Targeted Microglial Attenuation through Dendrimer-Drug Conjugates Improves Glaucoma Neuroprotection

Abstract

Retinal microglial/macrophage activation and optic nerve (ON) microglial/macrophage activation are glaucoma biomarkers and potential therapeutic targets for this blinding disease. We report targeting of activated microglia by PAMAM dendrimers in a rat glaucoma model and neuroprotection by N-acetylcysteine-conjugated dendrimer (D-NAC) conjugates in a post-injury rescue experiment. Intravitreally delivered fluorescently labeled dendrimer (D-Cy5) conjugates targeted and were retained in Iba-1-positive cells (90% at 7 days and 55% after 28 days) in the retina following intraocular pressure (IOP) elevation, while systemically delivered D-Cy5 targeted ON cells. A single intravitreal D-NAC dose given 1 week after IOP elevation significantly reduced transcription of pro-inflammatory (IL-6, MCP-1, IL-1β) and A1 astrocyte (Serping1, Fkbp5, Amigo2) markers and increased survival of retinal ganglion cells (39 ± 12%) versus BSS- (20 ± 15%, p = 0.02) and free NAC-treated (26 ± 14%, p = 0.15) eyes. These results highlight the potential of dendrimer-targeted microglia and macrophages for early glaucoma detection and as a neuroprotective therapeutic target.

Figure 1.

TLL activates microglia. (A) Schematic of circumferential laser application (trans limbal laser, TLL) to rat eyes. (B–D) Retinal flat mounts of control eye (non-TLL-treated fellow eye) demonstrating resting microglia with ramified morphology. (E–G) Retinal flat mount of TLL-treated eye (7 days) with increased ameboid microglia indicating an activated state. (H) Microglial counts indicating that the TLL-induced glaucoma results in a significant increase in microglial population (~3.3-fold), p < = 0.0003, t-test, n = 6. Scale bar 100 μm.

Figure 2.

Intravitreally administered dendrimers demonstrate pathology-dependent biodistribution. (A) Schematic diagram of experimental protocol for TLL-induced glaucoma, D-Cy5 intravitreal injection, and tissue collection schedule for analysis. (B) Retinal flat mounts of the healthy injected with D-Cy5 minimal or no dendrimer signals in microglial cells (Iba-1+, Green). (C) Retinal flat mount of glaucoma eye demonstrating dendrimer (D-Cy5, red) co-localization in retinal microglia (arrow marks). Retinal tissues were stained Iba-1 (green) to lectin (blue) to label microglia and blood vessels. Scale bar 100 μm.

Figure 3.

Biodistribution, colocalization, and retention of intravitreally administered dendrimer (D-Cy5) in retinal tissue after TLL. The retinal flat mounts were stained for microglia using Iba-1 (green) and blood vessels using lectin (blue). (A) Retinal flat mount at day 14 demonstrating dendrimer (D-Cy5, red) co-localization in activated microglia (arrow marks). (B) Retinal microglia (Iba-1+, Green) counts using IMARIS at day 14, demonstrating that ~90% of retinal microglia are positive for D-Cy5. (C) Confocal images of retinal tissue at day 35 demonstrating D-Cy5 retention in retinal microglia (arrows). (D) Quantification of retinal microglia demonstrating D-Cy5 retention intracellularly at day 35. Scale bar 100 μm for 20× and 20 μm for 40×, n = 6 for POD 7 and n = 5 for POD 28.

Figure 4.

Retinal cross sections after TLL-induced glaucoma at 28 days post intravitreal injection. (A–E) 20× magnification of retinal tissue near the optic nerve demonstrating D-Cy5 co-localization in retina microglia (white arrows) but not ON microglia (scale bar 50 μm). (F–I) Higher-magnification (40×) images of the retinal tissue (white box in E) confirming intracellular co-localization of Iba-1 and D-Cy5 (scale bar 20 μm). The sections were stained with DAPI for nucleus (orange), Brn3a for RGC (blue), Iba-1 for microglia/macrophages (green), and D-Cy5 (red).

Figure 5.

Systemic delivery of D-Cy5 targets the ON in TLL-treated eyes. (A) Experimental schematic. (B) Retinal flat mount after intravitreal dendrimer injection shows selective uptake of dendrimers at the ONH (white arrows) (scale bar 100 μm). (C) Cross sections showing dendrimer uptake within the ON (white arrows) (scale bar 50 μm). Both the flat mounts and sections were stained with DAPI for nucleus (orange), lectin for blood vessels (blue), Iba-1 for microglia/macrophages (green), and D-Cy5 (red).

Figure 6.

D-NAC reduces TLL-induced neuroinflammation. (A) Peak IOP elevation of TLL treatment groups compared to fellow eyes. Whole retinal expression of inflammatory (B) and astrocyte (C) markers in TLL-treated eyes normalized to non-TLL-treated fellow eyes at 28 days (n = 6).

Figure 7.

A single post-TLL treatment with D-NAC prevents RGC loss. (A) Schematic of rat glaucoma experiment with laser treatment occurring at day 0 and intravitreal injection of medications at day 7 (arrows = IOP measurements). (B) IOP elevation was equivalent between treatment groups (C) as was peak IOP. D-NAC treatment increased RGC survival significantly compared to vehicle-treated eyes when a fellow eye comparison was used (D) but did not reach p < 0.05 when compared to (E) a pooled average of control eyes. Representative images of fellow (C) and glaucomatous (G) eyes from vehicle (F), NAC (G), and D-NAC (H) treatment groups (red = RPBMS; blue = DAPI).