Effect of integrin ανβ3 on conjunctival scar formation after glaucoma filtration surgery in rats

Objective

To investigate the expression of integrin ανβ3 in conjunctival scar after glaucoma filtration surgery and to explore its role in the formation of filtering scar in rats.

Methods

Fifty-five SPF male SD rats aged 6-8 weeks were selected to establish the model of glaucoma filtration surgery, and 15 unsuccessful modeling rats were excluded.The remaining rats were divided into control group, transforming growth factor (TGF) group, integrin ανβ3 receptor antagonist LM609 group and TGF+ LM609 group, with 10 rats in each group.The conjunctival flap of different groups was treated with cotton piece containing 0.9% sodium chloride, TGF-β2 (2 mg/ml), LM609 (40 μg/ml), TGF-β2 (2 mg/ml)+ LM609 (40 μg/ml) solution for two minutes, respectively, according to grouping.The intraocular pressure (IOP) was monitored with an iCARE tonometer before operation and at 3 days, 1 week, 2 weeks after operation, and the average IOP ratio ([postoperative IOP/preoperative IOP]×100%) was calculated.At 3 days, 1 week and 2 weeks after operation, slit lamp microscopy was used to observe the morphological changes of filtering blebs and calculate the survival rate of filtering blebs.Two weeks after operation, the operative eyeball tissues were removed and made into paraffin section.The inflammatory changes, fibrosis and collagen deposition of bulbar conjunctiva in the operation area were observed by hematoxylin-eosin staining and picric acid-sirius red staining, respectively.Western blot was used to detect the expression of integrin αν and β3.Immunohistochemical staining was used to detect the expression of integrin ανβ3 in the conjunctival tissue of operation area.The use and care of the rats complied with the Instruction Notions with Respect to Care for Laboratory by State Ministry of Science and Technology.The study protocol was approved by an Ethics Committee of The Daping Hospital, Army Medical University (No.2017-06).

Results

The IOP of rats in each group was significantly decreased on the third day after operation compared with before operation, and gradually recovered to the preoperative level in the first to second week after operation.The mean IOP ratios of the control group, TGF group, LM609 group and TGF+ LM609 group were (101.80±4.44)%, (103.90±5.65)%, (94.10±7.45)% and (100.60±4.61)%, respectively, showing a statistically significant difference among them (F=4.571, P=0.010). The mean IOP ratio of LM609 group was lower than that of the control group, TGF group and TGF+ LM609 group (all at P<0.05). Two weeks after operation, the filter blebs became flat and the bulbar conjunctival tissue was slightly thickened in the control group and TGF+ LM609 group, and bulbar conjunctival tissue was significantly thickened with a large number of blood vessels growing in the TGF group, and localized small filter blebs as well as less vascularization were seen in the LM609 group.The survival rate of filtering bleb in the TGF group was significantly lower than that in the control group, LM609 group and TGF+ LM609 group, and the survival rate of filtering bleb in the LM609 group was significantly higher than that in the control group and TGF+ LM609 group (all at P<0.05). Two weeks after operation, there were fewer infiltrated inflammatory cells and looser collagen fiber deposition in the conjunctival tissue in the LM609 group and TGF+ LM609 group compared with the TGF group.The relative expression levels of integrin αν and β3 under conjunctival tissue in the TGF group were significantly higher than those in the control group, LM609 group and TGF+ LM609 group (all at P<0.05). Immunohistochemical staining showed that integrin ανβ3-positive cells were mainly in the conjunctival tissue around the operation area and microtubules.The number of integrin ανβ3-positive cells in the conjunctival tissue of the operation area in the TGF group were significantly greater than those of other groups.

Conclusions

Integrin ανβ3 is highly expressed in the scar of conjunctival tissue after filtering surgery in rats.The inhibition of integrin ανβ3 can reduce the deposition of fibers and prolong the survival of filtering blebs, suggesting that integrin ανβ3 may be involved in the formation of conjunctival scar.

Glaucoma; Filtering surgery; Disease models, animal; Rats; Transforming growth factor-β; Integrins; Filtering blebs; Scarring