VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model

Abstract

Purpose: To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model.

Methods: Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30 days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31).

Results: Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P < 0.001, P < 0.001) and vs. control (P < 0.001, P < 0.001). After 30 days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P < 0.001), and control (P < 0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P < 0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978).

Conclusions: Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Figure 1.

Methodological flow chart.

Figure 2.

Qualitative bleb assessment. Bleb appearances on postoperative day 10 (A–D), day 20 (E–H), and day 30 (I–L) for each treatment group. Qualitatively, the VEGF-C combined with 5-FU group (C, G, K) maintained better morphology with bulging, diffuse, fleshy blebs compared with the 5-FU (A, E, I), VEGF-C (B, F, J) and control (D, H, L) groups. (A–L) Black arrows denote the bleb margins. (M) Survival curves of filtration blebs. VEGF-C combined with 5-FU treatment group showed prolonged bleb survival.

Figure 3.

Quantitative bleb assessment and IOP. (A) Bleb length, (B) bleb width, and (C) IOP for the four treatment groups. VEGF-C combined with 5-FU treatment resulted in significantly (A) longer and (B) wider blebs compared with the other three treatments. (C) VEGF-C combined with 5-FU treatment showed the only negative trending IOP that was statistically significant.

Figure 4.

AS-OCT and hematoxylin and eosin staining of filtration blebs. (A–D) Hematoxylin and eosin staining and (E–H) bleb AS-OCT images at day 30. (E–H) The lower right corner inserts shows where the OCT was performed using the corneal line scan protocol. AC, anterior chamber; C, cornea; FB, filtering bleb; PE, iris pigment epithelium; S, sclera. Scale bars, 200 microns.

Figure 5.

Gene expression of lymphatic and blood vessel markers. Gene expression was assessed across the various conditions evaluating lymphatic (A; LYVE-1) and (B; podoplanin), as well as blood vessel endothelial (C; CD31) markers from blebs harvested on postoperative day 30. *P < 0.05, **P < 0.01, ***P < 0.001.