The effects of 3% diquafosol sodium application on the tear functions and ocular surface of the Cu,Zn-superoxide dismutase-1 (Sod1)-knockout mice

Abstract

Purpose: To investigate the role of a water and mucin secretagogue (3% diquafosol sodium eye drops) on the tear function and conjunctival ocular surface changes in Sod1(-/-) in comparison to the wild-type (WT) mice.

Methods: Fourteen eyes of 7 Sod1(-/-) male mice with C57BL/background and 14 eyes of 7 C57BL6 strain wild-type male mice were examined at 40 weeks in this study. All mice had application of 3% diquafosol ophthalmic solution six times a day for 2 weeks. Tear film stability and corneal epithelial damage was evaluated by fluorescein and Rose Bengal stainings. Anterior segment photography was performed before and after eye drop instillations. Aqueous tear quantity was measured with phenol red-impregnated cotton threads without anesthesia. Animals were sacrificed at 42 weeks after diquafosol treatment and the whole globe specimens were subjected to periodic acid Schiff staining. Goblet cell density was quantified by J Image software. Quantitative real-time PCR for conjunctival muc 5AC messenger RNA expression was also performed.

Results: Sod1(-/-) mice had significantly higher fluorescein staining scores compared to the WT mice before eye drop instillation. The mean tear film breakup time, Rose Bengal staining scores, and muc5 messenger RNA expression improved significantly with diquafosol treatment in both the WT and the knockout mice. The mean fluorescein staining score and aqueous tear quantity significantly improved in the Sod1(-/-) mice with treatment. A notable and consistent increase in goblet cells and decrease in inflammatory cell infiltrates could be confirmed in all specimens after 2 weeks of diquafosol eye drop application.

Conclusions: Three percent diquafosol ophthalmic solution appears to be effective in the treatment of ocular surface disease in this age-related dry eye disease mouse model.

Figure 1

Tear film breakup time changes with 3% topical diquafosol application in the Sod1^-/- mice and wild type (WT) mice. Note the significant improvement in tear stability with 2 weeks of diquafosol sodium treatment. * represents p < 0.01.

Figure 2

Anterior segment photographs showing changes in vital staining with 3% topical diquafosol application in the Sod1^-/- mice. Upper inserts: Note the improvement in fluorescein staining after diquafosol sodium application. Lower inserts: Note the improvement in Rose Bengal staining after diquafosol sodium application.

Figure 3

Anterior segment photographs showing changes in vital stainings with 3% topical diquafosol application in the wild-type (WT) mice. Upper inserts: Note the improvement in fluorescein staining after diquafosol sodium application. Lower inserts: Note the improvement in Rose Bengal staining after diquafosol sodium application.

Figure 4

Changes of fluorescein staining scores with 3% topical diquafosol application in the Sod1^-/- mice and wild type (WT) mice. Note the significant improvement in the fluorescein staining score in the Sod1^-/- mice with 2 weeks of diquafosol sodium treatment. * represents p < 0.01.

Figure 5

Changes in Rose Bengal staining scores with 3% topical diquafosol application in the Sod1^-/- mice and wild type (WT) mice. Note the significant improvement in the Rose Bengal staining score in the Sod1^-/- mice with 2 weeks of diquafosol sodium treatment. * represents p < 0.01.

Figure 6

Weight-adjusted tear quantity changes with two weeks of 3% topical diquafosol application in the Sod1^-/- mice and wild type (WT) mice. Note the significant improvement in tear quantity in both the Sod1^-/- and WT mice with 2 weeks of diquafosol sodium treatment. * represents p < 0.01.

Figure 7

Periodic acid Schiff stainings of conjunctival specimens showing changes in the Sod1^-/- mice before and after 3% topical diquafosol (DQS) application. A: Note the extensive inflammatory cell infiltration and lack of goblet cells in the conjunctival epithelium. B: Note the marked decrease in inflammatory cells and the presence of numerous goblet cells.

Figure 8

Real time reverse transcriptase polymerase chain reaction mucin 5AC glycoprotein messenger RNA expression changes with diquafosol application in the Sod1^-/- mice and wild type (WT) mice. Note the significant increase in muc 5Ac mRNA expression in both WT and the Sod1^-/- mice with 2 weeks of diquafosol sodium treatment. * represents p < 0.01.