Background: To compare two prolonged animal models of glaucoma over 24 weeks of follow-up. A novel pre-trabecular model of chronic glaucoma was achieved by injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (10–20 µm) (Ms20/10) into the ocular anterior chamber to progressively increase ocular hypertension (OHT). Methods: Rat right eyes were injected to induce OHT: 50% received a suspension of Ms20/10 in the anterior chamber at 0, 2, 4, 8, 12, 16 and 20 weeks, and the other 50% received a sclerosing episcleral vein injection biweekly (EPIm). Ophthalmological clinical signs, intraocular pressure (IOP), neuroretinal functionality measured by electroretinography (ERG), and structural analysis of the retina, retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) protocols using optical coherence tomography (OCT) and histological exams were performed. Results: Both models showed progressive neuroretinal degeneration (p < 0.05), and contralateral eye affectation. The Ms20/10 model showed a more progressive increase in IOP and better preservation of ocular surface. Although no statistical differences were found between models, the EPIm showed a tendency to produce thicker retinal and thinner GCL thicknesses, slower latency and smaller amplitude as measured using ERG, and more aggressive disturbances in retinal histology. In both models, while the GCL showed the greatest percentage loss of thickness, the RNFL showed the greatest and earliest rate of thickness loss. Conclusions: The intracameral model with biodegradable microspheres resulted more like the conditions observed in humans. It was obtained by a less-aggressive mechanism, which allows for adequate study of the pathology over longer periods.

Progressive degeneration of neuroretinal tissue with maintained elevated intraocular pressure (IOP) to simulate chronic glaucoma was produced by intracameral injections of poly (lactic-co-glycolic) acid (PLGA) microspheres (Ms) in rat eyes. The right eye of 39 rats received different sizes of PLGA-Ms (2 µL suspension; 10% w/v): 14 with 38–20 µm Ms (Ms38/20 model) and 25 with 20–10 µm particles (Ms20/10 model). This novel glaucoma animal model was compared to the episcleral vein sclerosis (EPI) model (25 eyes). Injections were performed at baseline, two, four and six weeks. Clinical signs, IOP, retina and optic nerve thicknesses (using in vivo optical coherence tomography; OCT), and histological studies were performed. An IOP increment was observed in all three groups, however, the values obtained from the PLGA-Ms injection resulted lower with a better preservation of the ocular surface. In fact, the injection of Ms20/10 created a gentler, more progressive, and more sustained increase in IOP. This IOP alteration was correlated with a significant decrease in most OCT parameters and in histological ganglion-cell count for the three conditions throughout the eight-week follow-up. In all cases, progressive degeneration of the retina, retinal ganglion cells and optic nerve, simulating chronic glaucoma, was detected by OCT and corroborated by histological study. Results showed an alternative glaucoma model to the well-known episcleral vein model, which was simpler to perform, more reproducible and easier to monitor in vivo.

Glaucoma causes blindness due to the progressive death of retinal ganglion cells. The immune response chronically and subclinically mediates a homeostatic role. In current clinical practice, it is impossible to analyse neuroinflammation non-invasively. However, analysis of vitreous images using optical coherence tomography detects the immune response as hyperreflective opacities. This study monitors vitreous parainflammation in two animal models of glaucoma, comparing both healthy controls and sexes over six months. Computational analysis characterizes in vivo the hyperreflective opacities, identified histologically as hyalocyte-like Iba-1+ (microglial marker) cells. Glaucomatous eyes showed greater intensity and number of vitreous opacities as well as dynamic fluctuations in the percentage of activated cells (50–250 microns²) vs. non-activated cells (10–50 microns²), isolated cells (10 microns²) and complexes (>250 microns²). Smaller opacities (isolated cells) showed the highest mean intensity (intracellular machinery), were the most rounded at earlier stages (recruitment) and showed the greatest change in orientation (motility). Study of vitreous parainflammation could be a biomarker of glaucoma onset and progression.

Intravitreal injection is the gold standard therapeutic option for posterior segment pathologies, and long-lasting release is necessary to avoid reinjections. There is no effective intravitreal treatment for glaucoma or other optic neuropathies in daily practice, nor is there a non-invasive method to monitor drug levels in the vitreous. Here we show that a glaucoma treatment combining a hypotensive and neuroprotective intravitreal formulation (IF) of brimonidine–Laponite (BRI/LAP) can be monitored non-invasively using vitreoretinal interface imaging captured with optical coherence tomography (OCT) over 24 weeks of follow-up. Qualitative and quantitative characterisation was achieved by analysing the changes in vitreous (VIT) signal intensity, expressed as a ratio of retinal pigment epithelium (RPE) intensity. Vitreous hyperreflective aggregates mixed in the vitreous and tended to settle on the retinal surface. Relative intensity and aggregate size progressively decreased over 24 weeks in treated rat eyes as the BRI/LAP IF degraded. VIT/RPE relative intensity and total aggregate area correlated with brimonidine levels measured in the eye. The OCT-derived VIT/RPE relative intensity may be a useful and objective marker for non-invasive monitoring of BRI/LAP IF.

Glaucoma is a multifactorial neurodegenerative disease that affects the retina and optic nerve. The aim of this work was to reach different therapeutics targets by co-encapsulating three neuroprotective substances with hypotensive (latanoprost), antioxidant (melatonin) and anti-inflammatory (ketorolac) activity in biodegradable poly (lactic-co-glycolic acid) (PLGA) microspheres (MSs) capable of releasing the drugs for months after intravitreal injection, avoiding the need for repeated administrations. Multi-loaded PLGA MSs were prepared using the oil-in-water emulsion solvent extraction-evaporation technique and physicochemically characterized. PLGA 85:15 was the polymer ratio selected for the selected formulation. Tri-loaded MSs including vitamin E as additive showed good tolerance in retinal pigment epithelium cells after 24 h exposure (>90% cell viability). The final formulation (KMLVE) resulted in 33.58 ± 5.44 µm particle size and drug content (µg/mg MSs) of 39.70 ± 5.89, 67.28 ± 4.17 and 7.51 ± 0.58 for melatonin, ketorolac and latanoprost respectively. KMLVE were able to release in a sustained manner the three drugs over 70 days. KMLVE were injected at 2 and 12 weeks in Long-Evans rats (n = 20) after the induction of chronic glaucoma. Ophthalmological tests were performed and compared to not treated glaucomatous (n = 45) and healthy (n = 17) animals. Treated glaucomatous rats reached the lowest intraocular pressure, enhanced functionality of bipolar and retinal ganglion cells and showed greater neuroretinal thickness by optical coherence tomography (p < 0.05) compared to not treated glaucomatous rats at 24 weeks follow-up. According to the results, the tri-loaded microspheres can be considered as promising controlled-release system for the treatment of glaucoma.

Glaucoma is a multifactorial pathology involving the immune system. The subclinical immune response plays a homeostatic role in healthy situations, but in pathological situations, it produces imbalances. Optical coherence tomography detects immune cells in the vitreous as hyperreflective opacities and these are subsequently characterised by computational analysis. This study monitors the changes in immunity in the vitreous in two steroid-induced glaucoma (SIG) animal models created with drug delivery systems (microspheres loaded with dexamethasone and dexamethasone/fibronectin), comparing both sexes and healthy controls over six months. SIG eyes tended to present greater intensity and a higher number of vitreous opacities (p < 0.05), with dynamic fluctuations in the percentage of isolated cells (10 µm²), non-activated cells (10–50 µm²), activated cells (50–250 µm²) and cell complexes (>250 µm²). Both SIG models presented an anti-inflammatory profile, with non-activated cells being the largest population in this study. However, smaller opacities (isolated cells) seemed to be the first responder to noxa since they were the most rounded (recruitment), coinciding with peak intraocular pressure increase, and showed the highest mean Intensity (intracellular machinery), even in the contralateral eye, and a major change in orientation (motility). Studying the features of hyperreflective opacities in the vitreous using OCT could be a useful biomarker of glaucoma.