Immune Analysis Using Vitreous Optical Coherence Tomography Imaging in Rats with Steroid-Induced 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.

Smart biodegradable hydrogels: Drug-delivery platforms for treatment of chronic ophthalmic diseases affecting the back of the eye.

This paper aims to develop smart hydrogels based on functionalized hyaluronic acid (HA) and PLGA-PEG-PLGA (PLGA,poly-(DL-lactic-co-glycolic acid); PEG,polyethylene glycol) for use as intraocular drug-delivery platforms. Anti-inflammatory agent dexamethasone-phosphate (0.2 %w/v) was the drug selected to load on the hydrogels. Initially, different ratios of HA-aldehyde (HA-CHO) and thiolated-HA (HA-SH) were assayed, selecting as optimal concentrations 2 and 3 % (w/v), respectively. Optimized HA hydrogel formulations presented fast degradation (8 days) and drug release (91.46 ± 3.80 % in 24 h), thus being suitable for short-term intravitreal treatments. Different technology-based strategies were adopted to accelerate PLGA-PEG-PLGA water solubility, e.g. substituting PEG1500 in synthesis for higher molecular weight PEG3000 or adding cryopreserving substances to the buffer dissolution. PEG1500 was chosen to continue optimization and the final PLGA-PEG-PLGA hydrogels (PPP1500) were dissolved in trehalose or mannitol carbonate buffer. These presented more sustained release (71.77 ± 1.59 % and 73.41 ± 0.83 % in 24 h, respectively) and slower degradation (>14 days). In vitro cytotoxicity studies in the retinal-pigmented epithelial cell line (RPE-1) demonstrated good tolerance (viability values > 90 %). PLGA-PEG-PLGA hydrogels are proposed as suitable candidates for long-term intravitreal treatments. Preliminary wound healing studies with PLGA-PEG-PLGA hydrogels suggested faster proliferation at 8 h than controls.

Chronic Glaucoma Induced in Rats by a Single Injection of Fibronectin-Loaded PLGA Microspheres: IOP-Dependent and IOP-Independent Neurodegeneration.

To evaluate a new animal model of chronic glaucoma induced using a single injection of fibronectin-loaded biodegradable PLGA microspheres (Ms) to test prolonged therapies. 30 rats received a single injection of fibronectin-PLGA-Ms suspension (MsF) in the right eye, 10 received non-loaded PLGA-Ms suspension (Control), and 17 were non-injected (Healthy). Follow-up was performed (24 weeks), evaluating intraocular pressure (IOP), optical coherence tomography (OCT), histology and electroretinography. The right eyes underwent a progressive increase in IOP, but only induced cohorts reached hypertensive values. The three cohorts presented a progressive decrease in ganglion cell layer (GCL) thickness, corroborating physiological age-related loss of ganglion cells. Injected cohorts (MsF > Control) presented greater final GCL thickness. Histological exams explain this paradox: the MsF cohort showed lower ganglion cell counts but higher astrogliosis and immune response. A sequential trend of functional damage was recorded using scotopic electroretinography (MsF > Control > Healthy). It seems to be a function-structure correlation: in significant astrogliosis, early functional damage can be detected by electroretinography, and structural damage can be detected by histological exams but not by OCT. Males presented higher IOP and retinal and GCL thicknesses and lower electroretinography. A minimally invasive chronic glaucoma model was induced by a single injection of biodegradable Ms.

Tunable degrees of neurodegeneration in rats based on microsphere-induced models of chronic glaucoma.

This study compares four different animal models of chronic glaucoma against normal aging over 6 months. Chronic glaucoma was induced in 138 Long-Evans rats and compared against 43 aged-matched healthy rats. Twenty-five rats received episcleral vein sclerosis injections (EPIm cohort) while the rest were injected in the eye anterior chamber with a suspension of biodegradable microspheres: 25 rats received non-loaded microspheres (N-L Ms cohort), 45 rats received microspheres loaded with dexamethasone (MsDexa cohort), and 43 rats received microspheres co-loaded with dexamethasone and fibronectin (MsDexaFibro cohort). Intraocular pressure, neuroretinal function, structure and vitreous interface were evaluated. Each model caused different trends in intraocular pressure, produced specific retinal damage and vitreous signals. The steepest and strongest increase in intraocular pressure was seen in the EPIm cohort and microspheres models were more progressive. The EPIm cohort presented the highest vitreous intensity and percentage loss in the ganglion cell layer, the MsDexa cohort presented the greatest loss in the retinal nerve fiber layer, and the MsDexaFibro cohort presented the greatest loss in total retinal thickness. Function decreased differently among cohorts. Using biodegradable microspheres models it is possible to generate tuned neurodegeneration. These results support the multifactorial nature of glaucoma based on several noxa.

Mimicking chronic glaucoma over 6 months with a single intracameral injection of dexamethasone/fibronectin-loaded PLGA microspheres.

To create a chronic glaucoma animal model by a single intracameral injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (Ms) co-loaded with dexamethasone and fibronectin (MsDexaFibro). MsDexaFibro were prepared by a water-in-oil-in-water emulsion method including dexamethasone in the organic phase and fibronectin in the inner aqueous phase. To create the chronic glaucoma model, an interventionist and longitudinal animal study was performed using forty-five Long Evans rats (4-week-old). Rats received a single intracameral injection of MsDexafibro suspension (10%w/v) in the right eye. Ophthalmological parameters such as clinical signs, intraocular pressure (IOP), neuro-retinal functionality by electroretinography (ERG), retinal structural analysis by optical coherence tomography (OCT), and histology were evaluated up to six months. According to the results obtained, the model proposed was able to induce IOP increasing in both eyes over the study, higher in the injected eyes up to 6 weeks (p < 0.05), while preserving the ocular surface. OCT quantified progressive neuro-retinal degeneration (mainly in the retinal nerve fiber layer) in both eyes but higher in the injected eye. Ganglion cell functionality decreased in injected eyes, thus smaller amplitudes in PhNR were detected by ERG. In conclusion, a new chronic glaucoma animal model was created by a single injection of MsDexaFibro very similar to open-angle glaucoma occurring in humans. This model would impact in different fields such as ophthalmology, allowing long period of study of this pathology; pharmacology, evaluating the neuroprotective activity of active compounds; and pharmaceutical technology, allowing the correct evaluation of the efficacy of long-term sustained ocular drug delivery systems.

Influence of Sex on Neuroretinal Degeneration: Six-Month Follow-Up in Rats With Chronic Glaucoma.

Purpose: To evaluate differences by sex in the neuroretina of rats with chronic glaucoma over 24 weeks of follow-up, and to assess by sex the influence on neurodegeneration of different methods of inducing ocular hypertension. Methods: Forty-six Long-Evans rats-18 males and 28 females-with induced chronic glaucoma were analyzed. Glaucoma was achieved via 2 models: repeatedly sclerosing the episcleral veins (9 male/14 female) or by injecting poly(lactic-co-glycolic acid) microspheres measuring 20 to 10 µm (Ms20/10) into the anterior chamber (9 male/14 female). The IOP was measured weekly by tonometer; neuroretinal function was recorded by dark/light-adapted electroretinography at baseline and weeks 12 and 24; and structure was analyzed by optical coherence tomography using the retina posterior pole, retinal nerve fiber layer and ganglion cell layer protocols at baseline and weeks 8, 12, 18, and 24. Results: Males showed statistically significant (P < 0.05) higher IOP in both chronic glaucoma models, and greater differences were found in the episcleral model at earlier stages. Males with episclerally induced glaucoma showed a statistically higher increase in retinal thickness in optical coherence tomography recordings than females and also when comparing Ms20/10 at 12 weeks. Males showed a higher percentage of retinal nerve fiber layer thickness loss in both models. Ganglion cell layer thickness loss was only detected in the Ms20/10 model. Males exhibited worse dark/light-adapted functionality in chronic glaucoma models, which worsened in the episcleral sclerosis model at 12 weeks, than females. Conclusions: Female rats with chronic glaucoma experienced lower IOP and structural loss and better neuroretinal functionality than males. Sex and the ocular hypertension-inducing method influenced neuroretinal degeneration.

Combination therapy and co-delivery strategies to optimize treatment of posterior segment neurodegenerative diseases.

Neurodegenerative diseases affecting the posterior segment of the eye are one of the major causes of irreversible blindness worldwide. The pathogenesis of these retinal pathologies is characterized by a multifactorial etiology, involving the complex interaction of different apoptotic mechanisms, suggesting that effective treatments will require a multimodal approach. Thus, combination therapy based on the potential synergistic activities of drugs with different mechanisms of action is currently receiving considerable attention. Here, we summarize several kinds of strategy for the co-administration of different drugs to the posterior segment of the eye, highlighting those that involve co-delivery from multiloaded drug delivery systems.