DRUG DELIVERY 2021, VOL. 28, NO. 1, 2427–2446 https://doi.org/10.1080/10717544.2021.1998245 RESEARCH ARTICLE Long-term corticosteroid-induced chronic glaucoma model produced by intracameral injection of dexamethasone-loaded PLGA microspheres MJ Rodrigoa,b,c, D Garcia-Herranzd,e, A Arago n-Navasd,e, M Subiasa,b, T Martinez-Rinco na,b, S Mendez-Martınez , MJ Cardiel , J Garcıa-Feijoo a,f f,g h,i,j , J Rubertek,l,m , R Herrero-Vanrellc,d,e, a,c,f a,c,f c,d,e L Pablo , E Garcia-Martin and I Bravo-Osuna a Department of Ophthalmology, Miguel Servet University Hospital, Zaragoza, Spain; bMiguel Servet Ophthalmology Research Group (GIMSO), Aragon Health Research Institute (IIS Aragon), Zaragoza, Spain; cNational Ocular Pathology Network (OFTARED), Carlos III Health Institute, Madrid, Spain; dComplutense University of Madrid. Innovation, Therapy and Pharmaceutical Development in Ophthalmology (InnOftal) Research Group, UCM 920415, Department of Pharmaceutics and Food Technology, Faculty of Pharmacy, Complutense University of Madrid, Spain; eHealth Research Institute, San Carlos Clinical Hospital (IdISSC), Madrid, Spain; fMiguel Servet Ophthalmology Research Group (GIMSO), University of Zaragoza, Aragon Health Research Institute (IIS Aragon), Zaragoza, Spain; gDepartment of Pathology, Lozano Blesa University Hospital, Zaragoza, Spain; hComplutense University of Madrid. Innovation, Therapy and Pharmaceutical Development in Ophthalmology (InnOftal) Research Group, UCM 920415. National Ocular Pathology Network (OFTARED), Carlos III Health Institute, Spain; i Servicio de Oftalmologıa, Hospital Clınico San Carlos, Madrid, Spain; jDepartamento de Inmunologıa, Oftalmologıa y ORL, Facultad de Medicina, Universidad Complutense de Madrid (UCM), IdISSC, Madrid, Spain; kAnimal Biotechnology and Gene Therapy Centre (CBATEG), Universitat Autonoma de Barcelona, Bellaterra, Spain; lNetworked Biomedical Research Centre for Diabetes and Associated Metabolic Diseases (CIBERDEM), Madrid, Spain; mDepartment of Animal Health and Anatomy, School of Veterinary Medicine, Universitat Autonoma de Barcelona, Bellaterra, Spain ABSTRACT ARTICLE HISTORY Purpose: To evaluate a new chronic glaucoma model produced by intracameral injection of dexa- Received 6 August 2021 methasone-loaded poly lactic-co-glycolic acid microspheres (Dex-PLGA-Ms) over six months. Revised 14 October 2021 Methods: Healthy rats received two injections (at baseline and Week 4) of Dex-PLGA-Ms into the Accepted 18 October 2021 anterior chamber of the right eye. Clinical signs and intraocular pressure (IOP) were weekly recorded. KEYWORDS The structure of the retina and optic nerve was in vivo evaluated using optical coherence tomography Glaucoma; animal model; (OCT) every two weeks and functionally using dark- and light-adapted electroretinography at PLGA microspheres; 0–12–24 weeks. Histological studies were also performed. neurodegeneration; Results: IOP progressively increased up to hypertension (23.22 ± 3.63 mmHg) in both eyes but did so dexamethasone later in left eyes. OCT quantified a decrease in full-thickness retina posterior pole (R), retinal-nerve-fiber layer (RNFL), and ganglion-cell layer (GCL) thickness up to 24 weeks. Right eyes showed higher neuro- retinal thickness loss up to week 8. RNFL experienced the highest percentage thickness loss at the inferior-superior axis, while in GCL the inner sectors of the horizontal axis (Nasal-Temporal) suffered the greatest decrease in thickness. Retinal ganglion cell, photoreceptor, and intermediate cell function- ality decreased over time. Increased deposition of collagen IV was also found in zonular fibers and the ciliary body. Conclusions: This work shows the usefulness of drug delivery systems, not to treat pathology but to induce it. Only two injections of Dex-PLGA-Ms in the anterior chamber of rat eyes were enough to progressively create ocular hypertension and subsequent functional and structural neuroretinal degen- eration, at least over 6 months. Introduction return to normal values in a few weeks after stopping the corticosteroid exposition, but sometimes patients develop a Ocular hypertension (OHT) has been associated with treat- ment with corticosteroids, such as dexamethasone, adminis- difficult-to-treat SIG (Kersey and Broadway, 2006) that in tered via topical eye drops or ointments (Chan and Salmon, many cases requires eye surgery. The incidence of iatrogenic 2019) or via subconjunctival, subtenon (Li et al., 2019) or SIG has increased in recent years due to the increase in cor- intravitreous injections (Zarei-Ghanavati et al., 2012; Storey ticosteroid treatments for posterior pole pathologies with et al., 2018). When OHT is maintained over time, in some macular edema. cases, this side effect produces what is known as steroid- SIG shares characteristics with primary open-angle glau- induced glaucoma (SIG). Most patients decrease IOP and coma (POAG). In fact, patients with POAG or susceptible to CONTACT Bravo-Osuna I ibravo@ucm.es National Ocular Pathology Network (OFTARED), Carlos III Health Institute, Madrid, Spain These authors contributed equally to this work. Supplemental data for this article can be accessed here. ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2428 M. J. RODRIGO ET AL. glaucoma (Fini et al., 2017), as well as their descendants, the corticosteroid in the proximity of the trabecular mesh- have a greater corticosteroid response, and a correlation has work but also additional mechanical blockage of the trabecu- been demonstrated between the response to corticosteroids lar meshwork produced by the presence of PLGA Ms in the and the risk of developing glaucoma over time (Chan intracameral space. To our knowledge, this is the first SIG et al., 2019). model that permits short- and long-term study and simulates To study SIG, ex vivo models were developed (Torrejon real clinical conditions in which chronic treatment et al., 2016; Rybkin et al., 2017). However, none of them fully with corticosteroid is needed, such as corneal transplant or simulate in vivo complexity. Several OHT models were there- pathologies of the posterior pole with macular edema fore created in 8 non-human species (Overby and Clark, (Razeghinejad and Katz, 2012; Kornmann and Gedde, 2016). 2015), but only the rat (Razali et al., 2015) and mouse (Zode et al., 2014; Li et al., 2019) models developed glaucoma with functional and structural neuroretinal degeneration over a Materials maximum follow-up of 2 months. These models have facili- tated understanding of the histological, proteomic, and gen- Dexamethasone (Dex) was supplied by Sigma-Aldrich (St. etic characteristics of SIG, but many unknowns remain. It is Louis Mo., USA) at the highest purity available (>98%). Poly known that corticosteroids increase the production of myoci- (D,L-lactide-co-glycolide) (PLGA) 50:50 (inherent viscosity: lin, fibronectin, or other proteins of the extracellular matrix 0.16–0.24 dl/g) was obtained from Evonik Espan ~a (Granollers, (Faralli et al., 2015; Shan et al., 2017), and inhibit the metallo- Spain). The other materials employed in microsphere (Ms) proteinase that leads to excessive accumulation of extracellu- manufacture were polyvinyl alcohol 67,000 g/mol (PVA) lar matrix and hence the clogging of the trabecular (purchased from Merck KGaA (Darmstadt, Germany) and meshwork. Corticosteroids also decrease phagocytic activity methylene chloride (obtained from PanReac AppliChem, (Zeng et al., 2019) (the function of trabecular meshwork Barcelona, Spain). cells), which leads to an increase in retained material and rigidity in the trabecular meshwork (Raghunathan et al., 2015), altering the motility/regulation of the aqueous humor Methods drainage pathways (Overby and Clark, 2015; Xin et al., 2017; Manufacture of dexamethasone-loaded PLGA Patel et al., 2019, Patel et al., 2018). However, all these stud- microspheres ies have only been conducted over short periods that pre- vent analysis of dynamic changes in the pathology over Dexamethasone-loaded PLGA Ms were obtained by applying time. For these reasons, recent studies demand better char- the solvent extraction-evaporation method to an oil-in-water acterization and improvement of the current SIG models, (O/W) emulsion. First, PLGA (400 mg) was dissolved in methy- with the aim of simulating human chronic glaucoma lene chloride (2 mL). Micronized dexamethasone (40 mg) was (Agarwal and Agarwal, 2017; Biswas and Wan, 2019; Pang added to the polymeric solution and dispersed by ultrasoni- and Clark, 2020). cation (Ultrasons; J.P. Selecta, Barcelona, Spain) for 5 minutes, In a recently published paper, a novel pre-trabecular then further sonicated (Sonicator XL; Heat Systems, Inc., model of chronic glaucoma was achieved by repeated injec- Farmingdale, NY, USA) for 1 minute at 4  C to obtain homo- tion of biodegradable poly lactic-co-glycolic acid (PLGA) genous dispersion. The so-formed O-phase was emulsified by microspheres into the ocular anterior chamber of healthy adding 5 mL of PVA MilliQV water solution 1% (w/v) via a R rats. The presence of blank microspheres across the iridocor- homogenizer (PolytronVRECO, Kinematica, GmbHT PT3000, R neal angle and the trabecular meshwork produced a mech- Lucerna, Switzerland) at 7,000 rpm for 1 minute. The resulting anical blockage able to induce sustained OHT over six O/W emulsion was added to 100 mL of PVA MilliQV water R months. This continuous elevation of intraocular pressure solution 0.1% (w/v) and stirred for 3 hours leading to (IOP) translated into progressive degradation of the retina in Ms maturation. a pattern similar to that found in human glaucoma, with the Once maturation was completed, MilliQV water was R advantage, over other animal models, that the ocular surface employed to wash the Ms, removing the remaining superfi- remained healthy throughout the study, causing less animal cial PVA. Subsequently, the 20–10 mm fraction was selected, pain and allowing in vivo retinal monitoring over the six freeze-dried (freezing: 60  C/15 min.; drying: 60  C/12 h/0.1 months (Rodrigo et al., 2021). mBar) and stored at 30  C in dry conditions (Figure 1). Long-term corticosteroid-induced chronic glaucoma requires delivery of small amounts of the active substance close to the trabecular meshwork over prolonged periods. Dexamethasone-loaded PLGA microsphere This can now be achieved thanks to the development of characterization drug delivery systems. This study presents a new animal model of chronic SIG Ms characterization was performed in terms of morphological induced using injections of biodegradable PLGA Ms loaded evaluation, mean particle size, particle size distribution, with dexamethasone in the anterior chamber of rat eyes. encapsulation efficiency, and in vitro release. The production This method combines not only pharmacological blockage of yield percentage of the selected granulometric fraction was aqueous humor exit induced by the constant presence of also determined. DRUG DELIVERY 2429 Figure 1. Dexamethasone-loaded PLGA microsphere manufacture. Production yield percentage (PY %) from the Ms. This method comprised 50% ammonium acet- ate 15 mM/1 mL formic acid in MilliQV water and 50% aceto- R The production yield percentage was calculated according to nitrile at a flow rate of 0.3 mL/min. the following Equation (1). amount of microspheres PY % ¼  100 Dexamethasone encapsulation efficiency Total amount of polymer þ Total amount of drug (1) A known quantity of dexamethasone-loaded PLGA Ms (1 mg) was dissolved in 2.5 mL of methylene chloride. Then, 6 mL of methanol were added to precipitate the dissolved polymer. External morphological evaluation After vortex mixing and centrifugation (5,000 rpm for Gold sputter-coated freeze-dried dexamethasone-loaded 5 minutes at 20  C), the upper methanolic supernatant was PLGA Ms were observed using scanning electron microscopy collected, filtered (0.22 mm), and analyzed using the previ- ously described HPLC/Ms method in order to quantify the (SEM; Jeol, JSM-6335F, Tokyo, Japan). dexamethasone. Mean particle size and particle size distribution In vitro study of dexamethasone release from Particle size and particle size distribution were measured dexamethasone-loaded PLGA Ms using dual light scattering (MicrotracV R S3500 Series Particle A dexamethasone-loaded PLGA Ms suspension (2.5 mg/mL) Size Analyzer, Montgomeryville, PA, USA). The volume means was prepared in quadruplicate using phosphate-buffered diameters ( ± standard deviation) obtained from 3 measure- saline (PBS, pH 7.4) with sodium azide (0.02% (w/v) as the ments were used to express the mean particle size. release media. These samples were placed in a water shaker bath (100 rpm, 37  C, Memmert Shaking Bath, Memmert, Dexamethasone quantification by HPLC/Ms Schwabach, Germany). Supernatants were periodically col- lected after gentle centrifugation (5,000 rpm for 5 min, 20  C) The HPLC/Ms system comprised a liquid chromatography and filtered (0.22 lm) for dexamethasone quantification by instrument (Waters 1525 binary HPLC pump and Waters HPLC/Ms employing the method previously mentioned. The 2707 autosampler) employing a Nova-Pak C18 column (4 lm, remaining Ms samples were refilled with fresh release media. ID 2.1 mm  150 mm) coupled to a guard column (4 lm, The same protocol was performed at each time point. 3.9 mm  20 mm), both maintained at 45  C. A Ms detector In order to mimic the animal study, the second dose of (Waters 3100 single quadrupole mass spectrometer) was con- 2.5 mg of dexamethasone-loaded PLGA Ms was added to nected to this system via an Empower 2 (Waters, Milford, each sample at 28 days post-study (time of the second injec- USA). The ESI source was adjusted in the positive ion mode tion), also increasing the amount of release media to main- (ESI(þ)) for DX detection. Selected ion recordings (SIR) dexa- tain the initial suspension concentration. The release study methasone mass (m/z) 393.40 was measured under mass was performed as explained above for 140 additional days spectrometer source conditions of 3.5 kV electrospray voltage (total time of in vitro release study: 168 days, i.e. 24 weeks). and 130  C heated capillary temperature. Nebulization (100 L/ h flow rate, 130  C source temperature, 5 V extractor voltage) Animals and desolvation (400 L/h flow rate, 300  C desolvation temperature) were performed employing nitrogen gas The work with animals was approved by the Ethics (>99.999%). An isocratic HPLC method was developed to Committee for Animal Research (PI34/17) and was carried quantify dexamethasone encapsulation efficiency and release n Biomedical Research Center (CIBA) in strict out at the Arago 2430 M. J. RODRIGO ET AL. accordance with the ARVO Statement on the Use of Animals Doble ColircusiVR , Alcon Cus ıV R SA, Barcelona, Spain) and lubri- in Ophthalmic and Vision Research. The animals were housed cated with hypromellose 2% (MethocelV R OmniVision, in a light/dark-cycled room (12 hours light/12 hours dark) Germany). Active electrodes were placed on the cornea, under controlled temperature (22  C) and relative humidity references were placed on either side under the skin and the (55%) conditions and in standard cages with environmental ground electrode was placed near the tail. Electrode imped- enrichment, water, and food ad libitum. Forty-three ance with a difference of <2 kX between electrodes was Long–Evans rats (40% males, 60% females; 4 weeks old; initial accepted. Both eyes were simultaneously tested using a weights ranging from 50 to 100 g) were used for this longitu- Ganzfeld Q450 SC sphere with white LED flashes for stimuli, dinal and interventionist study. and seven steps at increasing luminance intensity and inter- vals were performed. Scotopic test-examined rod response: step 1: 40 dB, 0.0003 cds/m2, 0.2 Hz [20 recordings aver- Ocular hypertension (OHT) induction aged]; step 2: 30 dB, 0.003 cds/m2, 0.125 Hz [18 recordings At baseline and Week 4, all animals received a right-eye (RE) averaged]; step 3: 20 dB, 0.03 cds/m2, 8.929 Hz [14 record- ocular hypertensive injection administered by superotempo- ings averaged]; step 4: 20 dB, 0.03 cds/m2, 0.111 Hz [15 ral corneal puncture using a micro-metered HamiltonV R syr- recordings averaged]; step 5: 10 dB, 0.3 cds/m2, 0.077 Hz inge with a glass micropipette to insert 2 microlitres of [15 recordings averaged]; mixed rod–cone response: step 6: dexamethasone-PLGA-Ms suspension into the anterior cham- 0 dB, 3.0 cds/m2, 0.067 Hz [12 recordings averaged]; and ber of the eye. oscillatory potentials: step 7: 0 dB, 3.0 cds/m2, 29.412 Hz [10 This was performed under the following conditions: a recordings averaged]). The PhNR protocol was performed sedative gas mixture containing 3% sevoflurane gas and after light adaptation to a blue background (470 nm, 25 cds/ m2), and a red LED flash (625 nm, 10 dB, 0.30 cds/m2, 1.5% oxygen, topical eye drops containing tetracaine (1 mg/ 1.199 Hz [20 recordings averaged]) was used as stimulus. ml) þ oxybuprocaine (4 mg/ml) (Anestesico Doble ColircusiV R, Latency (in milliseconds) and amplitude (in microvolts) were Alcon CusıVR SA, Barcelona, Spain), analgesic subcutaneous studied in a, b and PhNR waves. dilution 1/10 of buprenorphine (0.05 mg/kg), aseptic surgical conditions achieved with the iodized solution and antibiotic eye drops containing ofloxacin (3 mg/ml) (Exocin ColircusiVR, Optical coherence tomography Alcon CusıV R SA, Barcelona, Spain). The temperature was con- Neuroretinal structures were analyzed using optical coher- trolled with warm pads and after the procedures, the animals ence tomography (OCT Spectralis, HeidelbergV R Engineering, were left to recover in a 2.5% oxygen-enriched atmosphere. Germany) at 0, 2, 4, 6, 8, 12, 18, and 24 weeks applying a The ophthalmological examination methodology was as contact lens adapted to the rat cornea to acquire higher described in (Rodrigo et al., 2021) as follows: quality images. The full-thickness Retina posterior pole (R), Retina Nerve Fiber Layer (RNFL), and Ganglion Cell Layer (GCL) protocols were used with automatic segmentation. Ophthalmological studies These protocols analyzed a circled area, centered on the In vivo optic disk, by means of 61 b-scans. Subsequent follow-up Clinical signs such as ocular surface redness, corneal altera- examinations were performed at this same location using tions, intraocular inflammation, infection or cataract forma- the eye-tracking software and follow-up application. The tion, as well as IOP measurements using the TonolabV R Retina and GCL were analyzed via the 9 ETDRS areas, which tonometer (Tonolab; Tiolat Oy Helsinki, Finland), were included a central (C) 1 mm circle centered on the optic disk recorded by the same operator every week. A sedative mix- (though no fovea exists in rats) and inner (inferior -II-, super- ture of 3% sevoflurane gas and 1.5% oxygen was applied, as ior -IS-, nasal -IN-, temporal -IT-) and outer (inferior -OI-, recommended, for less than 3 minutes in order to avoid superior -OS, nasal -ON-, temporal -OT-) rings measuring 2 hypotensive effects (Ding et al., 2011). The IOP value was the and 3 mm in diameter, respectively. Total volume (TV) was average of three consecutive measurements, which resulted also measured. Retinal thickness comprises from the inner from the average of 6 rebounds. limiting membrane to the retinal pigment epithelium, and the GCL comprises from the RNFL to the inner nuclear layer boundaries. The RNFL protocol provided measurements of Electroretinography the 6 peripapillary sectors (inferotemporal -IT-, inferonasal Neuroretinal structure functionality was studied using elec- -IN-, superotemporal -ST-, superonasal -SN-, nasal -N- and troretinography (ERG) (Roland consultV R RETIanimal ERG, temporal -T-), measuring the thickness from the inner limit- Germany), applying the flash scotopic ERG and light-adapted ing membrane to the GCL boundaries. (LA) photopic negative response (PhNR) protocols at base- Each OCT scan was revised by the same researcher. The line, Week 12 and Week 24. To perform the scotopic ERG image was manually corrected if detected biased examina- test, the animals were dark-adapted for 12 hours and their tions due to errors in segmentation, excessive noise, or arti- pupils were fully dilated with tropicamide (10 mg/ml) and fact. It occurred very occasionally. To perform the ERG and phenylephrine (100 mg/ml), (Alcon CusıV R SA, Barcelona, OCT tests, the animals were intraperitoneally anesthetized Spain) topically anesthetized with eye drops containing tetra- with a mixture of ketamine (60 mg/kg) þ dexmedetomidine caine (1 mg/ml) þ oxybuprocaine (4 mg/ml) (Anestesico (0.25 mg/kg) and temperature controlled. At the end of the DRUG DELIVERY 2431 study, six unconscious animals were euthanized under (SPSS Inc., Chicago, IL). The Kolmogorov-Smirnov test was humane conditions with an intracardiac injection of sodium used to assess sample distribution, Student’s t-test was used thiopental (25 mg/ml) and their eyes were immedi- to evaluate the differences between eyes, and a paired ately enucleated. Student’s t-test was used to compare the changes recorded in each eye over the study period. All values were expressed as mean ± standard deviations. Values of p < .05 (expressed Histology as ) were considered to indicate statistical significance. The Paraffin-embedded eyes were sectioned (5 mm) along the eye Bonferroni correction for multiple comparisons was also cal- axis, deparaffinized, and rehydrated. After several washes in culated to avoid a high false-positive rate. The level of sig- PBS, the sections were incubated overnight at 4  C with the nificance for each variable was established according to following primary antibodies: mouse anti-Brn3a (Santa Cruz Bonferroni calculations (expressed as #). Biotechnology, Heidelberg, Germany) at 1:50 dilution; goat anti-mouse Collagen IV (Milllipore, Temecula, USA) at 1:20 Results dilution; and rabbit anti-mouse laminin (DAKO, Glostrup, Denmark) at 1:200 dilution. After washing the sections in Dexamethasone-loaded PLGA microsphere PBS, the slides were incubated for 2 hours at room tempera- characterization ture with the secondary rabbit anti-goat Alexa 488 and anti- rabbit Alexa 488 antibodies (Invitrogen, Carlsbad, CA, USA) at Dexamethasone-loaded PLGA Ms showed a production yield 1:100 dilution. Propidium iodide (Sigma-Aldrich Corp., St. of 77.34% for the 20–10 mm fraction. The particle size distri- Louis, MO, USA) diluted in PBS (1 : 10) was used for nuclear bution was unimodal, with a mean particle size of counterstaining. The slides were mounted in Fluoromount 13.13 ± 0.60 mm. Drug loading was 60.70 ± 1.03 mg dexa- (Sigma-Aldrich) medium for further analysis using a laser methasone/mg Ms, constituting 66.77 ± 1.14% encapsulation scanning confocal microscope (TCS SP5; Leica Microsystems efficiency. SEM images (Figure 2) evidenced the presence of GmbH, Heidelberg, Germany). Immunohistochemistry con- spherical non-porous Ms surfaces. trols were performed by omission of the primary antibody in a sequential tissue section. In vitro release studies For ganglion cell count, slides probed with mouse anti- Brn3a were incubated with biotinylated horse anti-mouse at The in vitro release profile of dexamethasone from PLGA 1:50 dilution (Vector Laboratories, Burlingame, CA, USA). microspheres showed the typical multiphasic shape combin- They were then incubated with ABC-HRP (Thermo Fisher ing rapid- and slow-release periods. From Day 0 to Day 7, Scientific, Waltham Massachusetts, USA) at 1:50 dilution at the rapid release occurred leading to a total release of 62 mg. room temperature. The sections were washed in PBS before This first phase was followed by slow release up to Day 28, and after every incubation. Finally, the sections were stained with an average release rate of 0.191 mg/day. After the inclu- with DAB (Sigma-Aldrich) for 3 minutes and counterstained sion of the additional amount of Ms in the release media, a with Harris’ hematoxylin (Sigma-Aldrich) for 20 minutes at second rapid-release occurred, delivering 94.5 mg in the fol- room temperature. Ganglion cells were counted in radial sec- lowing 3 days. Subsequently, a second slow-release rate of tions of the retina along 2 mm of a linear region of the gan- 0.390 mg/day was observed until Day 77 of the in vitro glion cell layer corresponding to four areas, two on each release study. No dexamethasone release was observed from side of the optic nerve head. The images were analyzed by Day 77 to study end at Day 168, but the release assay was an operator blinded to treatment groups. continued to cover the complete six-month study (Figure 3). Statistical analysis Ophthalmological clinical signs and intraocular pressure The data were recorded in an Excel database and statistical Ocular injections were generally well tolerated. The visual analysis was performed using SPSS software version 20.0 axis remained clear and the iridocorneal angle was open, Figure 2. SEM pictures of dexamethasone-loaded PLGA Ms. 2432 M. J. RODRIGO ET AL. Figure 3. In vitro dexamethasone release profile from dexamethasone-loaded PLGA Ms. appearing normal when examined by light microscopy. As more evident in the retina and at Week 12. REs experienced for disadvantages, four animals developed mild peripheral the same fluctuation tendency in the RNFL and GCL, which corneal leucomas that nevertheless did not preclude proper occurred 2 weeks later in LEs (Figure 5). testing and follow-up, but one rat developed cataracts with Figure 6 shows the percentage of thickness loss over pupillary seclusion and ocular hypotension and this animal time. The RNFL was the parameter that showed the biggest was discarded from the results. percentage loss in both eyes at every time point and on At baseline, no differences in IOP were found between average. It was followed by the GCL and the retina. Injected eyes and the IOP progressively increased over follow-up. REs showed a bigger loss than LEs. Injected REs experienced an increase (4 mmHg; 36.30% The neuroretinal percentage loss by OCT sectors from the increase) from Week 1 onwards, reached OHT (considered as retina, RNFL and GCL was quantified and the loss tendency >20 mmHg) at Week 5 (95.39% increase) and remained sig- was analyzed (see Supplementary Figures 1, 2 and 3, respect- nificantly higher than the contralateral left eye (LE) up to ively). Variability in retina alteration was observed in REs over Week 9 (23.22 ± 3.63 vs 19.68 ± 4.03 mmHg, p ¼ .013) time, alternating from outer to inner sectors. However, in LEs (102.42% increase). LEs reached OHT at Week 8 (79.25% the outer sectors tended to experience a greater percentage increase) and even surpassed it at later stages (16.46 ± 2.11 loss in thickness. The same loss trend was found between vs 21.88 ± 4.21 mmHg, p ¼ .029) (Figure 4(a)). This model eyes at Weeks 6 and 24 and on average. This also happened showed a sustained and increasing percentage of OHT in at Week 4 and on average in the RNFL, where the superior- both eyes, albeit occurring 3 weeks later in LEs. REs reached inferior sectors of the vertical axis were the most often the OHT percentage peak (87.5%) at Week 9 and an even altered. In the GCL, the inner sectors showed greater per- higher level (90%) was quantified in LEs at 15–16 weeks centage losses in both eyes at every time point; the nasal- (Figure 4(b)). Most rats experienced an IOP increase of temporal sectors of the horizontal axis were the most between 6 and 15 mmHg (medium corticosteroid response) affected and the inferior sector the least. and only 5% on average showed an increase higher than The average loss rate expressed in microns per mmHg 15 mmHg (high corticosteroid response) (Figure 4(c)). and day extracted from all sectors was also quantified in both eyes and at all stages. The highest levels of thickness In vivo neuroretinal examination loss were found in the retina in the early stages (up to 8 weeks) and in the RNFL in the intermediate and later Optical coherence tomography: stages. On average, the highest loss rate was found in the Both eyes experienced a progressive decrease in thickness retina, followed by the RNFL and then the GCL. REs showed (in microns) in the retina, RNFL, and GCL over 6 months of a higher loss rate in the RNFL but a lower one in the retina follow-up. REs showed lower thicknesses than LEs in all sec- than their non-injected contralateral LEs (Figure 7). tors explored (except nasal sectors), with statistical differen- ces up to Week 8, although this tendency inverted at Weeks 12 and 18. By the end of the study period (Week 24), REs Electroretinography (ERG): showed smaller thicknesses in all sectors in the RNFL and REs showed longer latency and smaller amplitude in all dark- GCL but higher thicknesses in the Retina (Table 1). adapted (DA) phases explored over time and the biggest Fluctuations were observed in both eyes, with these being decrease in signal was found from baseline to Week 12. LEs DRUG DELIVERY 2433 Figure 4. (a) Intraocular pressure curve over 6 months in the MsDex (dexamethasone-loaded microsphere) model. IOP: intraocular pressure; MsDex: microspheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week; p < 0.05; #p < 0.02. (b) Percentage of ocular hypertensive eyes (>20 mmHg) in MsDex model over 6 months of follow-up. OHT: ocular hypertension; %: percentage; MsDex: microspheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week. (c) Percentage of corticosteroid response in right and left eyes. Low: <6 mmHg increase; medium: 6–15 mmHg increase; high: >15 mmHg increase. followed a similar pattern but did not keep decreasing at material (Overby and Clark, 2015). Greater accumulation of Week 24. The light-adapted (LA) PhNR protocol also showed type IV collagen, heparin sulfate proteoglycan, and fibronec- diminished signal over time, with REs smaller than LEs (see tin at the trabecular meshwork has been observed in gluco- Figure 8). corticoid-induced human glaucoma (Tawara et al., 2008). REs showed statistically decreased amplitudes when com- In our experiment, rat eyes injected with dexamethasone pared to LE DA rod response (Phase 4: photoreceptors (a- microspheres showed increased deposition of collagen IV in wave): 14.23 ± 9.49 vs 47.38 ± 28.07 lV; p ¼ .021) and oscilla- zonular fibers and in the basement membrane of the non- tory potentials (Phase 7: intermediate cells (b-wave): pigmented epithelium at the ciliary body (Figure 10). In 66.88 ± 26.20 vs 135.13 ± 39.38 lV; p ¼ .005), and LA-PhNR contrast with the human ciliary body (Marshall et al., 1992), (intermediate cells (b-wave): 23.05 ± 21.17 vs 75.70 ± 48.78 lV; collagen IV was not detected by immunohistochemistry in p ¼ .036) at Week 12; and in DA rod response (Phase 1: inter- the pigmented epithelium basement membrane of the rat mediate cells (b-wave): 34.09 ± 22.14 vs 97.88 ± 45.69 lV; ciliary body (Figure 10). p ¼ .012) (Phase 2: photoreceptors (a-wave): 25.77 ± 16.84 vs The composition of zonular fibers has been studied using 60.87 ± 32.51 lV, p ¼ .041) and ganglion cell response both immunohistochemical and mass spectrometric LA-PhNR (intermediate cells (b-wave): 22.35 ± 12.81 vs approaches (Bassnett, 2021). The zonular fibers are com- 54.40 ± 30.21 lV; p ¼ .038) at Week 24. posed almost entirely of fibrillin-based microfibrils and three well-characterized microfibril-associated proteins: LTBP-2, MFAP2, and EMILIN-1 (29). Although De Maria et al. (De Histology Maria et al., 2017) identified hundreds of zonular proteins, The count of positive Brn3a cells along 2 mm of the retina laminin has not been detected in human and bovine zonular showed that the mean number of ganglion cells was not sig- fibers. Similarly, laminin was not detected by immunohisto- nificantly different at the study end in LEs when compared chemistry in left-eye zonular fibers (Figure 11). However, with REs injected with dexamethasone microspheres glucocorticoid-induced glaucomatous right eyes showed lam- (p ¼ .3787) (24 weeks) (Figure 9). inin deposition in zonular fibers (Figure 11). As a hypothesis, The mechanism of glucocorticoid-induced aqueous humor our results suggest that collagen IV and laminin deposition outflow obstruction is not well understood but appears to at zonular fibers in glucocorticoid-induced hypertensive rat be associated with the accumulation of extracellular matrix eyes could have disturbed aqueous humor flow and may Table 1. Structural neuroretinal analysis, measured by optical coherence tomography (OCT), using microspheres loaded with dexamethasone (MsDex) over 6 months. Baseline 2w 4w 6w 8w 12 w 18 w 24 w OCT parameters Mean ± SD p Mean ± SD %Ch p Mean ± SD %Ch p Mean ± SD %Ch p Mean ± SD %Ch p Mean ± SD %Ch p Mean ± SD %Ch P Mean ± SD %Ch p Retinal thickness Central 288.90 ± 16.32 0.373 267.00 ± 26.90 7.58 0.431 275.33 ± 18.49 4.69 0.580 255.33 ± 19.18 11.61 0.058 260.40 ± 19.61 9.87 0.190 292.50 ± 17.74 1.25 0.461 262.00 ± 14.56 9.31 0.780 260.60 ± 24.05 9.80 0.774 295.20 ± 14.45 276.50 ± 9.02 6.33 282.50 ± 24.45 4.30 276.00 ± 13.82 6.50 276.50 ± 18.06 6.76 285.60 ± 9.99 3.25 259.80 ± 8.75 11.99 263.83 ± 11.08 10.63 Inner inferior 267.90 ± 8.71 0.668 257.33 ± 17.31 3.94 0.254 262.33 ± 6.59 2.07 0.443 250.33 ± 6.25 6.55 0.316 255.60 ± 4.61 4.59 0.575 259.83 ± 7.62 3.01 0.709 255.20 ± 13.18 4.74 0.804 249.80 ± 17.65 6.76 0.980 266.30 ± 7.64 267.00 ± 9.12 0.26 259.67 ± 4.84 2.48 254.17 ± 6.33 4.55 253.83 ± 5.30 4.91 262.20 ± 12.59 1.54 258.60 ± 26.50 2.89 250.00 ± 6.63 6.12 Outer inferior 254.80 ± 6.59 0.955 244.50 ± 6.71 4.04 0.269 244.50 ± 2.07 4.04 0.561 240.17 ± 2.63 5.74 0.254 240.20 ± 6.30 5.73 0.856 244.00 ± 5.96 4.24 0.543 239.20 ± 6.26 6.12 0.875 241.40 ± 20.37 5.26 0.596 254.60 ± 8.82 248.50 ± 5.01 2.39 245.67 ± 4.27 3.50 242.33 ± 3.50 4.81 241.00 ± 7.64 5.64 247.40 ± 11.52 2.83 238.00 ± 15.28 6.52 236.50 ± 7.63 7.11 Inner superior 261.20 ± 7.81 0.897 245.50 ± 10.69 6.01 0.209 242.00 ± 6.00 7.35 0.039 238.83 ± 4.53 8.56 0.005# 240.80 ± 8.46 7.81 0.278 260.50 ± 14.30 0.27 0.588 243.40 ± 7.19 6.81 0.932 241.20 ± 12.55 7.66 0.812 261.60 ± 5.56 255.50 ± 14.78 2.33 254.00 ± 10.84 2.90 248.33 ± 4.63 5.07 246.67 ± 8.33 6.05 256.00 ± 11.76 2.14 243.80 ± 7.19 6.80 243.17 ± 13.84 7.05 Outer superior 260.00 ± 7.24 0.941 247.83 ± 8.03 4.68 0.311 242.50 ± 2.73 6.73 0.072 243.17 ± 4.66 6.47 0.098 246.60 ± 5.94 5.15 0.696 258.17 ± 7.78 0.70 0.634 242.20 ± 7.53 6.85 0.940 252.20 ± 12.35 3.00 0.249 259.70 ± 10.39 252.83 ± 8.18 2.64 248.83 ± 7.22 4.18 247.33 ± 3.07 4.76 248.33 ± 7.89 4.58 256.00 ± 6.55 1.42 242.60 ± 8.79 6.58 243.67 ± 10.63 6.17 Inner nasal 263.10 ± 7.63 0.810 250.00 ± 9.57 4.97 0.139 250.33 ± 6.37 4.85 0.010# 244.17 ± 6.52 7.19 0.035 247.40 ± 7.47 5.97 0.751 257.67 ± 10.13 2.06 0.646 247.00 ± 7.96 6.12 0.192 251.80 ± 7.49 4.29 0.748 264.00 ± 8.81 258.67 ± 9.11 2.01 261.50 ± 5.82 0.94 253.50 ± 6.74 39.727 249.17 ± 9.90 5.95 261.80 ± 18.29 0.83 254.80 ± 9.28 3.48 250.67 ± 3.55 5.05 Outer nasal 258.70 ± 7.11 0.659 246.83 ± 4.62 4.58 0.715 244.33 ± 2.87 5.55 0.536 239.33 ± 7.17 7.48 0.294 244.00 ± 5.24 5.68 0.882 250.17 ± 6.43 3.30 0.938 241.20 ± 8.22 6.76 0.973 248.75 ± 2.63 3.85 0.083 257.20 ± 7.81 248.00 ± 6.03 3.57 245.83 ± 4.95 4.42 242.83 ± 2.92 5.58 244.50 ± 5.54 5.19 250.60 ± 11.45 2.57 241.40 ± 9.94 6.14 241.00 ± 7.40 6.30 Inner temporal 261.60 ± 7.80 0.432 252.83 ± 9.90 3.35 0.444 248.50 ± 8.01 5.00 0.807 244.17 ± 5.19 6.66 0.723 240.60 ± 6.84 8.03 0.466 255.50 ± 11.45 2.33 0.409 252.20 ± 11.32 3.59 0.455 246.40 ± 11.03 5.81 0.681 258.80 ± 7.77 257.17 ± 8.88 62 249.67 ± 8.06 3.52 245.00 ± 2.09 5.33 243.83 ± 7.13 6.14 250.40 ± 7.02 3.25 247.00 ± 9.56 4.56 243.83 ± 9.04 5.78 Outer temporal 255.70 ± 7.36 0.761 243.83 ± 6.55 4.64 0.046 243.50 ± 3.27 4.77 0.021 241.50 ± 5.82 5.55 0.052 245.40 ± 8.41 4.03 0.874 250.67 ± 8.71 1.97 0.830 242.60 ± 9.18 5.12 0.836 248.60 ± 6.50 2.78 0.135 256.70 ± 7.10 252.00 ± 5.86 1.83 248.33 ± 2.80 3.26 247.33 ± 2.87 3.65 246.17 ± 7.19 4.28 251.60 ± 3.84 1.99 243.80 ± 8.52 5.03 241.83 ± 7.02 5.79 Total volume 1.85 ± 0.04 0.852 1.76 ± 0.06 4.90 0.127 1.76 ± 0.35 4.90 0.194 1.72 ± 0.02 7.23 0.017# 1.74 ± 0.05 6.03 0.549 1.82 ± 0.05 1.67 0.892 1.74 ± 0.05 6.14 0.999 1.71 ± 0.13 7.76 0.696 1.86 ± 0.04 1.81 ± 0.03 2.41 1.79 ± 0.43 3.40 1.76 ± 0.02 5.10 1.76 ± 0.05 5.38 1.82 ± 0.06 2.15 1.74 ± 0.06 6.34 1.73 ± 0.04 6.72 RNFL thickness GLOBAL 49.10 ± 5.34 0.963 43.00 ± 3.52 12.42 0.059 40.17 ± 3.06 18.18 0.122 39.50 ± 3.20 19.55 0.411 40.40 ± 1.14 17.72 0.368 40.33 ± 3.67 17.86 0.640 39.60 ± 4.33 19.35 0.575 33.40 ± 8.47 31.98 0.097 49.00 ± 3.94 49.67 ± 6.80 1.36 44.33 ± 5.20 9.53 41.00 ± 2.82 16.32 41.17 ± 1.47 19.02 41.60 ± 5.03 15.10 38.40 ± 1.51 21.63 41.83 ± 6.64 14.63 Inferior temporal 51.30 ± 6.53 0.758 44.50 ± 8.26 13.25 0.678 35.17 ± 6.82 31.44 0.042 32.67 ± 9.35 36.31 0.200 44.40 ± 6.42 13.45 0.214 42.67 ± 10.15 16.82 0.912 45.60 ± 27.68 11.11 0.559 37.80 ± 9.75 26.32 0.094 52.30 ± 7.68 46.50 ± 7.91 11.08 42.83 ± 4.26 18.10 38.17 ± 2.99 27.01 39.83 ± 4.91 31.31 43.40 ± 11.14 17.02 38.00 ± 3.39 27.34 51.33 ± 13.42 1.85 Inferior nasal 51.70 ± 4.83 0.513 43.67 ± 6.86 15.53 0.073 41.67 ± 2.58 19.40 0.026 42.67 ± 8.26 17.46 0.664 45.80 ± 5.35 11.41 0.568 50.50 ± 8.96 2.32 0.633 36.40 ± 6.02 29.59 0.506 43.00 ± 11.42 16.83 0.476 49.30 ± 10.29 55.33 ± 12.53 12.23 47.67 ± 5.00 3.30 40.67 ± 7.20 17.50 43.17 ± 8.61 14.20 47.40 ± 11.88 3.85 39.80 ± 9.09 19.27 49.33 ± 15.87 0.06 Superior temporal 51.90 ± 6.72 0.849 43.83 ± 12.28 15.54 0.166 51.33 ± 11.74 1.09 0.624 41.50 ± 11.52 20.03 0.207 41.20 ± 3.76 20.62 0.388 40.00 ± 13.19 22.93 0.859 42.60 ± 6.46 17.92 0.491 30.40 ± 18.91 41.43 0.486 52.60 ± 9.30 55.17 ± 13.93 4.88 48.33 ± 8.54 8.11 49.17 ± 7.78 6.52 46.00 ± 11.24 14.35 41.40 ± 12.03 21.29 45.20 ± 4.81 14.07 38.83 ± 19.38 26.18 Superior nasal 40.30 ± 12.07 0.725 41.67 ± 8.95 3.39 0.732 34.00 ± 3.74 15.63 0.075 37.67 ± 5.20 6.52 0.949 32.80 ± 6.14 18.61 0.401 33.60 ± 9.65 16.63 0.765 36.20 ± 3.11 10.17 0.270 25.40 ± 7.47 36.97 0.052 42.20 ± 11.67 43.17 ± 5.34 2.29 40.17 ± 6.61 4.81 37.83 ± 3.43 10.35 36.67 ± 8.01 15.08 35.20 ± 6.38 16.59 34.00 ± 2.73 19.43 33.83 ± 4.99 19.83 Nasal 48.20 ± 10.62 0.142 40.83 ± 4.11 15.29 0.359 40.50 ± 2.88 15.97 0.948 39.50 ± 3.56 18.04 0.156 41.40 ± 6.65 14.11 0.611 40.67 ± 3.77 15.62 0.398 37.00 ± 6.28 23.24 0.475 32.20 ± 10.28 33.20 0.194 42.50 ± 5.01 43.67 ± 5.92 27.576 40.33 ± 5.35 5.10 36.50 ± 3.20 14.11 39.50 ± 5.32 7.59 36.80 ± 9.93 13.41 34.60 ± 3.43 18.59 39.67 ± 7.36 6.66 TEMPORAL 51.40 ± 11.66 0.413 44.00 ± 3.74 14.39 0.037 38.67 ± 5.61 24.76 0.032 41.17 ± 10.96 19.90 0.498 39.00 ± 2.55 24.12 0.043 40.33 ± 4.32 21.54 0.162 40.40 ± 4.77 21.40 0.999 32.80 ± 11.03 36.19 0.125 55.30 ± 8.97 54.33 ± 9.85 1.75 47.83 ± 7.05 13.50 44.67 ± 5.31 19.22 42.83 ± 2.78 29.12 46.60 ± 8.96 15.73 40.40 ± 3.13 26.94 41.17 ± 4.75 25.55 GCL THICKNESS CENTRAL 22.00 ± 2.70 0.827 20.67 ± 4.27 6.04 0.999 18.67 ± 3.14 15.13 0.139 18.17 ± 2.78 17.40 0.225 18.00 ± 2.73 18.18 0.415 21.00 ± 2.82 4.55 0.067 18.60 ± 2.51 15.45 0.819 17.40 ± 2.40 20.91 0.291 21.70 ± 3.30 20.67 ± 1.86 4.74 21.00 ± 1.67 3.22 20.33 ± 3.01 6.31 19.50 ± 3.01 11.28 17.60 ± 2.51 18.89 19.00 ± 2.82 12.44 18.83 ± 1.83 13.23 INNER INFERIOR 27.10 ± 2.13 0.671 26.67 ± 3.67 1.58 0.497 26.83 ± 2.04 0.99 0.726 24.83 ± 1.32 8.37 0.172 25.40 ± 1.14 6.27 0.737 25.50 ± 1.64 5.90 0.749 25.60 ± 3.36 5.54 0.738 22.80 ± 2.38 15.87 0.022 27.50 ± 2.01 27.83 ± 1.72 1.2 27.17 ± .98 1.2 26.00 ± 1.41 5.45 25.67 ± 1.36 7.13 25.80 ± 1.30 6.18 26.20 ± 1.92 4.73 25.83 ± 1.16 6.07 OUTER INFERIOR 26.10 ± 1.79 0.819 25.33 ± 1.03 2.95 0.260 25.00 ± 1.54 4.21 0.172 23.50 ± 2.34 9.96 0.091 23.40 ± 2.60 10.34 0.967 24.50 ± 1.87 6.13 0.531 23.00 ± 3.53 11.88 0.324 21.40 ± 3.43 18.01 0.135 26.30 ± 2.05 26.00 ± 0.8 1.14 26.17 ± 1.16 0.49 25.33 ± 0.51 3.68 23.33 ± 2.58 12.73 25.20 ± 1.64 4.18 24.80 ± 1.48 5.70 23.83 ± 1.16 9.39 INNER SUPERIOR 25.70 ± 2.83 0.682 23.00 ± 2.75 10.50 0.165 22.67 ± 2.80 11.78 0.169 19.50 ± 2.16 24.12 0.012 21.20 ± 2.38 17.51 0.035 23.00 ± 1.78 10.51 0.999 21.20 ± 4.65 17.51 0.945 18.20 ± 0.83 29.18 0.653 26.10 ± 1.10 25.17 ± 2.22 3.56 24.67 ± 1.75 5.47 23.33 ± 2.16 10.61 23.67 ± 0.51 10.27 23.00 ± 3.74 11.88 21.00 ± 4.30 19.54 18.83 ± 2.92 27.85 OUTER SUPERIOR 24.70 ± 2.79 0.710 24.50 ± 2.074 0.80 0.892 23.83 ± 1.47 3.52 0.064 24.17 ± 2.71 2.14 0.528 24.60 ± 2.19 0.40 0.464 24.00 ± 3.79 2.83 0.615 25.00 ± 2.55 1.21 0.636 19.40 ± 2.60 21.46 0.104 25.10 ± 1.85 24.67 ± 2.06 1.711 25.83 ± 1.83 2.90 25.00 ± 1.54 0.39 25.33 ± 0.81 0.91 22.60 ± 5.12 9.96 24.20 ± 2.58 3.59 22.33 ± 2.73 11.04 INNER Nasal 25.60 ± 2.31 0.916 23.00 ± 4.19 10.15 0.215 23.50 ± 2.34 8.20 0.260 21.50 ± 3.93 16.01 0.401 22.80 ± 1.30 10.94 0.728 21.67 ± 3.26 15.35 0.498 22.80 ± 2.28 10.94 0.551 19.00 ± 1.41 25.78 0.293 25.70 ± 1.82 25.33 ± 1.03 1.43 25.33 ± 2.94 1.43 23.33 ± 3.26 9.22 23.17 ± 1.94 10.92 23.00 ± 2.91 10.51 21.80 ± 2.77 15.18 20.17 ± 1.94 21.52 Outer Nasal 26.10 ± 1.66 0.558 25.17 ± 1.47 3.56 0.169 25.50 ± 1.37 2.29 0.679 23.17 ± 2.48 11.22 0.178 24.40 ± 1.34 6.51 0.596 22.50 ± 3.01 13.79 0.559 23.00 ± 2.44 11.88 0.550 19.75 ± 1.25 24.33 0.337 25.60 ± 2.06 24.17 ± 0.7 5.58 25.17 ± 1.32 1.67 24.83 ± 1.32 3.00 23.83 ± 1.94 7.43 23.60 ± 2.96 7.81 23.80 ± 1.48 7.03 21.00 ± 2.19 17.97 Inner temporal 26.30 ± 2.26 0.777 24.00 ± 2.96 8.74 0.432 23.33 ± 1.86 11.29 0.056 22.00 ± 2.28 16.34 0.401 22.40 ± 2.60 14.83 0.527 22.33 ± 0.81 15.10 0.409 23.20 ± 2.77 11.79 0.497 21.40 ± 2.30 18.63 0.599 26.00 ± 2.40 25.17 ± 1.83 3.19 25.83 ± 2.13 0.65 23.33 ± 2.94 10.26 23.50 ± 2.88 10.64 21.20 ± 3.11 18.46 24.20 ± 1.48 6.92 20.67 ± 2.16 20.50 Outer temporal 26.30 ± 2.21 0.610 25.33 ± 2.42 3.68 0.144 24.67 ± 1.63 6.19 0.112 24.50 ± 2.07 6.84 0.746 24.60 ± 1.14 6.46 0.956 24.00 ± 2.28 8.75 0.808 25.00 ± 2.73 4.94 0.897 21.00 ± 3.53 20.15 0.455 26.80 ± 2.09 27.17 ± 1.47 1.38 26.17 ± 1.32 2.35 24.00 ± 3.03 10.44 24.67 ± 2.42 8.63 23.40 ± 5.36 12.69 24.80 ± 1.92 7.46 22.17 ± 0.98 17.28 Total volume 0.18 ± 0.01 0.999 0.17 ± 0.01 4.61 0.347 0.17 ± 0.00 5.55 0.207 0.16 ± 0.01 11.11 0.201 0.16 ± 0.01 10.00 0.259 0.16 ± 0.01 8.33 0.736 0.16 ± 0.01 8.89 0.820 0.13 ± 0.01 23.33 0.057 0.18 ± 0.00 0.17 ± 0.00 0.94 0.17 ± 0.00 1.83 0.17 ± 0.00 5.55 0.17 ± 0.01 5.88 0.16 ± 0.01 10.00 0.16 ± 0.01 7.78 0.15 ± 0.01 14.83 MsDex: microspheres loaded with dexamethasone; RNFL: retina nerve fibre layer; GCL: ganglion cell layer; thickness in microns (mm); mean ± SD (SD: standard deviation); p < 0.05 statistical significance; p < 0.020# statistical significance with Bonferroni correction for multiple comparisons. Cells coloured in grey when right eye (upper cell) showed thinner sectors and/or higher percentage loss compared to left eye (lower cell). DRUG DELIVERY 2435 Figure 5. Structural analysis of the neuroretina, measured by optical coherence tomography, of both eyes in the dexamethasone-loaded microsphere (MsDex) model. MsDex: microspheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week; OCT: optical coherence tomography; RNFL: Retina Nerve Fiber Layer; GCL: Ganglion Cell Layer; average thickness in microns (lm). Black arrows: from the right eye; gray arrows: from the left eye; down arrow: decreasing thickness; up arrow: increasing thickness. play a role in the development of glaucoma. According to of its pathophysiology, along with better-characterized mod- Figure 12, microspheres aggregates remained in the iridocor- els that reproduce it, are in demand. neal angle of eyes after intracameral injection of dexametha- The ideal glaucoma model (Biswas and Wan, 2019) is sone-loaded microspheres 6 months after injection. one that meets the following criteria: (1) easily induced, (2) biologically plausible and with minimal side effects, (3) repro- ducible, (4) predictable in onset and clinical course and, Discussion finally and most importantly, (5) simulates the pathophysi- ology of glaucoma, i.e. glaucomatous damage with A multitude of ocular hypertension models has been devel- decreased axonal fibers and RGC and increased cup/disk- oped for the study of glaucoma, including steroid-induced optic associated with increased IOP. However, current models glaucoma. SIG shares similarities with POAG. Patients with present complications or limitations (very high hypertension POAG present the increased hypertensive response to gluco- in episcleral vein sclerosis or cauterization, or corneal edema corticoids, characteristic histological alterations in the tra- and synechiae, in non-biodegradable microspheres) that hin- becular meshwork (Weinstein et al., 1985), and elevated der longitudinal in vivo monitoring and disease dynamics, corticosteroid levels in aqueous humor (Kasimov and and it is difficult to identify those time windows where early/ Aghaeva, 2017). In addition, a relationship between chronic intermediate/late intervention would be useful (Kowal et al., exposure to stress hormones and retinal (O’steen et al., 1987) 2021). Our model meets all the above requirements and min- and neuronal (Vyas et al., 2016) degeneration has been dem- imizes complications or limitations as much as possible. onstrated. The SIG model is therefore considered a suitable The microsphere model is a consequence of the physical model for the study of glaucomatous neurodegeneration blockage of the outflow of aqueous humor by clogging the (Agarwal and Agarwal, 2017). Furthermore, the interest in iat- trabecular meshwork. Its technical requirements are less rogenic SIG is growing due to the increase in ophthalmologic demanding than those of other models such as the episcleral pathologies associated with aging that require treatment model (Morrison et al., 2015). The non-biodegradable micro- with glucocorticoids (GC) (Phulke et al., 2017), so knowledge sphere model (Biswas and Wan, 2019) ranks second (with 2436 M. J. RODRIGO ET AL. Figure 6. Thickness percentage loss, measured by optical coherence tomography (OCT), in the dexamethasone-loaded microsphere (MsDex) model from baseline to 6 months of follow-up. MsDex: microspheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week; OCT: optical coherence tomography; RNFL: retina nerve fiber layer; GCL: ganglion cell layer; %: percentage. Figure 7. Neuroretinal loss rate, measured by optical coherence tomography (OCT), in the dexamethasone-loaded microsphere (MsDex) model. MsDex: micro- spheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week; RNFL: retina nerve fiber layer; GCL: ganglion cell layer. Each data is obtained from baseline time. DRUG DELIVERY 2437 Figure 8. Neuroretina functionality, measured by dark- and light-adapted electroretinography (ERG), in the dexamethasone-loaded microsphere (MsDex) model over 6 months of follow-up. MsDex: microspheres loaded with dexamethasone; RE: right eye; LE: left eye; w: week; DA: dark-adapted; LA: light-adapted; lV: micro- volts; ms: milliseconds. respect to the other hypertensive models) in terms of gener- delivery systems. An intraocular PLGA implant loaded with ating IOP elevation with RGC loss and appears to be the dexamethasone is already available in ophthalmological clin- only one that demonstrates a negative trend between OHT ical care (OzurdexV R ) (Chang-Lin et al., 2011). In addition, it is duration and RGC loss. However, it requires reinjections to well known that PLGA Ms can encapsulate and subsequently maintain an elevated IOP. Our group created a glaucoma sustainedly release corticosteroids such as dexamethasone model by administering biodegradable PLGA microspheres for several months (Barcia et al., 2009; Dawes et al., 2012; into the anterior chamber of rat eyes to generate ocular Barbosa-Alfaro et al., 2021). Depending on Ms particle size, hypertension (Garcia-Herranz et al., 2021). A subsequent 6- among other technical parameters, it is common to find an month study (Rodrigo et al., 2021) using 7 injections demon- initial rapid release of the active compound followed by one strated the reproducibility of the new model and produced or several low-rate release phases. In our case, as a second functional and structural damage while maintaining superb injection of the formulation was performed in the in vitro ocular tolerance that enabled serial in vivo functional and release study to mimic the in vivo administration, the two structural testing with reliable results. release profiles overlapped.It has already been shown in Over the past 30 years, lactic and glycolic acid copolymers many previous works that the degradation of PLGA matrices (PLGA) have been implemented in biomedicine for diagnosis is a homogeneous degradation, which, unlike the heteroge- and treatment. This biomaterial is approved by the U.S. Food neous degradation of polymers in the matrices, takes place and Drug Administration (FDA) and the European Medicines in the entire polymer chain at the same time. In aqueous Agency for use in human beings due to its biocompatibility media, although partially aggregated, PLGA microspheres and biodegradability (Martins et al., 2018). PLA, PGA, and maintain a spherical external appearance for weeks, despite PLGA polymers have been used to create controlled drug- the continuous degradation by hydrolysis of PLGA until a 2438 M. J. RODRIGO ET AL. Figure 9. Ganglion cell analysis in glaucomatous eyes. (A) Ganglion cells were counted in four areas (squares) of a radial section of the retina passing through the optic nerve. (B) Two representative images of the retina marked with anti-Brn3a, corresponding to a left control eye (LE) and a right eye (RE) from the same animal injected with dexamethasone microspheres. (B) The mean number of ganglion cells per linear mm of retina did not differ between non-injected eyes and injected eyes. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer. Scale bars: 50 lm. moment comes when the structures collapse, becoming Those models that require frequent reinterventions pro- polymeric aggregates without definite shape (Checa- duce more complications. To decrease intervention in the Casalengua et al., 2011; Barbosa-Alfaro et al., 2021). Although animal, and thanks to the sustained release of dexametha- a similar behavior can be assumed in vivo, it is very difficult sone from MsPLGA (Rodrıguez Villanueva et al., 2016), the to establish the exact behavior of these PLGA microspheres present model was developed in which only 2 injections of once injected into the anterior chamber. However, in this the MsPLGA formulation loaded with dexamethasone were context, the physical blockage can be explained by the pres- necessary to obtain a slow and sustained hypertensive curve ence of both the pellet (limiting the aqueous humor outflow) that produced functional and structural damage over and the remaining isolated PLGA material that infiltrates 6 months. Those models with rapid IOP elevations are the through the trabecular pores, that according to literature, least representative of POAG, and the key to success in trans- offer porous of around 25–27 mm in the uveal meshwork of lational studies is the choice of a model that closely repre- the TM, being significantly narrower in the corneoscleral sents the human disease (Agarwal and Agarwal, 2017) (Table meshwork of the TM (2–15 mm) (Abu-Hassan et al., 2014). In 2). In our case, it was decided to perform 2 ocular injections fact, remnants of microspheres were observed at the irido- to obtain an initial mechanical microsphere blockade and a corneal angle of eyes after intracameral injection of dexa- progressive alteration of the trabecular meshwork utilizing a methasone-loaded microspheres according to (Figure 12). pharmacological mechanism secondary to the sustained DRUG DELIVERY 2439 Figure 10. Increased deposition of collagen IV in the zonular fibers (arrowheads) and in the basement membrane of the non-pigmented epithelium (arrows) at the ciliary body in dexamethasone-injected rat eyes. LE: left eye; RE: right eye (injected with dexamethasone-loaded microspheres); CB: ciliary body; non-PE: non-pig- mented epithelium; PE: pigmented epithelium; TM: transmission mode. Scale bars: (A) 43.48 lm; (B, C) 18.18 lm. release of dexamethasone. This was performed to obtain a dexamethasone release for several weeks without interven- slow IOP rise curve that resembled human glaucoma, in add- tion but produced a high rate of deaths (Overby and Clark, ition to improving animal welfare, reducing costs, and reduc- 2015) (Table 3). However, few SIG studies in rats have sug- ing the risk of other complications such as infection or gested differences with mice. Very few studies have analyzed ocular toxicity due to high concentrations of PLGA (Park, the impact on IOP (Sawaguchi et al., 2005; Shinzato et al., 2017; Zhao et al., 2017). 2007; Miyara et al., 2008; Razali et al., 2016; Sato et al., 2016), Focusing on SIG models performed in rodents (Rybkin as well as the structural changes in the trabecular meshwork et al., 2017), most use mice and the effect of glucocorticoids (Sawaguchi et al., 2005; Shinzato et al., 2007; Razali et al., on OHT and ocular function and structure has been exten- 2016) and retinal structure (Miyara et al., 2008; Razali et al., sively studied in them (Overby et al., 2014; Zode et al., 2014; 2016). To our knowledge, there are none that explore the Faralli et al., 2018; Patel et al., 2018; Li et al., 2019; Patel effects of GCs on the structure using OCT or retinal function- et al., 2019). Topical administration produces the greatest ality and all are performed by daily topical administration OHT with retinal dysfunction and optic neuropathy but has with a maximum follow-up of 8 weeks. the disadvantage of requiring daily administration. Our SIG model reduces the repeated interventionism of Subconjunctival administration reduced outflow but required daily topical application and the potential negative side repeated injections to maintain elevated IOP (Li et al., 2019). effects of systemic administration or periocular administra- Systemic administration with a minipump allows tion (Li et al., 2019), which is considered the most dangerous 2440 M. J. RODRIGO ET AL. Figure 11. Laminin deposition in the zonular fibers (arrowheads) of dexamethasone-injected rat eyes. The ciliary body in (A) transmission mode (TM) and (B) in fluorescence. RE: right eye (injected with dexamethasone-loaded microspheres); LE: left eye; CB: ciliary body; non-PE: non-pigmented epithelium; PE: pigmented epithelium. Scale bars: (A) 43.48 lm; (B) 20.60 lm. because of its prolonged duration of action, non-predictabil- (data obtained from our previous work (Rodrigo et al., 2021), ity of response, and the inability to withdraw it in the event with respect to animals with microspheres loaded with dexa- of hyper-corticosteroid-responsiveness, in addition to the methasone (13.98 ± 3.08 mmHg vs. 15.3 9 ± 2.93 mmHg) since impossibility of visualization (Phulke et al., 2017). In contrast, the groups were comparable because they received only one anterior chamber administration allows visualization of the injection at baseline (same size of Ms and volume), the dif- Ms and control of the model by modulating the injections. ference in IOP was probably due to the dexamethasone Moreover, if required by the researcher, repeated injections released at that time. can modulate and control the hypertensive curve. In add- In addition, the corticosteroid response was analyzed, ition, our MsPLGA-Dex SIG model was well tolerated, as no being to our knowledge the first study to do so. OHT animal developed an infection and only one developed cata- response to corticosteroids varies between patients and SIG ract, even though it is known that chronic exposure to GC models, but typically there is an IOP increase after the third increases the risk of developing them (James, 2007; Migita week of exposure to corticosteroids. Between 30 and 40% of et al., 2013). humans respond to corticosteroid with an IOP increase of Our model produced a slightly earlier IOP increase (1 vs 6–15 mmHg (medium responders); 4–6% respond with an 2–3 weeks), possibly due to the synergy of the mechanical increase of more than 15 mmHg (high responders); and in blockage of PLGA microspheres combined with the pharma- 1–3% of patients, IOP remains high even after ceasing expos- cological action of the presumed presence of dexametha- ure to steroids (Clark, 1995; Kersey and Broadway, 2006; sone in the area, and reached OHT later (5 vs 2–4 weeks) Clark and Wordinger, 2009). The percentage of OHT cortico- than previous studies in rats (Sawaguchi et al., 2005; Miyara steroid response varies in other animals (50% of primates, et al., 2008). In fact, when comparing the IOP obtained at 80% of rats, and 100% of cows) (Danias et al., 2011). In our week 1 in animals induced with non-loaded microspheres rat model, the maximum percentage of hypertensive animals DRUG DELIVERY 2441 Figure 12. Iridocorneal angle hampering after intracameral injection of dexamethasone-loaded microspheres. A Iridocorneal angle LE Left eye RE Right eye CB Ciliary body Ir Iris Co Cornea Microsphere remnants Scale bars 77 mm (A) 38 mm (inset). was similar to previous studies (80–90%) (Razali et al., 2015) nevertheless suffered a progressive increase in IOP, meaning and mostly medium corticosteroid responsiveness was found, there are still unknowns to be resolved. Our model would be as in most humans. the first to analyze such an increase in IOP, even reaching An increase in IOP was also detected in the contralateral OHT in the contralateral eye over a long study period. eye at 8 weeks (later than other authors) (Rybkin et al., Neuroretinal degeneration was noninvasively assessed 2017). In this regard, in a mouse SIG model using the perioc- longitudinally and in vivo using OCT, allowing for longer ular injection of PLGA-Dex nanoparticles (Li et al., 2019), an tracking than in previous studies. Bilateral thickness loss was increase in IOP was detected in the contralateral eye, and detected; it was greater in the injected right eye and was possible perilymphatic communication was hypothesized, mainly in the RNFL (superior-inferior axis sectors) (Guo et al., but could not be fully explained (Zode et al., 2014; Patel 2010), followed by the GCL (nasal-temporal sectors) (Salinas- et al., 2017; 2018). Other reasons could be direct systemic Navarro et al., 2009). The reduction in the loss rate over time vascular communication and periocular lipophilic deposition was probably due to the floor effect. The bilateral loss meant with the subsequent systemic release of very low and that no statistically significant differences in ganglion cell chronic levels that end up in the aqueous humor as plasma counts at 24 weeks were found in histological studies. ultrafiltrate and alter the genomics and functionality of the However, fewer RGCs were counted than in our previous trabecular meshwork (see section Hypothesis of the effect of model with unloaded PLGA microspheres (Garcia-Herranz glucocorticoids in the glaucoma model and in the future). In et al., 2021). RNFL > GCL > R involvement may reflect the this regard, human studies have shown elevated levels of GC anterograde neurodegenerative pathway, as the same trend in the aqueous humor (Chang-Lin et al., 2011; Kasimov and of topographic loss in depth (from inner to outer layers) was Aghaeva, 2017). However, all of the above are hypotheses maintained throughout the 24 weeks. Likewise, the involve- and it would be beneficial to measure the levels of dexa- ment of the contralateral left eye could be due to the methasone in the aqueous humor of both eyes in our model increase in IOP (mentioned above) and/or the dissemination to corroborate this. Although our histological results found and activation of inflammatory factors due to transsynaptic no increased laminin deposition in the contralateral eye, it degeneration (Sapienza et al., 2016; de Hoz et al., 2018; 2442 M. J. RODRIGO ET AL. Lawlor et al., 2018). Immune activation has been detected in 9.81mm RNFL (OCT) 19.11mm RNFL (OCT) OHT: ocular hypertension; IOP: intraocular pressure; TM: trabecular meshwork; ON: optic nerve; RGC: retinal ganglion cell; RNFL: retinal nerve fiber layer; GCL: ganglion cell layer; OCT: optical coherence tomography; w: 12.88mm GCL (OCT) 10.76mm GCL (OCT) 2 injections ! 24 w. histological studies of glaucoma (Ramirez et al., 2017). In our Microspheres Corneal puncture. and sustained PLGA-DEX Periferal corneal study, OCT showed dynamic fluctuations over time and a leucoma, topographic variability in retinal thickness that could reflect Progressive Bi-injection cataract immune activation and infiltration in waves (Ramirez et al., Easy 2017). It cannot be ruled out that the observed fluctuations may be due in part to the variability of the device. However, 7 injections ! 24w the fact that follows up technology was used (which allows biodegradable Corneal puncture. Microspheres Periferal corneal measurement at the same exact points in each re-evaluation) and that the fluctuations were observed at the same study leucoma Progressive and sustained Injections. Multiple times as in previous work performed in healthy (Rodrigo Pretrabecular et al., 2020) and glaucomatous (Rodrigo et al., 2021) animals Easy with OCT, as well as in histological studies that related the weekly up to 30w. (Samsel et al. 2011; fluctuations to immune infiltration; makes the authors of the non-biodegradable 1 injection !1 w. 2 injections ! 8w. Hernandez, and Corneal puncture. Microspheres Vecino 2006) study consider it unlikely to be a failure or variability of the Corneal edema, 23-6%RGC loss Urcola, technique and they do consider it to be a sign of reproduci- Progressive and sustained sinechiaes, Injections. bility in the measurement, in agreement with the recognition ischemia Multiple of the influence of immunity in glaucoma (Geyer and Easy Levo, 2020). Weekly up to 10w. We found a greater thickness in the inner sectors of the R Corneal puncture. administration Benozzi et al. Intracameral in the contralateral left eye, which could reflect a possible of viscous 50–70% ON axon loss 13% ON axon loss 40% RGC loss No described agents 2002) infiltration from the central vessels and the optic nerve due Injections. Multiple to dissemination from the contralateral eye. This coincides Spikes Easy with an increase in RNFL observed at week 2 in the loss rate. On the other hand, the loss of inner sectors of the GCL Transduction of genetic (Kersey (Davis et al., 2016) was constant in both eyes, suggesting ini- animal genetics. inflammation and (Shepard et al. 2010) Gene edition. TM with tial damage close to the optic nerve (Howell et al., 2007). Gen edition. Challenging Progressive Modified Possible early immune infiltration of the retina in the Ocular induced right eye resulted in lower loss rates compared to 6w Trabecular – – the contralateral left eye. In the early weeks of the study (weeks 2–4), the animal’s eye is still growing, which was Ocular inflammation. (Levkovitch-Verbin photo-coagulation Transient elevation quantified as a higher rate of retinal thickness loss in the ocular injuries. Laser equipment et al. 2002) Unintended uninduced left eye (Rodrigo et al., 2020). Possible immune Laser Challenging needed. infiltration of the retina due to noxa (Sapienza et al., 2016; !1 year. Spikes. 1 laser Single de Hoz et al., 2018; Rodrigo et al., 2021) was quantified in Laser. week; %: percentage; min: minutes; h: hours; mm: microns (22) (Pang and Clark 2020). the right eye as a lower rate of loss and thus suggests being an underestimate measured with OCT. Rapid and sustained (Garcia-Valenzuela epiescleral veins vasocongection ERG scans revealed a loss of neuroretinal functionality Cauterization inflammation, et al. 1995) 1 cauterization Cauterization. over time. The injected right eye always showed lower func- 4% RGC loss tional signal under both scotopic and photopic conditions; per week !16w. Spectrum of IOP. Spikes. Ocular Single however, the left eye also suffered moderate functional limi- Burn. Easy tation as the signal enhancement caused by neuromodula- tion described in healthy animals 2–3 months old was not Single/ Multiple (J C Morrison Postrabecular et al. 1997) detected (Chaychi et al., 2015; Nadal-Nicolas et al., 2018; Cornea cloud, epiescleral Sclerosis 10–100%ON sustained axon loss Challenging veins Rapid and Rodrigo et al., 2020). This suggests that the hypertensive Injection. Multiple Surgery. ! 26w. cataract noxa in the left eye, lasting only 4 weeks, exerted a damag- ing/limiting influence. In this regard, in glaucoma, relatively few changes have been described in scotopic ERG, although Table 2. IOP-induced glaucoma models. (Crowston et al. 2015) there have been more significant alterations with pSTR and and transient Loop/cannulation Intermitent 7–52% RGC loss Ocular ischemia oscillatory potentials (Bach and Poloschek, 2013; Park et al., Challenging 30 min–8h. 2018). In our study, the phases with lower intensities (and Acute OHT Multiple Surgery. therefore close to the pSTR), as well as oscillatory potentials, Spikes. Short. were the most affected (Figure 8). Only one study with SIG in rodents (C57bL/6 mice) analyzed retinal function using degeneration Interventionism pattern ERG (specific for RGC), at 20 weeks finding a 55% loss Neuroretinal Iop increase Side effects of signal under photopic conditions (Zode et al., 2014). Our Glaucoma Induction Duration by OHT models study seems to be the first to analyze the effect of SIG on retinal function in rats, and for a longer time (24 weeks), DRUG DELIVERY 2443 Table 3. Steroid-induced glaucoma models. Systemic (Whitlock et Periocular (Wang et al. 2018; Topical (Shinzato et al. 2007; SIG models al. 2010) Li et al. 2019) Anterior chamber Sato et al. 2016) Induction Surgery Subconjuntival injection Corneal puncture Drops, ointment Subcutaneous minipump Nanoparticles PLGA-DEX Microspheres PLGA-DEX Challenging Easy Easy Easy Interventionism 1 3 2 Daily Side effects Systemic, Prolonged duration of action, Periferal corneal leucoma, Bodyweight loss body weight loss, non-predictability, cataract leucopenia, no visualization, infection, inability to withdraw death IOP increase Mild and progressive Progressive and sustained Progressive and sustained Progressive and sustained Neuroretinal degeneration -No data -No data 19.11 mm RNFL (OCT) 29% axon loss (at 15w) 10.76 mm GCL (OCT) Duration 4w 8w 24w 8w SIG: Steroid induced glaucoma; IOP: intraocular pressure; RNFL: retinal nerve fiber layer; GCL: ganglion cell layer; OCT: optical coherence tomography; w: week; %: percentage; mm: microns. detecting a decrease in the response under photopic condi- bioinformatic analysis of the anterior segment has recently tions of close to 50% of the PhNR at week 24, as well as sig- shown that dexamethasone-induced glaucoma involves nal deterioration under scotopic conditions from week genes related to different metabolic pathways, tyrosine or 12 onwards. cholesterol homeostasis (Song et al., 2011; Zhang et al., The hypothesis of the effect of glucocorticoids in the 2019). By virtue of its capacity to facilitate longitudinal study glaucoma model and in the future. GCs have distinct over time, our model would allow us to analyze possible effects on a multitude of cells, acting as activating or repres- connections between the pathways involved (with genomic/ sor gene modulators (Spies et al., 2011; Cain and Cidlowski, proteomic studies) and, therefore, to develop treatments 2017). GCs influence trabecular meshwork cells resulting in depending on the clinical situation of the patient. Likewise, cell death. This produces an increase in IOP that triggers an long-term formulations could be tested, since SIG treatments immune response (Chen et al., 2018). Recently, Glaucoma has have only been able to prove their short-term effects up to a been named an autoimmune disease (Geyer and Levo, 2020) maximum of 40 days (Ganekal et al., 2014; Borras et al., 2016; and, it has also been shown that GCs play a role in activating Razali et al., 2016). innate immunity (Cain and Cidlowski, 2017). It has been The main limitations of this study could also be its hypothesized that corresponding to physiological levels of potential strengths. While the rigidity of the trabecular mesh- GCs, macrophages (cells of the trabecular meshwork that work or outflow of aqueous humor has not been assessed have phagocytic action, and microglia resident in the retina) by noninvasive techniques such as OCT, neither have prote- are sensitized for rapid detection of damage and, in the omic, molecular or genetic studies been performed, or pos- inflammatory state, restrain inflammation (Spies et al., 2011). sible differences by sex evaluated. In our opinion, this study In our case, the chronic release of GC in the order of lg per would be the first that would make it possible to perform day could, to some extent, resemble the pathophysiology of the long-term assessment in a model that more closely glaucoma, where very low concentrations of GC in a chronic resembles the human condition. In future research, it would subinflammatory state could cause genomic and subse- be ideal to develop a model with minimal interventionism quently cellular/tissue/organ level changes. In this regard, on the animal, for example with a single intervention/injec- the modified release of prednisone at low doses over a long tion. Furthermore, it cannot be ruled out that the use of period of time seems to contribute to chronic changes made dexamethasone, used as an inducer of OHT, could partly to the genome (Spies et al., 2011). Cases of rebellious SIG counteract the proinflammatory effect secondary to the that end up requiring surgery could be a consequence of increase in IOP. It seems desirable to develop technologies already irreversible genomic changes in which conventional to obtain the same effect (gradual IOP increase with progres- treatment is insufficient. On the other hand, the greater the sive neuroretinal degeneration) without altering immunity. exposure to chronic GC, the greater the inflammatory In conclusion, this work explores the usefulness of drug response (Vyas et al., 2016), and greater inflammation has delivery systems, not to treat pathology but to induce it. We been associated with greater neuronal damage (Gonzalez have developed a biodegradable, biocompatible micro- et al., 2014; Noailles et al., 2014). sphere-based delivery system that creates a chronic gluco- In this sense, the initial slight increase in IOP due to corticoid- and mechanically induced glaucoma model that mechanical blockade by the Ms could trigger the immune mimics human steroid-induced glaucoma produced by response, and corticosteroids could further activate it. In add- increased IOP and retina and optic nerve degeneration. This ition, the changes that GCs cause in the trabecular meshwork model could be modified by increasing the number or injec- would further limit the outflow of aqueous humor, reaching tions, the quantity of dexamethasone loaded or even the OHT levels and perpetuating the damage to the neuroretinal rate of degradation, although in our case only two injections degenerative cascade. However, this mechanistic theory of were needed to obtain the outcomes at 24 weeks. As far as aqueous outflow limitation may not be so simple, since the we know, this is the first study that would allow the analysis 2444 M. J. RODRIGO ET AL. of the earliest and most chronic pathophysiological changes Chaychi S, Polosa A, Lachapelle P. (2015). Differences in retinal structure occurring in SIG. and function between aging male and female Sprague-Dawley rats are strongly influenced by the estrus cycle. PLOS One 10:e0136056. Checa-Casalengua P, Jiang C, Bravo-Osuna I, et al. (2011). Retinal gan- glion cells survival in a glaucoma model by GDNF/Vit E PLGA micro- Acknowledgements spheres prepared according to a novel microencapsulation procedure. The authors would like to acknowledge the contribution of the Servicio J Control Release 156:92–100. General de Apoyo a la Investigacio n (SAI) of the University of Zaragoza Chen H, Cho KS, Vu THK, et al. (2018). Commensal microflora-induced T and the technical SEM assistance of the Centro de Microscopıa cell responses mediate progressive neurodegeneration in glaucoma. nica Luis Bru (CAI) of the Complutense University of Madrid. Electro Nat Commun 9(1):3914. Clark AF, Wordinger RJ. (2009). The role of steroids in outflow resistance. Exp Eye Res 88:752–759. Disclosure statement Clark AF. (1995). Basic sciences in clinical glaucoma. J Glaucoma 4: 354–369. The authors declare no conflict of interest regarding the publication of Crowston JG, Kong YXG, Trounce IA, et al. (2015). An acute intraocular this paper. pressure challenge to assess retinal ganglion cell injury and recovery in the mouse. Exp Eye Res 141:3–8. Danias J, Gerometta R, Ge Y, et al. (2011). Gene expression changes in Funding steroid-induced IOP elevation in bovine trabecular meshwork. Invest Ophthalmol Vis Sci 52:8636–45. This study was supported by Rio Hortega research grants M17/00213, Davis BM, Guo L, Brenton J, et al. (2016). Automatic quantitative analysis PI17/01726, PI17/01946 and PI20/00437 (Carlos III Health Institute), and of experimental primary and secondary retinal neurodegeneration: by MAT2017-83858-C2-1, MAT2017-83858-C2-2 MINECO/AEI/ERDF, EU implications for optic neuropathies. Cell Death Discov 2: 16031. and PID2020-113281RB-C22 MINECO/AEI/ERDF, EU. Dawes G, Fratila-Apachitei LE, Necula BS, et al. (2012). Effects of dexa- methasone-loaded PLGA microspheres on human fetal osteoblasts. J Biomater Appl 27:477–83. de Hoz R, Ramırez AI, Gonzalez-Martın R, et al. (2018). Bilateral early acti- ORCID vation of retinal microglial cells in a mouse model of unilateral laser- induced experimental ocular hypertension. Exp Eye Res 171:12–29. J Garcıa-Feijoo http://orcid.org/0000-0002-7772-5718 De Maria A, Wilmarth PA, David LL, Bassnett S. (2017). Proteomic analysis L Pablo http://orcid.org/0000-0003-2389-8282 of the bovine and human ciliary zonule. Invest Ophthalmol Vis Sci 58: E Garcia-Martin http://orcid.org/0000-0001-6258-2489 573–585. Ding C, Wang P, Tian N. (2011). Effect of general anesthetics on IOP in elevated IOP mouse model. Exp Eye Res 92:512–20. References Faralli JA, Clark RW, Filla MS, Peters DM. (2015). NFATc1 activity regulates the expression of myocilin induced by dexamethasone. Exp Eye Res Abu-Hassan DW, Acott TS, Kelley MJ. (2014). The trabecular meshwork: a 130:9–16. basic review of form and function. J Ocul Biol. Faralli JA, Dimeo KD, Trane RM, Peters D. (2018). Absence of a secondary Agarwal R, Agarwal P. (2017). Rodent models of glaucoma and their glucocorticoid response in C57BL/6J mice treated with topical dexa- applicability for drug discovery. Expert Opin Drug Discov 12:261–70. methasone. PLOS One 13:e0192665–18. Bach M, Poloschek CM. (2013). Electrophysiology and glaucoma: current Fini ME, Schwartz SG, Gao X, et al. (2017). Steroid-induced ocular hyper- status and future challenges. Cell Tissue Res 353:287–96. tension/glaucoma: focus on pharmacogenomics and implications for Barbosa-Alfaro D, Andr es-Guerrero V, Fernandez-Bueno I, et al. (2021). precision medicine. Prog Retin Eye Res 56:58–83. Dexamethasone PLGA microspheres for sub-tenon administration: Ganekal S, Dorairaj S, Jhanji V, Kudlu K. (2014). Effect of topical calcium influence of sterilization and tolerance studies. Pharmaceutics 13: channel blockers on intraocular pressure in steroid-induced glaucoma. 228–1. J Curr Glaucoma Pract 8:15–9. Barcia E, Herrero-Vanrell R, Dıez A, et al. (2009). Downregulation of endo- Garcia-Herranz D, Rodrigo MJ, Subias M, et al. (2021). Novel use of PLGA toxin-induced uveitis by intravitreal injection of polylactic-glycolic microspheres to create an animal model of glaucoma with progres- acid (PLGA) microspheres loaded with dexamethasone. Exp Eye Res sive neuroretinal degeneration. Pharmaceutics 237. 89:238–45. Garcia-Valenzuela E, Shareef S, Walsh J, Sharma SC. (1995). Programmed Bassnett S. (2021). Zinn’s zonule. Prog Retin Eye Res 82:100902. cell death of retinal ganglion cells during experimental glaucoma. Exp Benozzi J, Nahum LP, Campanelli JL, Rosenstein RE. (2002). Effect of hya- Eye Res 61:33–44. Geyer O, Levo Y. (2020). Glaucoma is an autoimmune disease. luronic acid on intraocular pressure in rats. Invest Ophthalmol Vis Sci Autoimmun Rev 19:102535. 43:2196–200. Gonzalez H, Elgueta D, Montoya A, Pacheco R. (2014). Neuroimmune Biswas S, Wan KH. (2019). Review of rodent hypertensive glaucoma mod- regulation of microglial activity involved in neuroinflammation and els. Acta Ophthalmol 97:e331–e340. neurodegenerative diseases. J Neuroimmunol 274:1–13. Borras T, Buie LK, Spiga MG. (2016). Inducible scAAV2.GRE.MMP1 lowers Guo L, Normando EM, Nizari S, et al. (2010). Tracking longitudinal retinal IOP long-term in a large animal model for steroid-induced glaucoma changes in experimental ocular hypertension using the cSLO and gene therapy. Gene Ther 23:438–49. spectral domain-OCT. Invest Ophthalmol Vis Sci 51:6504–13. Cain DW, Cidlowski JA. (2017). Immune regulation by glucocorticoids. Howell GR, Libby RT, Jakobs TC, et al. (2007). Axons of retinal ganglion Nat Rev Immunol 17:233–47. cells are insulted in the optic nerve early in DBA/2J glaucoma. J Cell Chan HHL, Salmon JF. (2019). Glaucoma caused by topical corticosteroid Biol 179:1523–37. application to the eyelids. Med J Aust 210:152–3.e1. James ER. (2007). The etiology of steroid cataract. J Ocul Pharmacol Ther Chan W, Wiggs JL, Sobrin L. (2019). The genetic influence on corticoster- 23:403–20. oid-induced ocular hypertension: a field positioned for discovery. Am Kasimov EM, Aghaeva FA. (2017). [Cortisol levels in plasma and aqueous J Ophthalmol 202:1–5. humour of patients with steroid induced and other glaucomas]. Vestn Chang-Lin JE, Attar M, Acheampong AA, et al. (2011). Pharmacokinetics Oftalmol 133:39–45. and pharmacodynamics of a sustained-release dexamethasone intravi- Kersey JP, Broadway DC. (2006). Corticosteroid-induced glaucoma: a treal implant. Invest Ophthalmol Vis Sci 52:80–6. review of the literature. Eye 20:407–16. DRUG DELIVERY 2445 Kornmann HL, Gedde SJ. (2016). Glaucoma management after corneal Razali N, Agarwal R, Agarwal P, et al. (2015). Anterior and posterior seg- transplantation surgeries. Curr Opin Ophthalmol 27:132–9. ment changes in rat eyes with chronic steroid administration and Kowal TJ, Prosseda PP, Ning K, et al. (2021). Optogenetic modulation of their responsiveness to antiglaucoma drugs. Eur J Pharmacol 749: intraocular pressure in a glucocorticoid-induced ocular hypertension 73–80. mouse model. Transl Vis Sci Technol 10:10. Razali N, Agarwal R, Agarwal P, et al. (2016). Topical trans-resveratrol Lawlor M, Danesh-Meyer H, Levin LA, et al. (2018). Glaucoma and the ameliorates steroid-induced anterior and posterior segment changes brain: trans-synaptic degeneration, structural change, and implications in rats. Exp Eye Res 143:9–16. for neuroprotection. Surv Ophthalmol 63:296–306. Razeghinejad MR, Katz LJ. (2012). Steroid-induced iatrogenic glaucoma. Levkovitch-Verbin H, Quigley HA, Martin KRG, et al. (2002). Translimbal Ophthalmic Res 47:66–80. laser photocoagulation to the trabecular meshwork as a model of Rodrigo MJ, Garcia-Herranz D, Subias M, et al. (2021). Chronic glaucoma glaucoma in rats. Invest Ophthalmol Vis Sci 43:402–10. using biodegradable microspheres to induce intraocular pressure ele- Li G, Lee C, Agrahari V, et al. (2019). In vivo measurement of trabecular vation. Six-month follow-up. Biomedicines 9:682. meshwork stiffness in a corticosteroid-induced ocular hypertensive Rodrigo MJ, Martinez-Rincon T, Subias M, et al. (2020). Effect of age and mouse model. Proc Natl Acad Sci USA 116:1714–22. sex on neurodevelopment and neurodegeneration in the healthy eye: Marshall GE, Konstas AGP, Abraham S, Lee WR. (1992). Extracellular longitudinal functional and structural study in the long–evans rat. Exp matrix in aged human ciliary body: an immunoelectron microscope Eye Res 200: 108208. study. Investig Ophthalmol Vis Sci 33:2546–60. Rodrıguez Villanueva J, Bravo-Osuna I, Herrero-Vanrell R, et al. (2016). Martins C, Sousa F, Ara ujo F, Sarmento B. (2018). Functionalizing PLGA Optimising the controlled release of dexamethasone from a new gen- and PLGA derivatives for drug delivery and tissue regeneration appli- eration of PLGA-based microspheres intended for intravitreal adminis- cations. Adv Healthcare Mater 7:1701035. tration. Eur J Pharm Sci 92:287–97. Migita K, Arai T, Ishizuka N, et al. (2013). Rates of serious intracellular Rybkin I, Gerometta R, Fridman G, et al. (2017). Model systems for the infections in autoimmune disease patients receiving initial gluco- study of steroid-induced IOP elevation. Exp Eye Res 158:51–8. corticoid therapy. PLOS One 8:e78699. Salinas-Navarro M, Mayor-Torroglosa S, Jim enez-Lopez M, et al. (2009). A Miyara N, Shinzato M, Yamashiro Y, et al. (2008). Proteomic analysis of computerized analysis of the entire retinal ganglion cell population rat retina in a steroid-induced ocular hypertension model: potential and its spatial distribution in adult rats. Vision Res 49:115–26. vulnerability to oxidative stress. Jpn J Ophthalmol 52:84–90. Samsel PA, Kisiswa L, Erichsen JT, et al. (2011). A novel method for the Morrison JC, Cepurna WO, Johnson EC. (2015). Modeling glaucoma in induction of experimental glaucoma using magnetic microspheres. rats by sclerosing aqueous outflow pathways to elevate intraocular Invest Ophthalmol Vis Sci 52:1671–5. pressure. Exp Eye Res 141:23–32. Sapienza A, Raveu A-L, Reboussin E, et al. (2016). Bilateral neuroinflam- Morrison JC, Moore CG, Deppmeier LM, et al. (1997). A rat model of matory processes in visual pathways induced by unilateral ocular chronic pressure-induced optic nerve damage. Exp Eye Res 64:85–96. hypertension in the rat. J Neuroinflammation 13:44. Nadal-Nicolas FM, Vidal-Sanz M, Agudo-Barriuso M. (2018). The aging rat Sato K, Nishiguchi KM, Maruyama K, Moritoh S, et al. (2016). Topical ocu- retina: from function to anatomy. Neurobiol Aging 61:146–68. lar dexamethasone decreases intraocular pressure and body weight in Noailles A, Fernandez-Sanchez L, Lax P, Cuenca N. (2014). Microglia acti- rats. J Negat Results BioMed 15:1–6. vation in a model of retinal degeneration and TUDCA neuroprotective Sawaguchi K, Nakamura Y, Nakamura Y, et al. (2005). Myocilin gene effects. J Neuroinflammation 11:1–15. expression in the trabecular meshwork of rats in a steroid-induced O’steen WK, Sweatt AJ, Eldridge JC, Brodish A. (1987). Gender and ocular hypertension model. Ophthalmic Res 37:235–42. chronic stress effects on the neural retina of young and mid-aged Shan SW, Do CW, Lam TC, et al. (2017). New insight of common regula- Fischer-344 rats. Neurobiol Aging 8:449–55. tory pathways in human trabecular meshwork cells in response to Overby DR, Bertrand J, Tektas OY, et al. (2014). Ultrastructural changes dexamethasone and prednisolone using an integrated quantitative associated with dexamethasone-induced ocular hypertension in mice. proteomics: SWATH and MRM-HR mass spectrometry. J Proteome Res Invest Ophthalmol Vis Sci 55:4922–33. Overby DR, Clark AF. (2015). Animal models of glucocorticoid-induced 16:3753–65. glaucoma. Exp Eye Res 141:15–22. Shepard AR, Millar JC, Pang I-H, et al. (2010). Adenoviral gene transfer of Pang IH, Clark AF. (2020). Inducible rodent models of glaucoma. Prog active human transforming growth factor-fbetag2 elevates intraocular Retin Eye Res 75:100799. pressure and reduces outflow facility in rodent eyes. Invest Park K. (2017). Tolerance levels of PLGA microspheres in the eyes. J Ophthalmol Vis Sci 51:2067–76. Control Release 266:365. Shinzato M, Yamashiro Y, Miyara N, et al. (2007). Proteomic analysis of Park SJ, Paik SS, Lee J-Y, et al. (2018). Blue-on-green flash induces max- the trabecular meshwork of rats in a steroid-induced ocular hyperten- imal photopic negative response and oscillatory potential and serves sion model: downregulation of type I collagen C-propeptides. as a diagnostic marker for glaucoma in rat retina. Exp Neurobiol 27: Ophthalmic Res 39:330–7. 210–6. Song Z, Gao H, Liu H, Sun X. (2011). Metabolomics of rabbit aqueous Patel GC, Liu Y, Millar JC, Clark AF. (2018). Glucocorticoid receptor GRb humor after administration of glucocorticosteroid. Curr Eye Res 36: regulates glucocorticoid-induced ocular hypertension in mice. Sci Rep 563–70. 8:1–13. Spies CM, Strehl C, Van Der Goes MC, et al. (2011). Glucocorticoids. Best Patel GC, Millar JC, Clark AF. (2019). Glucocorticoid receptor transactiva- Pract Res Clin Rheumatol 25:891–900. tion is required for glucocorticoid-induced ocular hypertension and Storey PP, Obeid A, Pancholy M, et al. (2018). Ocular hypertension after glaucoma. Invest Ophthalmol Vis Sci 60:1967–78. intravitreal injection of 2-Mg triamcinolone. Retina. Patel GC, Phan TN, Maddineni P, et al. (2017). Dexamethasone-induced Tawara A, Tou N, Kubota T, et al. (2008). Immunohistochemical evalu- ocular hypertension in mice: effects of myocilin and route of adminis- ation of the extracellular matrix in trabecular meshwork in steroid- tration. Am J Pathol 187:713–23. induced glaucoma. Graefes Arch Clin Exp Ophthalmol 246:1021–8. Phulke S, Kaushik S, Kaur S, Pandav SS. (2017). Steroid-induced glau- Torrejon KY, Papke EL, Halman JR, et al. (2016). Bioengineered glau- coma: an avoidable irreversible blindness. J Curr Glaucoma Pract 11: comatous 3D human trabecular meshwork as an in vitro disease 67–72. model. Biotechnol Bioeng 113:1357–68. Raghunathan VK, Morgan JT, Park SA, et al. (2015). Dexamethasone stif- Urcola JH, Hernandez M, Vecino E. (2006). Three experimental glaucoma fens trabecular meshwork, trabecular meshwork cells, and matrix. models in rats: comparison of the effects of intraocular pressure ele- Invest Ophthalmol Vis Sci 56:4447–59. vation on retinal ganglion cell size and death. Exp Eye Res 83:429–37. Ramirez AI, de Hoz R, Salobrar-Garcia E, et al. (2017). The role of micro- Vyas S, Rodrigues AJ, Silva JM, et al. (2016). Chronic stress and glucocor- glia in retinal neurodegeneration: Alzheimer’s disease, Parkinson, and ticoids: from neuronal plasticity to neurodegeneration. Neural Plast glaucoma. Front Aging Neurosci 9:1–21. 2016:6391686–15. 2446 M. J. RODRIGO ET AL. Wang K, Li G, Read AT, et al. (2018). The relationship between outflow Zeng W, Wang W, Wu S, et al. (2019). Mitochondria and autophagy dys- resistance and trabecular meshwork stiffness in mice. Sci Rep 8:5848. function in glucocorticoid-induced ocular hypertension/glaucoma Weinstein BI, Munnangi P, Gordon GG, Southren AL. (1985). Defects in mice model. Curr Eye Res 00:1–9. cortisol-metabolizing enzymes in primary open-angle glaucoma. Zhang Y, Yang A, Huang J. (2019). Identification of gene changes Invest Ophthalmol Vis Sci 26:890–3. induced by dexamethasone in the anterior segment of the human Whitlock NA, McKnight B, Corcoran KN, et al. (2010). Increased intraocu- eye using bioinformatics analysis. Med Sci Monit 25:5501–9. lar pressure in mice treated with dexamethasone. Invest Ophthalmol Zhao M, Rodrıguez-Villagra E, Kowalczuk L, et al. (2017). Tolerance of Vis Sci 51:6496–503. high and low amounts of PLGA microspheres loaded with mineralo- Xin C, Wang RK, Song S, et al. (2017). Aqueous outflow regulation: corticoid receptor antagonist in retinal target site. J Control Release Optical coherence tomography implicates pressure-dependent tissue 266:187–97. motion. Exp Eye Res 158:171–86. Zode GS, Sharma AB, Lin X, et al. (2014). Ocular-specific ER stress reduc- Zarei-Ghanavati S, Malaekeh-Nikouei B, Pourmazar R, Seyedi S. (2012). tion rescues glaucoma in murine glucocorticoid-induced glaucoma. J Preparation, characterization, and in vivo evaluation of triamcinolone Clin Invest 124:1956–65. acetonide microspheres after intravitreal administration. J Ocul Pharmacol Ther 28:502–6.