Induction Of Ocular Hypertension Research Articles

Effect of nadolol in treatment of induced ocular hypertension in rabbits

In glaucoma, as optic neuropathy gradually proceeds unnoticed by the patient, early detection and treatment is of paramount importance in arresting or controlling the progress of damage. To explore effects of topical nadolol on intraocular pressure (IOP) ocular hypertensive eyes of rabbits. A group of 36 males of the rabbits were included in this study. Induction of ocular hypertension was achieved by injection of hydroxy propyl methylcellulose in the anterior chamber of rabbits right eye. The present study was designed to evaluate the possible beneficial therapeutic effect. The included rabbits were divided into distilled water group, timolol (0.25% and 0.5%) groups, and nadolol (0.25% and 0.5%). Each of drug eye drops (including distilled water) were instilled into right eyes 3 times/day for 10 days therapeutically. The rabbits had been examined for the IOP, pupil diameter, light reflex, corneal reflex, and conjunctival redness prior to instillation of drugs and along the trial period. Results: Ocular hypotensive effects of nadolol (0.25%) and (0.5%) eye drops were more efficient than that of distilled water (P<0.01). Furthermore, nadolol eye drop was more efficient than timolol eye drop (0.01>P>0.05) in its ocular hypotensive effect in both concentrations along the trial period. In both parts of the present study and regarding each of mean pupil diameter, light reflex, corneal reflex and conjunctival redness, nadolol (0.25% or 0.5%) eye drops had no significant adverse effect (P > 0.05). Conclusions: Nadolol eye drops instilled 3 times / day had an obvious beneficial, safe, and tolerable therapeutic ocular hypotensive effects on hydroxy propyl methyl cellulose - induced ocular hypertension in rabbits.

Minocycline Administration Does Not Have an Effect on Retinal Ganglion Cell Survival in a Murine Model of Ocular Hypertension.

This study aims to investigate two key aspects in a mouse model of ocular hypertension (OHT): first, the time course of retinal ganglion cell (RGC) death and the parallel activation of caspase-3 (a-Casp3+ cells) to narrow the therapeutic window; and second, the effect of caspase-3 and microglia inhibition by minocycline on RGC rescue in this model. RGC loss after OHT induction was significant at day 7 and progressed to 30 days. However, anatomical RGC death was preceded by significant Casp3 activation on day 3. Microglial inhibition by minocycline did not alter the course of OHT or rescue RGCs but resulted in a decrease in a-Casp3+ cells and phagocytic and total microglia. Therefore, RGC death commitment occurs earlier than their loss of Brn3a expression, microglial cells do not exacerbate RGC loss, and while this death is primarily apoptotic, apoptosis inhibition does not rescue RGCs, suggesting that alternative death pathways play a role in glaucomatous injury.

Axonal Protection by Oral Nicotinamide Riboside Treatment with Upregulated AMPK Phosphorylation in a Rat Glaucomatous Degeneration Model.

Nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), has been studied to support human health against metabolic stress, cardiovascular disease, and neurodegenerative disease. In the present study, we investigated the effects of oral NR on axonal damage in a rat ocular hypertension model. Intraocular pressure (IOP) elevation was induced by laser irradiation and then the rats received oral NR of 1000 mg/kg/day daily. IOP elevation was seen 7, 14, and 21 days after laser irradiation compared with the controls. We confirmed that oral NR administration significantly increased NAD+ levels in the retina. After 3-week oral administration of NR, morphometric analysis of optic nerve cross-sections showed that the number of axons was protected compared with that in the untreated ocular hypertension group. Oral NR administration significantly prevented retinal ganglion cell (RGC) fiber loss in retinal flat mounts, as shown by neurofilament immunostaining. Immunoblotting samples from the optic nerves showed that oral NR administration augmented the phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) level in rats with and without ocular hypertension induction. Immunohistochemical analysis showed that some p-AMPK-immunopositive fibers were colocalized with neurofilament immunoreactivity in the control group, and oral NR administration enhanced p-AMPK immunopositivity. Our findings suggest that oral NR administration protects against glaucomatous RGC axonal degeneration with the possible upregulation of p-AMPK.

Mesenchymal stem cells differentiate to retinal ganglion-like cells in rat glaucoma model induced by polystyrene microspheres

AimThe study aimed to evaluate the differentiation ability of intravitreally injected rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to retinal ganglion-like cells in a polystyrene microsphere induced rat glaucoma model. Materials and MethodsThe glaucoma rat model was generated via intracameral injection of 7 microliter polystyrene microspheres. Green fluorescence protein-labeled (GFP) rBM-MSCs were transplanted intravitreally at or after induction of ocular hypertension (OHT), depending on the groups. By the end of the fourth week, flat-mount retinal dissection was performed, and labeled against Brn3a, CD90, GFAP, CD11b, Vimentin, and localization of GFP positive rBM-MSCs was used for evaluation through immunofluorescence staining and to count differentiated retinal cells by flow cytometry. From 34 male Wistar albino rats, 56 eyes were investigated. ResultsFlow cytometry revealed significantly increased CD90 and Brn3a positive cells in glaucoma induced and with rBM-MSC injected groups compared to control(P = 0.006 and P = 0.003 respectively), sham-operated (P = 0.007 and P < 0.001 respectively), and only rBM-MSCs injected groups (P = 0.002 and P = 0.009 respectively). Immunofluorescence microscopy revealed differentiation of GFP labeled stem cells to various retinal cells, including ganglion-like cells. rBM-MSCs were observable in ganglion cells, inner and outer nuclear retinal layers in rBM-MSCs injected eyes. ConclusionIntravitreally transplanted rBM-MSCs differentiated into retinal cells, including ganglion-like cells, which successfully created a glaucoma model damaged with polystyrene microspheres. Promisingly, MSCs may have a role in neuro-protection and neuro-regeneration treatment of glaucoma in the future.

Retinal glial activation in the contralateral eyes in a mouse model of unilateral laser‐induced glaucoma

AbstractGlaucoma is a neurodegenerative chronic pathology, characterized by the loss of retinal ganglion cells (RGC), which leads to an irreversible vision field loss. The glaucomatous optic neuropathy is usually a bilateral disease, though asymmetric in time: this means that the damage initially present in one of the eyes, eventually also appears with a time delay in the contralateral eye. Notably, recent experimental models of unilateral ocular hypertension (OHT) have shown that the fellow normotensive contralateral eyes to hypertensive eyes exhibit changes in glial cells with little or no damage to RGCs.It has been reported that although increased intraocular pressure (IOP) may be the main trigger for this pathology, other pathogenic mechanisms such as neuroinflammation are involved in the RGCs death. Microglial cells constitute the population of immune resident cells, and they play an important role in the physiology and survival of RGC.One of the most widely used experimental models of glaucoma is the unilateral laser‐induced OHT model. In these animals, OHT induces an alteration of retrograde axonal transport in RGCs and a subsequent degeneration of these cells. In this model, at different time‐points after OHT induction (1, 3, 5, 8 and 15 days), the microglia exhibited different signs of activation, such as proliferation and migration to areas of damage, retraction, reorientation, and hyper‐ramification of their processes, soma thickening and the appearance of amoeboid‐ and rod‐like microglia with macrophagic capacity, related to RGC degeneration. All these changes were most intense at 3–5 days after OHT induction in the OHT eye. This was confirmed by the decreased expression of the purinergic receptor P2RY12, which would indicate higher microglial activation.In this model, at all time‐points after OHT induction, the activation of the macroglia (astrocytes and Müller cells) has also been demonstrated, showing morphological changes, as well as an overexpression of glial fibrillary acidic protein (GFAP).In this laser‐induced OHT model, an MHC‐II overexpression was observed in the microglia, astrocytes, and Müller cells, allowing these cells to induce a T‐cell mediated immune response.Significant changes in the expression of proinflammatory (IL‐1β, IL‐6, INF‐γ, IL‐17), anti‐inflammatory (IL‐4, IL‐10) and myokines (Fractalkine, BDNF, VEGF) cytokines were also observed.The same changes in glial cells (microglia, astrocytes and Müller cells) as well as in the expression of different cytokines and myokines were observed in the contralateral normotensive eyes at the different time points after OHT induction although with a lower intensity.Based on the alterations found in the contralateral eye, it can be concluded that this eye should not be used as a control eye in experimental models of unilateral glaucoma. Changes in contralateral normotensive eyes could exhibit the initial events of glaucomatous neurodegeneration, probably mediated by inflammatory mechanisms. Therefore, studies focusing on contralateral eyes could improve our understanding of glaucomatous pathophysiology. The study of the contralateral eye appears to have the potential for discovering points of intervention to which future neuroprotective therapeutics can be directed.

Administration of minocycline reduces microglia and Caspase‐3 activation but does not mitigate retinal ganglion cell loss after ocular hypertension in mice

AbstractPurpose: To study the effect of minocycline administration on the evolution of retinal ganglion cell (RGC) loss, the number of microglial cells and the activation of the apoptotic signal Caspase‐3 after ocular hypertension (OHT) in mice.Methods: In left eyes of Swiss albino mice, OHT was induced by diode laser photocoagulation of the limbal and episcleral veins and the intraocular pressure (IOP) evolution was monitored with a TonoLab®. Mice received daily intraperitoneal injections of minocycline hydrochloride (45 mg/kg) diluted in saline starting the day before OHT induction until the day of sacrifice (4 or 15 days, n = 12–14 per group/time point). Control groups received saline injections. Retinas were divided into two groups. Retinas of the first group were double immunodetected against Brn3a and active Caspase‐3 to identify surviving (Brn3a+RGCs) and apoptotic (a‐Casp3+RGCs) RGCs. In the second group, RGCs were retrogradely traced with Fluorogold 7 days before OHT induction and retinas were immunodetected against Iba‐1. Total Brn3a+RGCs, a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells in the RGC layer were manually or automatically quantified, and the distributions were analysed by topographical maps.Results: IOP was significantly elevated 24 hours after OHT induction (33.2 ± 6.1 mmHg) and returned to baseline at 1 week (17.3 ± 5.3 mmHg). This evolution was not altered by minocycline administration. Although minocycline treatment was not successful in protecting Brn3a+RGCs at 4 (40 878 ± 4860 vs 43 526 ± 2714) nor at 15 (16 161 ± 12 650 vs 12 919 ± 6085) days after OHT, the numbers of a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells were significantly lower in the minocycline‐treated groups at both 4 (58.6%, 46.8% and 24.5%, respectively) and 15 (33.6%, 54.2% and 37.3%, respectively) days compared to the saline‐treated groups (100%).Conclusions: Minocycline treatment decreases the number of microglial cells and caspase‐3 activation but does not reduce RGC loss after OHT.

Macroglial activation over time in an experimental glaucoma model

AbstractGlaucoma is a neurodegenerative retinal disease characterized by irreversible loss of retinal ganglion cells (RGCs), leading to visual loss. Increased intraocular pressure (IOP) is the only risk factor for which treatment is available. However, other factors such as glial activation and dysfunction can also induce RGC death in glaucoma.Astrocytes and Müller glia (macroglial cells) play important roles in neuronal activity by providing physical and metabolic support to neurons. In glaucoma, when tissue damage occurs, macroglial cells undergo reactive gliosis, to defend the nerve tissue against damage and attempt to maintain its homeostasis. However, alterations in the function of these cells could cause damage and even neuronal death. In reactive macrogliosis, cells undergo complex biochemical and functional remodelling and exhibit morphological changes characterized by cell body thickening, increased number and length of cell processes, increased cell number and up‐regulation of cytoskeletal components such as gliofibrillary acidic protein (GFAP), which is considered one of the main markers of this process and may act as antigen‐presenting cells by expressing major histocompatibility complex class II (MHC‐II). Reactivation of macroglia can initially be beneficial, as it increases their metabolic activity, increases the expression of antioxidant defence, and restores ion, water and neurotransmitter balance. However, if macroglia become chronic, it is detrimental by directly or indirectly damaging the tissue and preventing its repair. Astrocytes may act in concert with microglia during the inflammatory process, so inflammatory mediators produced by astrocytes can chronically activate microglial cells, contributing to neuronal death, and similarly, inflammatory mediators released by microglia can chronically activate astrocytes.One of the most widely used models to elevate IOP is the unilateral laser‐induced ocular hypertension (OHT) model. Studies in this model have analysed the temporal pattern of RGC death and microglial cell activation at time points: 1, 3, 5, 8 and 15 days. Molecular changes in proinflammatory and anti‐inflammatory cytokines involved in the neuroinflammatory process, released by glial cells (microglia, astrocytes, and Müller cells) at the same time points mentioned above, have also been identified.Given the close relationship between the activation of macroglia and microglia and their involvement in the death of RGCs in this pathology, the present work analysed how the activation of retinal macroglial cells occurred at different time points (1, 3, 5, 8 and 15 days) after the induction of OHT in an experimental model of laser‐induced OHT in mice. For this purpose, we have analysed the GFAP‐labelled retinal area, the intensity of GFAP labelling, and the expression of MHC‐II in both OHT eyes and normotensive contralateral eyes compared to naïve eyes.In this study, in both OHT and contralateral eyes, macroglial cells showed morphological changes as well as in GFAP and MHC‐II expression at all time points analysed, being slightly more intense at 3–5 days, coinciding with the pattern of microglial activation previously studied, demonstrating this fact, a bilateral inflammatory process maintained over time in which microglia, astrocytes and Müller glia are involved.

The application of lentiviral vectors for the establishment of TGFβ2-induced ocular hypertension in C57BL/6J mice

Elevated levels of TGFβ2 in the aqueous humor is associated with the pathological changes in the trabecular meshwork (TM). These changes lead to ocular hypertension (OHT), the most important risk factor for the development and progression of primary open angle glaucoma (POAG), a leading cause of blindness worldwide. Therefore, TGFβ2 is frequently used to develop OHT models including in perfusion cultured eyes and in mouse eyes. Adenovirus-mediated overexpression of human mutant TGFβ2 has demonstrated great success in increasing intraocular pressure (IOP) in mouse eyes. However, adenoviruses have limited capacity for a foreign gene, induce transient expression, and may cause ocular inflammation. Here, we explored the potential of using lentiviral vectors carrying the mutant human TGFβ2C226S/C228S (ΔhTGFβ2C226S/C228S) gene expression cassette for the induction of OHT in C57BL/6J mice. Lentiviral vectors using CMV or EF1α promoter to drive the expression of ΔhTGFβ2C226S/C228S were injected into one of the mouse eyes and the fellow eye was injected with the same vector but expressing GFP/mCherry as controls. Both intravitreal and intracameral injection routes were tested in male and female mice. We did not observe significant IOP changes using either promoter or injection route at the dose of 8 × 105 PFU/eye. Immunostaining showed normal anterior chamber angle structures and a slight increase in TGFβ2 expression in the TM of the eyes receiving intracameral viral injection but not in those receiving intravitreal viral injection. At the dose of 2 × 106 PFU/eye, intracameral injection of the lentiviral vector with the CMV-ΔhTGFβ2C226S/C228S cassette induced significant IOP elevation and increased the expression of TGFβ2 and fibronectin isoform EDA in the TM. Our data suggest that lentiviral doses are important for establishing the TGFβ2-induced OHT model in the C57BL/6J strain.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 μm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13–15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Retinal Molecular Changes Are Associated with Neuroinflammation and Loss of RGCs in an Experimental Model of Glaucoma.

Signaling mediated by cytokines and chemokines is involved in glaucoma-associated neuroinflammation and in the damage of retinal ganglion cells (RGCs). Using multiplexed immunoassay and immunohistochemical techniques in a glaucoma mouse model at different time points after ocular hypertension (OHT), we analyzed (i) the expression of pro-inflammatory cytokines, anti-inflammatory cytokines, BDNF, VEGF, and fractalkine; and (ii) the number of Brn3a+ RGCs. In OHT eyes, there was an upregulation of (i) IFN-γ at days 3, 5, and 15; (ii) IL-4 at days 1, 3, 5, and 7 and IL-10 at days 3 and 5 (coinciding with downregulation of IL1-β at days 1, 5, and 7); (iii) IL-6 at days 1, 3, and 5; (iv) fractalkine and VEGF at day 1; and (v) BDNF at days 1, 3, 7, and 15. In contralateral eyes, there were (i) an upregulation of IL-1β at days 1 and 3 and a downregulation at day 7, coinciding with the downregulation of IL4 at days 3 and 5 and the upregulation at day 7; (ii) an upregulation of IL-6 at days 1, 5, and 7 and a downregulation at 15 days; (iii) an upregulation of IL-10 at days 3 and 7; and (iv) an upregulation of IL-17 at day 15. In OHT eyes, there was a reduction in the Brn3a+ RGCs number at days 3, 5, 7, and 15. OHT changes cytokine levels in both OHT and contralateral eyes at different time points after OHT induction, confirming the immune system involvement in glaucomatous neurodegeneration.

Biomechanics of the optic nerve head and peripapillary sclera in a mouse model of glaucoma.

The deformation of the mouse astrocytic lamina (AL) and adjacent peripapillary sclera (PPS) was measured in response to elevated intraocular pressure. We subjected explanted mouse eyes to inflation testing, comparing control eyes to those 3 days and 6 weeks after induction of ocular hypertension (OHT) via ocular microbead injection. Laser scanning microscopy was used with second harmonic generation to image the collagenous PPS and two-photon fluorescence to image transgenic fluorescent astrocytes in the AL. Digital volume correlation was applied to calculate strains in the PPS and AL. The specimen-averaged strains were biaxial in the AL and PPS, with greater strain overall in the x- than y-direction in the AL and greater strain in the θ- than the r-direction in the PPS. Strains increased after 3-day OHT, with greater strain overall in the 3-day AL than control AL, and greater circumferential strain in the 3-day PPS than control PPS. In the 6-week OHT eyes, AL and PPS strains were similar overall to controls. This experimental glaucoma model demonstrated a dynamic change in the mechanical behaviour of the AL and PPS over time at the site of neuronal injury and remodelling in glaucoma.

Using retinal function to define ischemic exclusion criteria for animal models of glaucoma

Most animal models of glaucoma rely on induction of ocular hypertension (OHT), yet such models can suffer from high IOPs leading to undesirable retinal ischemia. Thus, animals with IOPs exceeding a threshold (e.g. > 60 mmHg) are often excluded from studies. However, due to the intermittent nature of IOP measurements, this approach may fail to detect ischemia. Conversely, it may also inappropriately eliminate animals with IOP spikes that do not induce ischemic damage. It is known that acute ischemia selectively impairs inner retinal function, which results in a reduced b-wave amplitude. Here, we explore the potential of using electroretinography (ERG) to detect ischemic damage in OHT eyes. 74 Brown Norway rats received a unilateral injection of magnetic microbeads to induce OHT, while contralateral eyes served as controls. IOP was measured every 2–3 days for 14 days after microbead injection. Retinal function was evaluated using dark-adapted bright flash ERG (2.1 log cd•s/m2) prior to, and at 7 and 14 days after, injection. We investigated two criteria for excluding animals: (IOP Criterion) a single IOP measurement > 60 mmHg; or (ERG Criterion) a b-wave amplitude below the 99.5% confidence interval for naïve eyes. 49 of 74 rats passed both criteria, 7 of 74 failed both, and 18 passed one criterion but not the other. We suggest that ERG testing can detect unwelcome ischemic damage in animal models of OHT. Since brief IOP spikes do not necessarily lead to ischemic retinal damage, and because extended periods of elevated IOP can be missed, such ERG-based criteria may provide more objective and robust exclusion criteria in future glaucoma studies.

Release of cytokines/myokines by retinal tissue at different times in a mouse model of glaucoma

AbstractDiverse cytokines released by retinal glial cells (IL‐1β, IL‐4, IL‐6, IL‐10, IL‐17, IFN‐, BDNF, VEGF and Fractalkine) were analyzed in order to determine the changes in the expression, at different times after unilateral laser‐induced ocular hypertension (OHT), both in OHT eyes and their contralateral eyes respect to naïve eyes.At each of time point after OHT induction (days 1, 3, 5, 7 and 15), 12 OHT retinas, 12 contralateral retinas and 12 naïve retinas (lasered group n = 60, naïve n = 12) from Albino Swiss mice were analyzed. The samples obtained were processed and multiarray kits (MILLIPLEX MAP Mouse Cytokine/Myokine Magnetic Bead Panel) were used to quantify the expression of cytokines.In OHT eyes, the expression of the following cytokines: BDNF (1, 3, 7 and 15 days), VEGF (1 day), IL‐4 (at all‐time points except day 15), IL‐6 (1, 3 and 5 days), IFN‐ (3, 5 and 15 days), IL‐10 (3 and 5 days), and Fractalkine (1 day) were significant higher than in naïve. In contralateral eyes, the following cytokines: IL‐6 (1, 5 and 7 days), IL‐1β (1 and 3 days), IL‐4 (7 and 15 days), IL‐10 (7 days) and IL‐17 (15 days) showed a significant increase in comparison with naïveAfter unilateral laser‐induced ocular hypertension, OHT eyes and contralateral eyes showed changes in the expression of the anti‐ and pro‐inflammatory cytokines associated with inflammatory process, mainly 3 days after OHT induction. These factors, which may be released by activated macroglia and microglia, may exert neuroprotective and neurodegenerative effects.This work was supported by Ophthalmological Network OFTARED (RD12‐0034/0002 and RD12/0034/0014. Prevención, Detección Precoz y Tratamiento de la Patología Ocular Prevalente Degenerativa y Crónica), the Institute of Health of Carlos III of the Spanish Ministry of Economy; PN I+D+i 2008‐2011; ISCIII, General Subdirection of Networks and Cooperative Research Centers; by the FEDER European program. Grants to J.A. Fernández‐Albarral are currently supported by a Predoctoral Fellowship (FPU) from the Spanish Ministry of Science, Innovation and University.

Posttreatment Intervention With Lycium Barbarum Polysaccharides is Neuroprotective in a Rat Model of Chronic Ocular Hypertension.

To investigate the neuroprotective effects of Lycium barbarum polysaccharides (LBP) against chronic ocular hypertension (OHT) in rats and to consider if effects differed when treatment was applied before (pretreatment) or during (posttreatment) chronic IOP elevation. Sprague-Dawley rats (10-weeks old) underwent suture implantation around the limbus for 15 weeks (OHT) or 1 day (sham). Four experimental groups were studied, three OHT groups (n = 8 each) treated either with vehicle (PBS), LBP pretreatment or posttreatment, and a sham control (n = 5) received no treatment. LBP (1 mg/kg) pre- and posttreatment were commenced at 1 week before and 4 weeks after OHT induction, respectively. Treatments continued up through week 15. IOP was monitored twice weekly for 15 weeks. Optical coherence tomography and ERG were measured at baseline, week 4, 8, 12, and 15. Eyes were collected for ganglion cell layer (GCL) histologic analysis at week 15. Suture implantation successfully induced approximately 50% IOP elevation and the cumulative IOP was similar between the three OHT groups. When compared with vehicle control (week 4: -23 ± 5%, P = 0.03), LBP pretreatment delayed the onset of retinal nerve fiber layer (RNFL) thinning (week 4, 8: -2 ± 7%, -11 ± 3%, P > 0.05) and arrested further reduction up through week 15 (-10 ± 4%, P > 0.05). LBP posttreatment intervention showed no significant change in rate of loss (week 4, 15: -25 ± 4.1%, -28 ± 3%). However, both LBP treatments preserved the retinal ganglion cells (RGC) and retinal functions up to week 15, which were significantly reduced in vehicle control. LBP posttreatment arrested the subsequent neuronal degeneration after treatment commencement and preserved RGC density and retinal functions in a chronic OHT model, which was comparable with pretreatment outcomes.

The effects of intravitreal H2 S application on apoptosis in the retina and cornea in experimental glaucoma model.

One of themost important causes of visual loss (blindness) is glaucoma, which occurs due to the degeneration of the ganglion cells in retina. It has been shown that hydrogen sulphide (H2 S) acts an antioxidant, neuroprotective and neuromodulator and provides protection against oxidative stress and apoptosis. This study aims to examine through which apoptotic pathway H2 S acts in experimental glaucoma model. Twenty-two male wistar albino rats were used in this study. Group 1 (n=6, control group): Intravitreal saline was given in the third week without inducing ocular hypertension (OHT) with laser photocoagulation. Group 2 (n=8): After the induction of OHT with laser photocoagulation, intravitreal saline was given in the third week. Group 3 (n=8): After the induction of OHT with laser photocoagulation, intravitreal H2 S's donor sodium hydrosulphide (NaSH) 100nmol/L was given in the third week. At the end of the 6th week, the eyes of the rats were sacrified under anaesthesia and extracted and then routine tissue follow-up was undertaken. Besides haematoxylin & eosin (H&E) staining, Bax, Bcl-2, p53 and caspase-3 activation were examined immunohistochemically in the retina and the cornea. This showed that ocular hypertension caused apoptosis through the intrinsic pathway, due to Bax and caspase-3 activation, in both retina and cornea, and that this led to DNA damage due to p53 activation. Also, we found that H2 S exposure in glaucoma distinctly suppressed Bax, caspase-3 and p53 activations in retina but that it has a limited effect on the cornea. According to these results, glaucoma caused apoptosis in the retina through intrinsic pathway, and the damage to the retina could be compensated partially by H2 S but would have limited on the cornea.

Menopause exacerbates visual dysfunction in experimental glaucoma

Glaucoma is the leading cause of irreversible blindness worldwide. Recently, estrogen deficiencies caused by early menopause, alterations in estrogen signaling via mutations in estrogen receptors, and polymorphisms along estrogen metabolic pathways have all been linked to an increased risk of developing glaucoma. Here, we examined how menopause and age impact visual function and retinal structure in an experimental model of glaucoma. Young (3–4 months) and aged (9–10 months) female Brown Norway rats were divided into pre- and post-menopausal cohorts by surgically inducing menopause via ovariectomy (OVX). After six weeks, ocular hypertension (OHT) was induced unilaterally for a period of eight weeks. Four cohorts were successfully followed to eight weeks: young sham (n = 8), young OVX (n = 9), aged sham (n = 10), and aged OVX (n = 11) animals. Intraocular pressure (IOP) was monitored weekly in all groups. Prior to inducing OHT (baseline) and at four and eight weeks after inducing OHT, we assessed visual acuity via the optomotor response (OMR) and retinal structure using optical coherence tomography (OCT). OHT decreased the OMR in all cohorts. We found that spatial frequency thresholds decreased by 54% in OVX animals after OHT compared to sham animals after OHT, regardless of age (p < 0.001). We also found thinning of the retinal nerve fiber layer (RNFL) and loss of total retinal thickness after induction of OHT. Aged animals had more thinning of the RNFL and loss of total retinal thickness compared to young animals (p < 0.001). Overall, OHT caused significant changes in visual function and retinal structure. Observing that OVX in young and aged animals further decreased spatial frequency thresholds after OHT suggests that an estrogen deficiency may intensify visual impairment after OHT.

Role of Histamine H₃ Receptor Antagonists on Intraocular Pressure Reduction in Rabbit Models of Transient Ocular Hypertension and Glaucoma.

Intraocular pressure (IOP) has a tendency to fluctuate throughout the day, reaching its peak in the early morning in healthy subjects or glaucoma patients. Likewise, histamine tone also fluctuates over time, being lower at nighttime. Numerous studies have demonstrated a correlation between short-term IOP fluctuation and glaucoma progression; however, it has not yet been determined whether histamine plays a role in IOP fluctuations. The aim of this research was to establish the distribution of the histamine receptor proteins and respective mRNAs in the eye by western blot, immunohistochemistry and RT-PCR in New Zealand rabbits. Furthermore, we used a transient ocular hypertension (OHT) model induced by injection of 50 µL of 5% hypertonic saline into the vitreous and a stable OHT model (100 µL 0.1% carbomer in the anterior chamber) to address the potential IOP-lowering ability of H3 receptor (H3R) antagonists (ciproxifan, DL76 and GSK189254). IOPs were performed with a Tono-Pen at baseline and 60, 120 and 240 min post treatment after transient OHT induction and, every day for 12 days in the stable OHT model. All histamine receptor subtypes were localized in the rabbit retina and ciliary body/trabecular meshwork. All the treatments lowered IOP in a dose-dependent fashion between 0.3% and 1%. More specifically, the effects were maximal with ciproxifan at 60 min post-dose (IOP60 change = −18.84 ± 4.85 mmHg, at 1%), remained stable until 120 min (IOP120 change = −16.38 ± 3.8 mmHg, at 1%) and decayed thereafter to reach baseline values at 240 min. These effects were highly specific and dependent on histamine release as pre-treatment with imetit (H3R agonist, 1%) or pyrilamine (H1R antagonist, 1%) largely blocked ciproxifan-mediated effects. Color Doppler ultrasound examination was performed to evaluate changes in ophtalmic artery resistivity index (RI) before and after repeated dosing with DL 76, GSK189254, ciproxifan and timolol. Chronic treatments with H3R antagonists and timolol improved the vascular performance of ophthalmic arteries and reduced retinal ganglion cell death. Oxidative stress was also reduced and measured 8-Hydroxy-2′-deoxyguanosine (8OHdG) expression, and by dihidroethydium (DHE) staining. These results demonstrated that the histamine system participates in IOP regulation and that H3R antagonists could represent a future promising therapy for glaucoma. Further studies should be focused on the long-term IOP circadian fluctuations.

Simultaneous co-delivery of neuroprotective drugs from multi-loaded PLGA microspheres for the treatment of glaucoma

Glaucoma is a multifactorial neurodegenerative disorder and one of the leading causes of irreversible blindness globally and for which intraocular pressure is the only modifiable risk factor. Although neuroprotective therapies have been suggested to have therapeutic potential, drug delivery for the treatment of ocular disorders such as glaucoma remains an unmet clinical need, further complicated by poor patient compliance with topically applied treatments. In the present study we describe the development of multi-loaded PLGA-microspheres (MSs) incorporating three recognised neuroprotective agents (dexamethasone (DX), melatonin (MEL) and coenzyme Q10 (CoQ10)) in a single formulation (DMQ-MSs) to create a novel sustained-release intraocular drug delivery system (IODDS) for the treatment of glaucoma. MSs were spherical, with a mean particle size of 29.04 ± 1.89 μm rendering them suitable for intravitreal injection using conventional 25G–32G needles. >62% incorporation efficiency was achieved for the three drug cargo and MSs were able to co-deliver the encapsulated active compounds in a sustained manner over 30-days with low burst release. In vitro studies showed DMQ-MSs to be neuroprotective in a glutamate-induced cytotoxicity model (IC50 10.00 ± 0.94 mM versus 6.89 ± 0.82 mM in absence of DMQ-MSs) in R28 cell line. In vivo efficacy studies were performed using a well-established rodent model of chronic ocular hypertension (OHT), comparing single intravitreal injections of microspheres of DMQ-MSs to their equivalent individual single-drug loaded MSs mixture (MSsmix), empty MSs, no-treatment OHT only and naïve groups. Twenty one days after OHT induction, DMQ-MSs showed a significantly neuroprotective effect on RGCs compared to OHT only controls. No such protective effect was observed in empty MSs and single-drug MSs treated groups. This work suggests that multi-loaded PLGA MSs present a novel therapeutic approach in the management of retinal neurodegeneration conditions such as glaucoma.

Bilateral early activation of retinal microglial cells in a mouse model of unilateral laser-induced experimental ocular hypertension

The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system.

Macrogilal retinal cells show a bilateral early activation in a mouse model of unilateral laser‐induced experimental glaucoma

PurposeTo describe macroglial morphological features of activation after 24 h of unilateral laser‐induced ocular hypertension (OHT) in OHT‐eyes and their contralateral eyes.MethodsAlbino Swiss mice were divided into two groups, naïve (n = 6) and lasered (n = 6). Retinal whole‐mounts were immunolabeled with antibodies against GFAP and MHC‐II.ResultsWith respect to naïve, OHT eyes and contralateral eyes showed different features of macroglial activation in some retinal areas: i) Müller cells were reactive and expressed GFAP; ii) astrocytes were more robust and had more primary and secondary processes; and iii) MHCII was up‐regulated in astrocytes and in some Müller cells. In contralateral eyes, these signs were observed mainly in the periphery. In addition, in the OHT eyes: i) in some retinal vessels near the optic disc, the astroglial reactivity was so intense that the entire vascular walls were sheathed by astroglial cells; ii) in some areas of the retina, astrocytes and Müller cells formed glial scars. iii) MHC‐II immunoreaction in astrocytes varied depending on the retinal area; in those in which MHC‐II immunostaining was less intense, only astrocyte primary processes could be recognized;ConclusionsAt 24 h after unilateral OHT, macroglia showed features of activation not only in OHT eyes, but also in untreated contralateral eyes. Early up‐regulation of MHC‐II in macroglial cells in both OHT retinas and contralateral retinas, suggest an immune system‐related inflammation early after the induction of ocular hypertension.