Inhibitory effects of bevacizumab on corneal neovascularization and scarring in mice with herpes simplex keratitis

Authors: Zhou Hongwei,  Wang Tongsong,  Zhang Songmei,  Xie Lixin

DOI: 10.3760/cma.j.issn.2095-0160.2017.12.007
Published 2017-12-10
Cite as Chin J Exp Ophthalmol, 2017,35(12): 1085-1091.

Abstract

Background

Corneal neovascularization and inflammation occur in herpes simplex keratitis (HSK). Aciclovir (ACV) is an antiviral medication which is primarily used for the treatment of HSV infection.Bevacizumab is an angiogenesis inhibitor which has the ability to slow the growth of corneal neovascularization.However, whether bevacizumab play treating effects on HSK is worth studing.

Objective

This study attempted to study the effects of bevacizumab on cornea lesion in mouse models of HSK.

Methods

The solution containing herpes simplex virus type-1 (HSV-1) of Mckrae strain was induced by cultured and infectious Vero cells and prepared by ten-times step dilution with free-serum DMEM, and plaque assay was used to detect the viral titers.HSV-1 of 1×107 plaque-forming unit (PFU) in 0.6 μl was injected into the corneal stroma of 6 to 8- week-old SPF male C57BL/6 mice using a microliter syringe to establish latent HSK mouse models.The models were examined under the slit lamp microscope at day 5, 7, 11, 14 and 17 after modeling as well as day 0, 2, 4 and 6 after recurrence, and the central cornea touch sensitivity was recorded.The models were divided into ACV-injected group, ACV+ bevacizumab-injected group and normal saline-injected group, and 5 μl normal saline with 50 μg ACV, 50 μg ACV+ 5 μl bevacizumab or 10 μl normal saline was subconjunctivally injected according to grouping in 4 eyes of each group, respectively.Twelve model eyes were exposed to ultraviolet (UV)-B to induce the recurrent HSK.Corneal wholemounts were prepared at day 9 after modeling for the assessment of corneal neovascularization and nerve fiber distribution by immunofluorescence assay of CD31 and βⅢ Tubulin antibodies.The areas of corneal neovascularization and scarring were measured with Image J software.The change rate of lesion was calculated and described as a ratio of lesion size at day 8 with day 0 after induction recurrence.

Results

The modeling success rate was over 80%, and all infected mice showed latent period at day 45 after modeling.Corneal opacification was the most serious at day 7 after modeling and day 2 after recurrence, and the largest corneal neovascular area was seen at day 15 after modeling and at day 2 after recurrence, and the central cornea touch sensitivity was the worst at day 9 after initial infection.The mean corneal lesion area was 3.348 mm2 in the ACV+ bevacizumab-injected group, which was smaller than 3.930 mm2 in the ACV-injected group (Z=-2.309, P =0.021). The central corneal sensitivity in the ACV+ bevacizumab-injected group was significantly higher than that in the normal saline-injected group (5.50±0.71 versus 0.50±1.41, Z=-2.397, P=0.029). The increase rate of corneal lesion area in the ACV+ bevacizumab-injected group was evidently lower than that in the normal saline-injected group ([167.10±52.53]% versus [312.30±74.18]%, Z=-1.992, P=0.046). At the 7th day after modeling, the relative expressing levels of thymidine kinase (TK) and infected-cell protein-27 (ICP-27) mRNA in the corneal tissue and trigeminal ganglion were significantly increased at day 7 and reduced at day 45 after modeling, and the factors raised again at day 2 and retreated at day 7 after induction of recurrence.In addition, the expression of LAT mRNA peaked at day 45 after modeling and reduced gradually at day 2 after recurrence until a new increasing peak at day 7 after recurrence (all at P<0.01). Immunofluorescence showed that compared with the normal saline-injected group, the corneal new vessels were lessened and corneal never fibers were increased in the ACV-injected group and ACV+ bevacizumab-injected group.

Conclusions

The combination of bevacizumab with ACV can inhibit corneal neovascularization and scarring in HSK mice, and bevacizumab exhibits a synergistic effect with ACV in management of HSK.

Key words:

Keratitis, herpetic; Herpes simplex virus type-1; Bevacizumab; Corneal neovascularization/drug therapy; Ultraviolet rays; Recurrence; Disease models, animal; Mice, inbred C57BL

Contributor Information

Zhou Hongwei
Shandong Eye Institute, Qingdao 266071, China
Wang Tongsong
Zhang Songmei
Xie Lixin
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Updated: September 4, 2019 — 2:16 pm