Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve

Abstract

Purpose: Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve.

Methods: A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining.

Results: The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group.

Conclusions: AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

Fig 1. Establishment and confirmation of AECs/hbFGF with lentiviral vectors.

A: Enzyme digestion analysis of lentiviral vector. Lane 1: SpeⅠand XhoⅠdouble digestion of lentiviral vector; Lane 2: Undigested lentiviral vector; M, DL2000 marker. B: Expression of bFGF in transfected AECs by RT-PCR. Lane 1 and 2: bFGF segment(222 bp); M: DL2000 marker. C: Morphology of AECs after blasticidin selection. Left: AECs in the untransfected group died and detached from the bottom; Right: Most AECs transfected with target fragment survived. Bar = 20μm D: Concentration of bFGF secreted by transfected AECs in different time groups by ELISA. (*: P<0.01 vs Control. ▲: P<0.01 vs 3d).

Fig 2. Rat model of optic nerve transection.

A: Exposed rat optic nerve (arrows). B: The transverse gap in the transected optic nerve (arrows). Bar = 500μm.

Fig 3. Tracing transplanted cells with Hoechst dyes within the injured optic nerve 28d after transplantation.

A: The transected optic nerve without cell transplantation. B: The transected optic nerve with AECs transplantation. AECs survived within the injury site. C: The transected optic nerve with AECs/hbFGF transplantation. AECs/hbFGF survived and migrated within the injury site (40×). D: The transected optic nerve with AECs/hbFGF transplantation. AECs/hbFGF survived and migrated within the injury site (100×). Bar = 300μm. (I: Injured area; P: Proximal part; D: Distal part).

Fig 4. RGCs labeling and counting in retina flattening of different groups.

A: RGCs labeled with BDA in different groups. Bar = 20μm. B: RGCs of Nissl staining in different groups. Bar = 20μm. C: Comparisons of RGCs counting in different groups (HP: High power field). (*: P<0.01 vs Normal group; ▲: P<0.01 vs Control group; ★: P<0.01 vs AECs group).

Fig 5. GAP-43 immunohistochemical staining in the injury site of optic nerve in different groups.

A: GAP-43 staining in control group. B: GAP-43 staining in AECs group. C: GAP-43 staining in AECs/hbFGF group. (Arrowhead indicates GAP-43 immunoreactive substance; The dotted line indicates the junction of lesion zone and the distal part; “D” indicates “the distal part”, and “P” indicates “the proximal part”). D: Comparisons of the GAP-43 integral optical density value among different groups. (*: P<0.01 vs The control group; ▲: P<0.01 vs The AECs group).