Ian Pitha

Losartan Treatment Protects Retinal Ganglion Cells and Alters Scleral Remodeling in Experimental Glaucoma

Purpose To determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice. Methods We produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by β-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry. Results Losartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001). The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01). Both losartan and enalapril significantly lowered blood pressure (p< 0.001), but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9). Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP. Conclusions The neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head.

Subconjunctival Delivery of Dorzolamide-Loaded Poly(ether-anhydride) Microparticles Produces Sustained Lowering of Intraocular Pressure in Rabbits

Topical medications that inhibit the enzyme carbonic anhydrase (CAI) are widely used to lower intraocular pressure in glaucoma; however, their clinical efficacy is limited by the requirement for multiple-daily dosing, as well as side effects such as blurred vision and discomfort on drop instillation. We developed a biodegradable polymer microparticle formulation of the CAI dorzolamide that produces sustained lowering of intraocular pressure after subconjunctival injection. Dorzolamide was ion paired with sodium dodecyl sulfate (SDS) and sodium oleate (SO) with 0.8% and 1.5% drug loading in poly(lactic-co-glycolic acid) (PLGA), respectively. Encapsulating dorzolamide into poly(ethylene glycol)-co-poly(sebacic acid) (PEG3-PSA) microparticles in the presence of triethylamine (TEA) resulted in 14.9% drug loading and drug release that occurred over 12 days in vitro. Subconjunctival injection of dorzolamide-PEG3-PSA microparticles (DPP) in Dutch belted rabbits reduced IOP as much as 4.0 ± 1.5 mmHg compared to untreated fellow eyes for 35 days. IOP reduction after injection of DPP microparticles was significant when compared to baseline untreated IOPs (P < 0.001); however, injection of blank microparticles (PEG3-PSA) did not affect IOP (P = 0.9). Microparticle injection was associated with transient clinical vascularity and inflammatory cell infiltration in conjunctiva on histological examination. Fluorescently labeled PEG3-PSA microparticles were detected for at least 42 days after injection, indicating that in vivo particle degradation is several-fold longer than in vitro degradation. Subconjunctival DPP microparticle delivery is a promising new platform for sustained intraocular pressure lowering in glaucoma.

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria

&NA; We developed an explant model of the mouse eye and optic nerve that facilitates the study of retinal ganglion cell axons and mitochondria in the living optic nerve head (ONH) in an ex vivo environment. Two transgenic mouse strains were used, one expressing yellow fluorescent protein in selected axons and a second strain expressing cyan fluorescent protein in all mitochondria. We viewed an explanted mouse eye and optic nerve by laser scanning microscopy at and behind the ONH, the site of glaucoma injury. Explants from previously untreated mice were studied with the intraocular pressure (IOP) set artificially at normal or elevated levels for several hours. Explants were also studied from eyes that had undergone chronic IOP elevation from 14 h to 6 weeks prior to ex vivo study. Image analysis in static images and video of individual mitochondria or axonal structure determined effects of acute and chronic IOP elevation. At normal IOP, fluorescent axonal structure was stable for up to 3 h under ex vivo conditions. After chronic IOP elevation, axonal integrity index values indicated fragmentation of axon structure in the ONH. In mice with fluorescent mitochondria, the normal density decreased with distance behind the ONH by 45% (p = 0.002, t‐test). Density increased with prior chronic IOP elevation to 21,300 ± 4176 mitochondria/mm2 compared to control 16,110 ± 3159 mitochondria/mm2 (p = 0.025, t‐test), but did not increase significantly after 4 h, acute IOP elevation (1.5% decrease in density, p = 0.83, t‐test). Mean normal mitochondrial length of 2.3 ± 1.4 &mgr;m became 13% smaller after 4 h of IOP elevation ex vivo compared to baseline (p = 0.015, t‐test, N‐10). Normal mitochondrial speed of movement was significantly slower in the anterograde direction (towards the brain) than retrograde, but there were more mitochondria in motion and traveling longer lengths in anterograde direction. The percent of mitochondria in motion decreased by >50% with acute IOP increase to 30 mm Hg after 60 min. A new ocular explant model implemented with eyes from transgenic mice with fluorescent cellular components provided real time measurement of the early events in experimental glaucoma and quantitative outcomes for neuroprotection therapy experiments. HighlightsAn explant model of the mouse eye was developed in the living optic nerve head, the site of glaucoma injury.This model will facilitate the study of retinal ganglion cell axons (RGC) and mitochondria via laser scanning microscopy.Two transgenic mouse strains; one fluorescing selected RGC and another all mitochondria were imaged.Glaucoma bead model increase mitochondria density, but decrease in mitochondrial length & mitochondrial movement.Glaucoma caused fragmentation of axon structures at the ONH, increasing in severity with longer periods of glaucoma exposure.

The effects of age on mitochondria, axonal transport, and axonal degeneration after chronic IOP elevation using a murine ocular explant model

ABSTRACT The purpose of this study was to compare younger and older mice after chronic intraocular pressure (IOP) elevation lasting up to 4 days with respect to mitochondrial density, structure, and movement, as well as axonal integrity, in an ex vivo explant model. We studied 2 transgenic mouse strains, both on a C57BL/6J background, one expressing yellow fluorescent protein (YFP) in selected axons and one expressing cyan fluorescent protein (CFP) in all mitochondria. Mice of 4 months or 14 months of age were exposed to chronic IOP by anterior chamber microbead injection for 14h, 1, 3, or 4 days. The optic nerve head of globe‐‐optic nerve explants were examined by laser scanning microscopy. Mitochondrial density, structure, and movement were quantified in the CFP explants, and axonal integrity was quantified in YFP explants. In control mice, there was a trend towards decreased mitochondrial density (# per mm2) with age when comparing younger to older, control mice, but this was not significant (1947±653 vs 1412±356; p=0.19). Mitochondrial density decreased after IOP elevation, significantly, by 31%, in younger mice (p=0.04) but trending towards a decrease, by 22%, in older mice (p=0.82) compared to age matched controls. Mitochondrial mean size was not altered after chronic IOP elevation for 14h or more (p≥0.16). When assessing mitochondrial movement, in younger mice, 5% were mobile at any given time; 4% in the anterograde direction and 1% retrograde. In younger untreated tissue, only 75% of explants had moving mitochondria (mean=15.8 moving/explant), while after glaucoma induction only 24% of explants had moving mitochondria (mean=4.2 moving/explant; difference from control, p=0.03). The distance mitochondria traveled in younger mice was unchanged after glaucoma exposure, but in older glaucoma explants the distance traveled was less than half of older controls (p<0.0003). In younger mice, mitochondrial speed increased after 14h of elevated IOP (p=0.006); however, in older glaucoma explants, movement was actually slower than controls (p=0.02). In RGC‐YFP explants, axonal integrity declined significantly after 4 days of IOP elevation to a similar degree in both younger and older mice. Older mice underwent greater loss of mitochondrial movement with chronic IOP elevation than younger mice, but suffered similar short‐term axonal fragmentation in C57BL/6J mice. These transgenic strains, studied in explants, permit observations of alterations in intracellular structure and organelle activity in experimental glaucoma. HIGHLIGHTSExplant model of the mouse eye was used to study retinal ganglion cell axons and mitochondria changes in the optic nerve head, in both older and younger mice.Using an LSM, imaging at the ONH was possible in 2 transgenic mouse strains; 1 fluorescently expressing select RGC axons & another all mitochondria.Glaucoma caused fragmentation of axon structures at the ONH, increasing in severity with longer periods of glaucoma exposure.Comparable fragmentation of the RGCs was found between young and older glaucoma exposed mice.Glaucoma bead model caused a significant decrease in mitochondria density in both younger and older exposed mice.A significant decrease in mitochondrial movement also occurred after glaucoma bead model exposure in younger and older mice.