effectively reduced corneal haze during corneal healing processes after corneal laceration.

Keywords: Corneal laceration, Corneal opacity, TGF-β inhibitor, Transforming growth factor-beta

References

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Figure 1.

Slit-lamp biomicroscopic photographs of the corneas at 2 weeks after corneal laceration. (A) Control group (B) Group I (treated with TGF-β inhibitor 10 μg) (C) Group II (treated with TGF-β inhibitor 25 μg) (D) Group III (treated with TGF-β inhibitor 50 μg). Linear corneal opacities were noted in the center of the cornea the severity of the corneal opacity in group II and group III was less severe than that in the control group and group I.

Figure 2.

Graphs of the overall opacity rate according to the fibrosis score. Group I=treated with TGF-β inhibitor 10 μg; Group II=treated with TGF-β inhibitor 25 μg; Group III=treated with TGF-β inhibitor 50 μg (Asterisk: clinically significant compared with the control group, p<0.05).

Figure 3.

Light microscopic findings of the corneas at 2 weeks after corneal laceration (H&E stain, ×400). (A) Control group (B) Group I (treated with TGF-β inhibitor 10 μg) (C) Group II (treated with TGF-β inhibitor 25 μg) (D) Group III (treated with TGF-β inhibitor 50 μg). The increasing density of keratocytes in the stormal layer was observed in the control group and group I compared with groups II and III.

Figure 4.

Light microscopic findings of the corneas at 2 weeks after corneal laceration (Masson trichrome stain, ×400). (A) Control group (B) Group II (treated with TGF-β inhibitor 25 μg) (C) Group III (treated with TGF-β inhibitor 50 μg). The irregular arrangement of the collagen lamella in the stromal layer was observed. In group III, however, the irregularity of the collagen lamella was less and thickness of the epithelial plug was thinner than in the other groups.

Figure 5.

Light microscopic findings of the corneas at 4 weeks after corneal laceration (Masson trichrome stain, ×400). (A) Control group (B) Group II (treated with TGF-β inhibitor 25 μg) (C) Group III (treated with TGF-β inhibitor 50 μg). The irregularity of the regenerated collagen lamella was significantly less in group III than in the other groups.

Figure 6.

Transmission electron photomicrographs of corneal keratocytes at 4 weeks after corneal laceration (×10,000). (A) Control group, (B) Group III (treated with TGF-β inhibitor 50 μg). The well- developed rough endoplasmic reticulum (rER) was observed in keratocytes within the corneal laceration wound site. However, the keratocytes in group III showed decreased number and density of the rough endoplasmic reticulum than in the control group.

Figure 7.

The fluorescence photomicrographs of the corneas at 4 weeks after corneal laceration (DTAF stain, ×200). (A) Control group (B) Group III (treated with TGF-β inhibitor 50 μg). The area of the regenerated corneal stroma is thinner in group III than control group; bright green fluorescent area represents original stromal bed (arrow) and dark non-fluorescent area represents regenerated corneal stroma.

Figure 8.

The immunocytochemical stainings of fibronectin at 2 weeks after corneal laceration (×400, Scale bar=50 μm). (A) Control group (B) Group II (treated with TGF-β inhibitor 25 μg) (C) Group III (treated with TGF-β inhibitor 50 μg). The immunoreactivity of fibronectin was observed at subepithelial (arrowhead) and regenerated stromal lamellar area in all groups. However, the expression of fibronectin decreased according to increasing concentration of TGF-β inhibitor.

Figure 9.

The immunocytochemical stainings of alpha-smooth muscle actin at 2 weeks after corneal laceration (×400, Scale bar=50 μm). (A) Corneal semithin section of control group (B) Corneal semithin section of group III (treated with TGF-β inhibitor 50 μg). (C) Semithin section of the iris in group III (Asterisk: blood vessels; arrow: iris muscle). The immunoreactivity of alpha-smooth muscle actin (arrowhead) was observed in stromal keratocytes within healing area. However, the number of immunoreactive keratocytes in group III was less than in control group. No significant differences of immunoreactivity between keratocytes (arrowhead) and iris muscle (arrow).

Table 1.

Comparison of the keratocyte number in the stroma around corneal laceration site

	2 weeks			4 weeks
	Anterior stroma^*	Posterior stroma^*	Total^†	Anterior stroma^* P	Posterior stroma^*	Total^†
Control	17.7±2.5	9.4±1.9	27.2±4.0	25.1±4.6	13.6±2.1	38.8±6.8
Group I	17.9±5.8	8.6±2.5	26.4±7.9	24.3±6.5	12.9±2.4	37.2±9.0
Group II	13.2±3.4^‡	8.0±0.8	21.2±4.1^‡	22.9±3.9	13.2±1.0	36.0±4.7
Group III	11.6±1.4^‡	5.1±1.3^‡	16.8±2.7^‡	18.4±3.1^‡	8.1±2.2^‡	26.6±5.5^‡

^*Mean± standard deviation (cells/5,000 μm²)

^†Total keratocytes within full thickness of the stroma, Mean± standard deviation (cells/10,000 μm²)

^‡Clinically significant comparing with control group (p<0.05).

Table 2.

Mean thickness of the regenerated corneal stroma at 4 weeks after corneal laceration

	Anterior stroma^*	Posterior stroma^†	Mean thickness^‡
Control	85.6±24.5 μm	61.4±29.0 μm	78.3±32.1 μm
Group I	80.1±38.8 μm	62.5±30.4 μm	74.0±43.9 μm
Group II	63.9±31.2 μm	54.8±20.8 μm	61.2±33.6 μm
Group III	45.1±17.4 μm^§	39.3±12.3 μm^§	42.5±19.0 μm^§

^*Thickness at anterior 1/3 position of the regenerated corneal stroma (Mean± SD)

^†Thickness at posterior 2/3 position of the regenerated corneal stroma (Mean± SD)

^‡Mean thickness of anterior 1/3, centeral and posterior 2/3 position of the regenerated corneal stroma (Mean± SD)

^§Clinically significant comparing with control group (p<0.05).

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