Intravitreal Injection of Liposomes Loaded with a Histone Deacetylase Inhibitor Promotes Retinal Ganglion Cell Survival in a Mouse Model of Optic Nerve Crush

Abstract

Various neuroprotective agents have been studied for the treatment of retinal ganglion cell (RGC) diseases, but issues concerning the side effects of systemically administered drugs and the short retention time of intravitreally injected drugs limit their clinical applications. The current study aimed to evaluate the neuroprotective effects of intravitreally injected trichostatin A (TSA)-loaded liposomes in a mouse model of optic nerve crush (ONC) and determine whether TSA-loaded liposomes have therapeutic potential in RGC diseases. The histone deacetylase inhibitor, TSA, was incorporated into polyethylene glycolylated liposomes. C57BL/6J mice were treated with an intravitreal injection of TSA-loaded liposomes and liposomes loaded with a lipophilic fluorescent dye for tracking, immediately after ONC injury. The expression of macroglial and microglial cell markers (glial fibrillary acidic protein and ionized calcium binding adaptor molecule-1), RGC survival, and apoptosis were assessed. We found that the liposomes reached the inner retina. Their fluorescence was detected for up to 10 days after the intravitreal injection, with peak intensity at 3 days postinjection. Intravitreally administered TSA-loaded liposomes significantly decreased reactive gliosis and RGC apoptosis and increased RGC survival in a mouse model of ONC. Our results suggest that TSA-loaded liposomes may help in the treatment of various RGC diseases.

Keywords: histone deacetylase inhibitor; intravitreal injection; liposome; optic nerve crush; retinal ganglion cell.

Figure 1

Concentration–time profile in the vitreous of mouse eye following the intravitreal injection of 1 μM trichostatin A (TSA).

Figure 2

Schematic illustration of liposome for tracking and TSA-loaded liposome. Two liposome particles were synthesized. (Left) The liposome was synthesized for the purpose of tracking the location of the drug when it was administered to the vitreous cavity. (Right) The liposome was loaded with TSA and synthesized for the purpose of treatment. DMPC = 1,2-dimyristoyl-sn-glycero-3-phosphocholine; PEG 2000-PE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; DOTAP = 1,2-dioleoyl-3-trimethylammonium propane.

Figure 3

Liposome characteristics. (A) Transmission electron microscopy (TEM) image of nonloaded liposomes. (B) Cumulative TSA release profile of TSA-loaded liposomes.

Figure 4

Temporal distribution of intravitreally injected liposomes. Images with lower magnification of the central retina including optic nerve head are presented in Supplementary Figure S1. Fluorescence microscopic images of the retina and fluorescence intensity at different time points after intravitreal injection of liposomes loaded with lipophilic fluorescence dye (red) are presented. The fluorescence intensity peaked at 3 days postinjection and declined gradually over a period of 10 days. D = days; Blue color = 4′,6-diamidino-2-phenylindole (DAPI) staining of retinal cell nuclei; Scale bar = 100 μm.

Figure 5

Effect of intravitreally injected TSA-loaded liposomes on gliosis in retina after optic nerve crush (ONC). The ONC procedure markedly increased GFAP (A,C) and Iba1 (B,D) protein expression in retinas at 3 days. The intravitreal injection of TSA-loaded liposomes significantly attenuated this response. The relative chemiluminescence intensity for each protein band was normalized using β-actin as a calibrator. TSA-Lip 50 µM = liposomes loaded with 50 µM TSA; TSA-Lip 500 µM = liposomes loaded with 500 µM TSA. Error bars represent the SD (n = 6 retinas per group); * p < 0.05; one-way ANOVA with post-hoc LSD test.

Figure 6

Immunohistochemical staining of GFAP and Iba1 in retinas at 7 days after ONC. (Upper) Sections from vehicle-injected retinas after ONC show increased GFAP activity on the RGC layer and the radial process of macroglial cells spanning the inner plexiform layer. The intravitreal injection of TSA-loaded liposomes significantly attenuated the GFAP activity in retinas after ONC. (Lower) Immunolabeling for Iba1 in retinal sections showed prominent activation of microglia in vehicle-injected retinas after ONC. This activation was ameliorated in the retinas injected with TSA-loaded liposomes. Images with lower magnification are presented in Supplementary Figure S2. TSA-Lip 50 µM = liposomes loaded with 50 µM TSA; TSA-Lip 500 µM = liposomes loaded with 500 µM TSA.

Figure 7

TUNEL staining was performed on retinal sections 7 days after ONC. Vehicle-injected retinas demonstrated significantly higher TUNEL-positive apoptotic cells in RGC layer after ONC (** p < 0.001, compared to the uninjured normal eyes). The intravitreal injection of TSA-loaded liposomes immediately after ONC injury, suppressed RGC apoptosis (** p < 0.001, compared to the vehicle group). There were no significant differences in TUNEL-positive apoptotic cells between the groups with 50 μM and 500 μM TSA-loaded liposomes. Images with lower magnification are presented in Supplementary Figure S3. TSA-Lip 50 µM = liposomes loaded with 50 µM TSA; TSA-Lip 500 µM = liposomes loaded with 500 µM TSA. All comparisons were performed using one-way ANOVA with post-hoc LSD test.

Figure 8

Effect of TSA-loaded liposomes on RGC survival after ONC. Retinal flat mounts are shown for the uninjured normal retinas, vehicle-injected retinas, and retinas injected with 50 μM and 500 μM TSA-loaded liposomes (n = 5 retinas per group). A quantitative analysis of RGC survival in center, middle, and peripheral zones of each retinal quadrant is also shown as a graph. TSA-Lip 50 µM = liposomes loaded with 50 µM TSA; TSA-Lip 500 µM = liposomes loaded with 500 µM TSA. Scale bar = 100 µm; ** p < 0.001, compared to the uninjured normal retina; * p < 0.05 compared to the vehicle-injected retina; one-way ANOVA with post-hoc LSD test.