Effects of Honghua preserved amniotic membrane on scar healing in experimental glaucoma surgery

Abstract

Aim: To investigate the efficacy and safety of Honghua preserved amniotic membrane (AM) for preventing scar formation of the filtering bleb in a rabbit model of glaucoma trabeculectomy surgery.

Methods: Totally 36 rabbits (36 eyes) were randomly divided into 3 groups: the experimental group (ocular trabeculectomy in combination with Honghua preserved AM transplantation), the control group (ocular trabeculectomy surgery in combination with AM implantation), and the blank group (single trabeculectomy). Clinical observations [including intraocular pressure (IOP), filtering blebs and complications], Masson-Trichrome staining, real-time quantitative reverse transcription-polymerase chain reaction (real-time PCR), Western blot were performed on different time points (D1, D7, D14, D21 and D56) after the surgery.

Results: After operated for 14d, there were statistically significant differences in the filtering blebs compared to the situation before operation (P<0.05), whereas no statistically difference on that among three groups (P>0.05). After 21d, the IOP of experimental group was lowest (P<0.05). There was significant difference between control group and blank group (P<0.05). On postoperative D14, the mean number of fibroblasts in the experimental group was significantly lower (40.6±10.2) compared to those in the control group (54.4±10.8) and blank group (68.2±11.6) (P<0.05, respectively). The mean numbers of the macrophage in the experimental and control groups were respcitively significantly lower versus the blank group (P<0.05, P<0.05, respectively). Compared to that in blank group, the level of transforming growth factor-β (TGF-β1) expression in sclera and conjunctival areas was reduced in the experimental and control groups on protein and mRNA level (P<0.05), but not significant difference between these two groups (P>0.05).

Conclusion: The trabeculectory surgery with Honghua preserved AM can control IOP, sustain the functional filtration bleb, inhibit the proliferation of fibroblasts and open the filtrating pathway on the rabbit glaucoma models.

Keywords: Honghua injection; amniotic membrane; implant; scar formation; trabeculectomy; transforming growth factor-β.

Figure 1. Filtering bleb condition and IOP measurement in three groups after surgery

A: Filtering bleb condition at postoperative four weeks, the experimental group (A1), the control group (A2) and the blank group (A3); B: Filtering bleb score at each observation time points after operation in three groups; C: IOP measurement at each observation time points in three groups (pairwise comparison using the LSD method). ^aP<0.05 vs blank group, ^cP<0.05 vs control group. Red: the experimental group (ocular trabeculectomy in combination with Honghua preserved AM implantation; Blue: the control group (trabeculectory surgery and AM implantation); Green: the blank group (single trabeculectomy).

Figure 2. Number of fibroblast (A) and macrophage (B) in surgery area in three groups on postoperative day 14

^aP<0.05 vs blank group, ^cP<0.05 vs control group.

Figure 3. Western blot analysis TGF-β1 in surgery area in three groups on postoperative D7

TGF-β1 protein was increased significantly after trabeculectomy and decreased with AM application in surgery area. TGF-β1 expression returned to preoperative levels after preserved AM application in sclera (A) and conjunctiva area (B). The Western blot gray value quantitative is analyzed (ImageJ software). ^aP<0.05 vs blank group, ^cP<0.05 vs control group.

Figure 4. Expression of CTGF m RNA in surgery area in three groups on postoperative D14

^aP<0.05 vs blank group, ^cP<0.05 vs control group.