Dual-functional nanoparticle formulations for simultaneous intraocular pressure reduction and neuroprotection in glaucoma: a review.

To investigate the effects of high infusion pressure in conjunction with pars plana vitrectomy (PPV) on retinal morphology and function in rabbits. Pars plana vitrectomy was performed under urethane (0.8 mg/kg) anaesthesia in the right eye of albino rabbits following phacoemulsification and aspiration (PEA). The left eyes were not touched. After PEA, the animals were divided into two groups. In six eyes, intraocular pressure (IOP) was increased to 80 mmHg for 30 mins (high-pressure group) and in five eyes IOP was maintained at 40 mmHg for 30 mins (low-pressure group). The IOPs were regulated by the height of the bottle of balanced salt solution (BSS) and monitored with a pressure transducer. After the pressure elevation, vitreous fluid was collected to measure the glutamate concentration. Then, PPV was performed for 15 mins in both groups under an infusion pressure of 40 mmHg. In five additional rabbits, PEA alone was performed in the right eye, and vitreous fluid was collected (PEA group). Functional alterations were assessed by recording visual evoked potentials (VEPs) and electroretinograms (ERGs). Ten days after the IOP changes, the animals were killed with intravenous pentobarbital sodium and the eyes were prepared for histological analysis. Damage to retinal ganglion cells (RGCs) was quantified by counting the number of cells in the ganglion cell layer (GCL). The contralateral eyes in the high-pressure group served as controls (n = 6). The mean implicit time (IT) of the VEPs in the high-pressure group was significantly longer than that before the IOP elevation, by 114-124% (p < 0.05, paired t-test), and also than that of control eyes (p < 0.05, anova followed by t-test). No significant changes in the VEPs were detected in either the low-pressure group or the PEA group. There were significantly fewer cells in the GCL in the high-pressure group (24.7/mm) than in the control animals (41.4/mm; p < 0.05, Dunnett's test). The number of cells in the GCL in the low-pressure and PEA groups did not significantly differ to that in the controls. The amplitudes of the ERG a- and b-waves were not significantly changed (p > 0.05, paired t-test). These results suggest that high infusion pressure in conjunction with PPV leads to morphological and functional changes in the retina. The absence of ERG changes and presence of VEP changes suggest that these changes were due to damage to RGCs, which supports the morphological observations.

The alternative complement pathway drives neuroinflammation and neurodegeneration in mouse models of glaucoma and optic nerve injury.

BackgroundGlaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP.MethodsC57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT.ResultsProlonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss).ConclusionsThese findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.

BackgroundPrimary open angle glaucoma is a heterogeneous group of optic neuropathies that results in optic nerve degeneration and a loss of retinal ganglion cells (RGCs) ultimately causing blindness if allowed to progress. Elevation of intraocular pressure (IOP) is the most attributable risk factor for developing glaucoma and lowering of IOP is currently the only available therapy. However, despite lowering IOP, neurodegenerative effects persist in some patients. Hence, it would be beneficial to develop approaches to promote neuroprotection of RGCs in addition to IOP lowering therapies. The endothelin system is a key target for intervention against glaucomatous neurodegeneration. The endothelin family of peptides and receptors, particularly endothelin-1 (ET-1) and endothelin B (ETB) receptor, has been shown to have neurodegenerative roles in glaucoma. The purpose of this study was to examine changes in endothelin A (ETA) receptor protein expression in the retinas of adult male Brown Norway rats following IOP elevation by the Morrison’s model of ocular hypertension and the impact of ETA receptor overexpression on RGC viability in vitro.ResultsIOP elevation was carried out in one eye of Brown Norway rats by injection of hypertonic saline through episcleral veins. After 2 weeks of IOP elevation, immunohistochemical analysis of retinal sections from rat eyes showed an increasing trend in immunostaining for ETA receptors in multiple retinal layers including the inner plexiform layer, ganglion cell layer and outer plexiform layer. Following 4 weeks of IOP elevation, a significant increase in immunostaining for ETA receptor expression was found in the retina, primarily in the inner plexiform layer and ganglion cells. A modest increase in staining for ETA receptors was also found in the outer plexiform layer in the retina of rats with IOP elevation. Cell culture studies showed that overexpression of ETA receptors in 661W cells as well as primary RGCs decreases cell viability, compared to empty vector transfected cells. Adeno-associated virus mediated overexpression of the ETA receptor produced an increase in the ETB receptor in primary RGCs.ConclusionsElevated IOP results in an appreciable change in ETA receptor expression in the retina. Overexpression of the ETA receptor results in an overall decrease in cell viability, accompanied by an increase in ETB receptor levels, suggesting the involvement of both ETA and ETB receptors in mediating cell death. These findings raise possibilities for the development of ETA/ETB dual receptor antagonists as neuroprotective treatments for glaucomatous neuropathy.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid in vivo crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.

More than 5,000 abstracts were presented at the 2001 Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, Fort Lauderdale, FL, April 29–May 4, 2001 (Invest Ophthalmol Vis Sci 2001;42 (4). We highlight some of the investigations of interest to neuro-ophthalmologists. Abstracts are referenced by number (#), sections, and symposia by the letter S. Apoptosis and regeneration The week began with two symposia, one devoted to molecular mechanisms of apoptosis and cytoprotection and the other to tissue injury, repair, and regeneration. The first symposium was dedicated to Richard Lolley, PhD, former Dean of Research at the USC School of Medicine, and an important contributor to the field of the neurobiology of inherited degenerative disorders who died last year. The symposium reviewed how mouse mitochondrial mutant strains owing to a mitochondrial genome knock-out are used to investigate mitochondrial dysfunction. The pathophysiology of these disorders goes beyond that of energy deficiency to involve problems with reactive oxygen species and the early initiation of apoptosis. In the second symposium, investigators described how cultured neural stem cells are implanted in the mature nervous system to facilitate regeneration. The implantation of these stem cells leads to interactions whereby the host tissues modify the stem cells. Stem cells cause release of factors and alteration of the mature nervous system (S1). Stem cells release factors that alter the environment and interaction with other stems cells and mature neurons. Cataract and color vision Nuclear opacification of the crystalline lens is correlated with color vision loss owing to selective wavelength absorption (S7). Axonal imaging Imaging of the optic nerve and the peripapillary nerve fiber layer has improved with technical advances but is still limited in the precise longitudinal analysis of axonal loss. Media clarity, corneal birefringence, an intraocular lens, and ocular movement are confounding factors (S15–S19). The GDx nerve fiber analyzer may not recognize nerve fiber layer swelling when caused by ischemia (#110). Eye movements The neural control of eye movements was reviewed in a mini-symposium (S111). The superior colliculus appears to be important in providing a motor map for sequential saccades. Target distance modulates the vestibuloocular reflex. Three short-latency mechanisms normally compensate for subject movement to maintain fixation before the subject is aware of a disturbance in vision. Neuroprotection Aspects of the molecular regulation of nitric oxide synthase 2 expression in astrocytes in the optic nerve head were demonstrated and associated with a number of cellular signals and cytokines (#128). In a rat model of retinal ischemia (high intraocular pressure), systemic administration of brimonidine was more effective than topical administration in mitigating the extent of retinal ganglion cell (RGC) drop out and electroretinogram (ERG) abnormalities (#130). However, as in previous models of central nervous system ischemia, a beneficial effect required that brimonidine be administered at least 1 hour before onset of ischemia. The administration of brain-derived neurotrophic factor could mitigate RGC death after crush injury to the optic nerve in rats (#132). A similar experiment demonstrated the utility of the trophic factor CTNF (#134). The glial-derived neurotrophic factors (GDNF) and glomerular filtration rate (GFRalpha-1), as well as neurturin and brain-derived neurotrophic factor (BDNF), have receptors located on RGCs; these receptors become apparent after axotomy in the rat (#138). Because these are separate receptors, their trophic effects may be additive. A different type of study (#141) demonstrated the neuroprotective effects of memantine in two types of rat models of RGC injury: optic nerve crush injury and RGC ischemic injury through induced intraocular hypertension. Multifocal ERG Two months after optic nerve section, the multifocal ERG is unchanged from controls, suggesting RGCs make little or no contribution to this signal (#786). Frequency doubling perimetry The danger of accepting new technology uncritically was highlighted in a study showing that frequency doubling time (FDT) perimetry was poor at detecting the visual field defects of brain lesions, especially in the posterior visual pathways (#807). FDT detected fewer than 50% of the quadrantanopic defects detected by Humphrey 24-2 testing. The sensitivity of FDT perimetry can be improved by offsetting the stimulus by 3 degrees from the vertical meridian (#820). Retinal ischemia In mice developed with N-methyl d-aspartate (NMDA) receptor subunit knock-out, experimentally induced transient ischemia caused significantly less RGC death than it did in controls, which suggests excitatory amino acid–related injury acts through these receptors (#1055). In a patient with anticardiolipin antibodies, a central retinal vein occlusion developed despite the patient's being already treated with warfarin (#1270). Retrograde axonal flow can be demonstrated by careful ophthalmoscopy in retinal artery occlusion. In these cases, swelling and opacification similar to cotton wool spots can be seen. This phenomenon probably accounts for some of the swelling of the optic disk seen with central retinal artery occlusion (#1281). An intravenous treatment of 50 mg rt-PA followed by eight days of heparin treatment improved the vision in three patients with macula-involving branch artery occlusion of less than 12 hours' duration (#1296). Gene therapy A mini-symposium discussed progress in gene therapy and the issues facing future development (S302). In a canine model of Leber's congenital amaurosis, the known deleted gene was transfected into three animals using a methodology that not only got the gene into the retinal cells, but altered the promotor region of the existing genes. A functional gene resulted, as evidenced improvement of behavioral vision and the ERG, and a recordable visual evoked potential and pupillary response to light were demonstrated at 2.5 months and maintained for at least nine months (#1624). Retinal ganglion cells A mini-symposium on RGCs discussed how glial cells, which provide the normal supportive environment for neurons, can become a destructive influence when reactive astrocytes evolve (S305). The Müller glial cell is particularly important in maintaining RGC function. The notion of selective loss of parvocellular RGCs in early glaucoma was demonstrated to be erroneous. Congenital nystagmus Increased foveation time followed horizontal rectus tenotomy in 10 adults with congenital nystagmus. Binocular visual acuity improved by five letters or more in three subjects (#1720). Leber's hereditary optic neuropathy In a Leber's hereditary optic neuropathy (LHON)/11778 pedigree with 80 members and six generations, environmental factors—particularly tobacco, alcohol, and toxic exposures—increased the disease expression (#1754). Furthermore, this susceptibility to environmental factors was increased in the subset with haplo group J. More than two thirds of 44 patients visually impaired by ethambutol toxicity showed dramatic recovery, but recovery was less likely in older patients (#1755). Intracranial hypertension Immunofluorescent antibody studies revealed somatostatin receptors on arachnoid granulation and choroid plexus cells. These receptors might play a role in the iatrogenically induced increased intracranial pressure after growth hormone therapy (#1762). Giant cell arteritis In a series of patients with giant cell arteritis treated with corticosteroids, reduction of the prednisone dose to 40 mg daily by the fourth week of therapy had a low a rate of drug complications and only one recurrence of giant cell arteritis symptoms. In this single instance, increasing the prednisone dose reversed the symptoms (#1763). Goldfish axons In actively growing goldfish optic nerve axons, mitochondria migrate toward the growth cone, possibly owing to a demand for a high-energy supply (#1779). In an elderly patient with LHON/3460, there was depletion of Purkinje cells largely limited to the superior cerebellar vermis (#1781). This depletion may reflect the cellular architectural and surface antigen homology between RGCs and Purkinje cells. von Hippel-Lindau Genetic testing for the von Hippel-Lindau gene was negative in 10 patients with an isolated retinal capillary hemangioma, which suggests that this test could be used to screen patients for the complete disorder (#1807). New retinal hemangioblastomas developed in 22% of eyes of 68 patients with the von Hippel-Lindau gene over a median follow-up period of 16 months (#1808). Functional MRI Functional MRI (fMRI) was used to demonstrate that stereopsis is dependent on a network of functional fields found in the occipital, parietal, and prefrontal cortex regions (#2160). Single fMRI studies inconsistently demonstrated the retinotopic mapping of the V1-V2 border. Signal averaging of repeated measures is necessary to accomplish this (#2192). The voxel (three-dimensional pixel) size needs to be optimized and is an important parameter in detecting brain activation in fMRI with T2-weighted echo-planar imaging (#2197). MRI coordinates A new method for specifying cortical location on MRI used a coordinate system with the fundamental axis in the sagittal plane extending from the anterior corpus callosum to the posterior aspect of the parietooccipital sulcus. This system seemed to be easier to use than previous systems (#2193). Optic nerve injury The RGC biologic basis for ischemic injury and neuroprotection was reported. Glutamate-nitric oxide signaling during development may influence the circuitry between RGCs, amacrine cells, and bipolar cells (#2025). Schwann cell-derived neurotrophic agent, administered intraocularly, can protect RGCs from optic nerve crush injury (#2035). Nitric oxide plays a major role in retinal damage after ischemia/reperfusion injury in the rat (#2211). This observation fits well with the report that inhibiting nitric oxide synthase with aminoguanidine partially protected the optic nerve axons after ischemia injury from increased intraocular pressure in the rat eye (#2224). Glaucoma In an experimental unilateral glaucoma monkey model, the lateral geniculate nucleus developed atrophy not only in layers with connections to the glaucomatous eye, but also in layers receiving input from the unaffected eye. This finding suggests a more complex relationship for glaucomatous progression than merely injury at the ganglion cell level and that still unclear central mechanisms play a role (#2212). “Filling in” The perceptual completion effect (“filling in”) in a patient with an occipital injury and hemianopic scotoma was demonstrated to be a retinotopic phenomenon and not attributable to inattention (#2776). Completion of filling in also occurs within the scotomas of age-related macular degeneration. Modeling that enlarges the receptive fields of adjacent normal retina appears to account for some of the observed improvement in the field defect, including retention of pattern detection (#2777). Retinal bipolar cells A mini-symposium reported that the bipolar cell is the starting point for parallel processing of vision (S519). On bipolar cells have sustained or transient responses as well as on or off responses. These functions are modulated through subtypes of γ-aminobutyric acid receptors. Multiple types of bipolar cells, each with unique axons, provide the basis for complex interactions with photoreceptor input and interaction with amacrine and ganglion cells. Amacrine cells might provide the modulation that results in differential tuning of bipolar cells. Parkinson's disease Parkinson's disease causes saccadic multistepping worse with upward saccades. l-dopa significantly reduces this dysfunction (#3353). Histology of ischemic optic neuropathy In a well-preserved histopathologic specimen of an optic nerve that had had anterior ischemic optic neuropathy 20 days pre-mortem, there was severe axonal loss but no evidence of an infarct in a single vascular territory (#3360). Post-operative ischemic optic neuropathy In a retrospective survey by anesthesiologists of 27 patients with postoperative anterior or posterior ischemic optic neuropathy, visual loss after spinal or cardiac surgery was associated with low intraoperative blood pressure and/or hematocrit, vigorous fluid management to maintain perfusion of the ocular tissues, and patient positioning (#3363). Leber's congenital amaurosis Numerous investigators reported progress in uncovering genetic defects in Leber's congenital amaurosis (S640, S644–645). Brimonidine Brimonidine mitigated the apoptosis induced by tumor necrosis factor (TNF) on retinoblastoma cells in culture, but only in the faster dividing cell lines (#3557). The same dose of brimonidine caused death of some of these cells. Curiously, TNF in conjunction with brimonidine led to less cell death, proving that two wrongs sometimes make a right. Pupils and retinal ganglion cells A morphologically distinct RGC type mediated the pupillary light reflex in rhesus monkey (#3639). This finding has interesting clinical implications, explaining perhaps why the pupillary reflexes are spared in some optic neuropathies (see #5020 below). Thrombolysis of retinal vein occlusion Direct cannulation of the retinal venous system with infusion of rt-PA via a pars plana approach with vitrectomy was performed in approximately 60 patients with central retinal vein occlusion, most of whom had prolonged reduction central acuity beforehand. The authors claimed three-line improvement in more than 50% and no major complications. Vitreous hemorrhage occurring two months after the procedure cleared spontaneously. One retinal detachment that did not affect the macula was surgically repaired (#3860). Retinal ischemia Adenosine A2a and A1 receptors were increased in the inner nuclear layer of the retina six hours after ischemia induced by 45 minutes of elevated intraocular pressure. The A1 receptor is predominantly expressed on ganglion cells; the A2a receptor is predominantly expressed in the nerve fiber layer and ganglion cell layer (#3984). In a primate model of transient central retinal artery occlusion induced by clamping the CRA for 190 minutes, vitreous sampling failed to demonstrate the expected elevation of glutamate or glycine (#3986). Pretreatment with carvedilol, a beta blocker that has antiapoptotic properties, and MK-801, an NMDA antagonist, preserves more retinal function than either agent alone (#3993). Pretreatment with trimetazide, an agent used for prophylaxis of angina pectoris, reduced the effects of ischemia on retinal morphology (#3998). In vitro hypothermia (25°C) prevented a drop in adenosine triphosphate levels and reduced morphologic changes in the rat retina (#3998). Homocysteine toxicity Intravitreal administration of homocysteine appears to be toxic to RGCs; the combination of homocysteine and glutamate induces even more ganglion cell loss (#4004). Batten's disease In a murine knock-out gene model of Batten's disease, the optic nerve develops significant loss of axons (#4112). Vigabatrin A wide-field (57–90 degrees) multifocal ERG can demonstrate loss of peripheral retinal dysfunction in patients on long-term vigabatrin treatment (#4206). Pupil studies Topical brimonidine causes the pupil to be approximately 1 mm smaller but has no effect on the clinical determination of the pupillary response to light (#4415). A study using computerized infrared pupillography confirmed the clinical observation that a brighter light stimulus produced a greater distinction between normal subjects and patients with unilateral anterior visual pathway lesions (#4518). MRA and third nerve palsy A statistical analysis of the literature on magnetic resonance angiography (MRA) in patients with isolated third nerve palsy concluded that MRA is not adequate to rule out an aneurysm in most patients younger than 70 years. However, the authors presented no original data. Their model did not distinguish between extradural and intradural aneurysms or consider MRA that includes high-resolution techniques or review of the source image data (#4531). Myasthenia gravis If patients with ocular myasthenia gravis are treated with corticosteroids, the development of generalized myasthenia gravis at two years appears to be significantly reduced. In diagnostic intravenous edrophonium testing for myasthenia, most patients require no more than 3 mg to demonstrate an ameliorative effect on ptosis or ocular motor dysfunction (#4532). Optical coherence tomography Optical coherence tomography appears to be the best imaging technique to view the nerve fiber layer. Asymmetry analysis of comparable areas in both eyes is useful in overcoming interindividual variations (#4548). Rat model of ischemic optic neuropathy A model of nonarteritic ischemic optic neuropathy was created in rats by using a laser activating an injected photosensitizing dye to thrombose small vessels supplying the optic nerve. Pale swelling of the optic disk, reduced visual evoked potentials, and a loss of RGCs were demonstrated (#5019). Pupil pathways Histopathology using Dil, a fluorescent dye, in a patient with LHON demonstrated preservation of optic nerve input to the pretectal region in the brain. This provides anatomic evidence for the clinical observation of relative preservation of the pupillary light response in these patients and supports the notion that some axons in the afferent loop of the pupillary reflex are not related to conscious vision (#5020).

Sirt6 protects retinal ganglion cells and optic nerve from degeneration during aging and glaucoma

To establish a rat glaucoma model with chronic intraocular pressure (IOP) elevation induced by microbeads suspended in sodium sulfate-sodium hyaluronate. Chronic elevation of IOP was induced unilaterally by injecting polystyrene microbeads, suspended in 4 % sodium sulfate and 3 % sodium hyaluronate, into the anterior chamber. The microbead suspension was injected through either the clear corneal (CC) or sclerocorneal (SC) tunnel. IOP changes were monitored up to 8 weeks after injection. The loss of retinal ganglion cells (RGCs) was assessed using fluorogold retrograde labeling of RGCs. RGC axons were evaluated by immunohistochemistry and immunoblotting. The resulting IOP elevation was maintained up to 3 weeks after the intracameral injection of microbeads through the CC route and up to 4 weeks after injection through the SC route. The density of RGCs was significantly reduced at 4 weeks after injection, with the SC route leading to more RGC loss than the CC route (p = 0.037). The neurofilament immunoreactivity and protein levels in the optic nerve were also significantly reduced at 4 weeks after injection. Some eyes in the SC route cohort received re-injection of the microbead suspension at 4 weeks after the initial injection, which led to an elevated IOP more than 8 weeks after the initial injection, and eventually a 27.5 % loss of RGC density compared with the control eyes. The intracameral injection of microbeads suspended in hyaluronate effectively produced chronic IOP elevation and subsequent RGC degeneration in rat eyes. The sclerocorneal tunnel approach yielded a longer period of IOP elevation than the clear corneal approach. Our modified microbead injection offers a reliable high-pressure glaucoma model.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ETB) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ETB receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ETB receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ETB receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ETB receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

We acquired age-matched and sex-matched Sprague-Dawley rats from 2 independent breeding establishments. Serendipitously, we observed that constitutive, and bacterial toxin-induced, expression of major histocompatibility complex (MHC) class II RT1B chain in the uveal tract was much lower in one of the cohorts. Activated microglia are known to upregulate MHC II RT1B expression during optic nerve (ON) degeneration induced by raised intraocular pressure (IOP). We investigated whether, in a model of experimental glaucoma, microglial upregulation of MHC II RT1B was less efficacious and ON degeneration correspondingly less severe in the cohort of rats with low MHC II RT1B expression. Experimental glaucoma was induced by lasering the trabecular meshwork using a standard protocol. After 2 weeks of elevated IOP, retinal ganglion cells (RGC) survival, ON degeneration, and microglial responses were determined in both cohorts of rats. Raised IOP-induced expression of MHC II RT1B by microglia was muted in the "Low" cohort compared with the "High" cohort. Axonal degeneration, RGC loss, and microgliosis were all significantly lower in the cohort of rats with low basal and induced expression of MHC II RT1B, despite both cohorts displaying IOP responses that were indistinguishable in terms of peak IOP and IOP exposure. Expression of MHC II RT1B by activated microglia in the ON during experimental glaucoma was associated with more severe RGC degeneration. Further studies are needed to elucidate the role of MHC II during experimental glaucoma.

Glaucoma is a leading cause of irreversible blindness, characterized by progressive retinal ganglion cell (RGC) loss and optic nerve degeneration. Mitochondrial dysfunction plays a central role in this neurodegeneration, yet effective targeted therapies remain limited. Here, we identify the mitochondrial scaffold A-kinase anchoring protein 1 (AKAP1) as a critical regulator of RGC resilience and axon regeneration. AKAP1 expression is diminished in human glaucomatous retinas and experimental glaucoma models, correlating with elevated intraocular pressure, disrupted mitochondrial dynamics, oxidative stress, and synaptic instability. Restoration of AKAP1 via adeno-associated virus serotype 2-mediated gene therapy preserves RGC survival, promotes mitochondrial fusion and cristae integrity, enhances ATP production, and mitigates oxidative and apoptotic stress in mouse models of glaucoma and optic nerve injury. Transcriptomic profiling of AKAP1 knockout retinas reveals widespread dysregulation of mitochondrial and synaptic gene networks. Mechanistically, AKAP1 stabilizes synapses by promoting mitochondrial biogenesis, modulating calcium/calmodulin-dependent kinase II and synapsin phosphorylation, maintaining synaptophysin expression, and suppressing complement component C1q expression, thereby preventing early synaptic loss in glaucomatous neurodegeneration. Moreover, restoring AKAP1 expression facilitates axonal regeneration, preserves the central visual pathway, and maintains visual function. Collectively, these findings establish AKAP1 as a master regulator of mitochondrial and synaptic homeostasis and axonal regeneration and a promising therapeutic target for vision preservation in glaucomatous neurodegeneration.

Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal ganglion cells (RGCs) and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. We evaluated the effects of NGF eye drops in a rat model of glaucoma. We also treated 3 patients with progressive visual field defects despite IOP control. Glaucoma was induced in rats through injection of hypertonic saline into the episcleral vein. Initially, 2 doses of NGF (100 and 200 mug/mL) were tested on 24 rats, and the higher dose was found to be more effective. Glaucoma was then induced in an additional 36 rats: half untreated and half treated with 200 mug/mL NGF QID for 7 weeks. Apoptosis/survival of RGCs was evaluated by histological, biochemical, and molecular analysis. Three patients with advanced glaucoma underwent psychofunctional and electrofunctional tests at baseline, after 3 months of NGF eye drops, and after 3 months of follow-up. Seven weeks of elevated IOP caused RGC degeneration resulting in 40% cell death. Significantly less RGC loss was observed with NGF treatment (2,530 +/- 121 vs. 1,850 +/- 156 RGCs/mm(2)) associated with inhibition of cell death by apoptosis. Patients treated with NGF demonstrated long lasting improvements in visual field, optic nerve function, contrast sensitivity, and visual acuity. NGF exerted neuroprotective effects, inhibiting apoptosis of RGCs in animals with glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF improved all parameters of visual function. These results may open therapeutic perspectives for glaucoma and other neurodegenerative diseases.

PurposeGlaucoma is characterized by progressive retinal ganglion cell (RGC) death and optic nerve degeneration. Chemokines are a family of small, secreted proteins that mediate cell-cell communication, an essential process for maintaining microenvironmental homeostasis and regulating pathophysiological changes in multicellular organisms. However, the contribution of retina-derived chemokines to RGC loss in glaucoma remains poorly understood.MethodsWe reanalyzed a publicly available retinal bulk RNA sequencing dataset from a mouse model of glaucoma to identify differentially expressed chemokines. A mouse model of microbead-induced glaucoma and primary RGCs and astrocytes were used to assess the role of the C–X–C motif chemokine ligand 10 (CXCL10)/C–X–C motif chemokine receptor 3 (CXCR3) axis in disease.ResultsSeveral chemokines were significantly upregulated during disease progression, including CXCL10, previously implicated in neurodegeneration. In the microbead model, CXCL10 expression increased markedly by day 5 post-injection. At 6 weeks, deletion of CXCR3, the receptor for CXCL10, significantly prevented RGC loss and axonal degeneration without affecting intraocular pressure (IOP). Visual function, assessed by pattern electroretinography and visual acuity, was preserved in CXCR3-deficient mice. Mechanistically, CXCL10/CXCR3 signaling upregulated complement component 3 (C3) in astrocytes and C3a receptor (C3aR) in RGCs, driving toxic astrocyte–RGC crosstalk. Gene therapy using intravitreal injection of adeno-associated virus-mediated dominant-negative CXCL10 or pharmacological blockade of C3aR effectively reduced RGC loss.ConclusionsCXCL10/CXCR3 signaling is a key mediator of RGC loss in glaucoma. Targeting this pathway, along with its upregulated C3/C3aR axis, represents a promising IOP–independent therapeutic strategy for glaucoma.

PurposeGlaucoma is a neurodegenerative disease associated with elevated intraocular pressure and characterized by optic nerve axonal degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The endothelin (ET) system of vasoactive peptides (ET-1, ET-2, ET-3) and their G-protein coupled receptors (ETA and ETB receptors) have been shown to contribute to the pathophysiology of glaucoma. The purpose of this study was to determine whether administration of the endothelin receptor antagonist macitentan was neuroprotective to RGCs and optic nerve axons when administered after the onset of intraocular pressure (IOP) elevation in ocular hypertensive rats.MethodsMale and female Brown Norway rats were subjected to the Morrison model of ocular hypertension by injection of hypertonic saline through the episcleral veins. Following IOP elevation, macitentan (5 mg/kg body wt) was administered orally 3 days per week, and rats with IOP elevation were maintained for 4 weeks. RGC function was determined by pattern electroretinography (PERG) at 2 and 4 weeks post-IOP elevation. Rats were euthanized by approved humane methods, and retinal flat mounts were generated and immunostained for the RGC-selective marker Brn3a. PPD-stained optic nerve sections were imaged by confocal microscopy. RGC and axon counts were conducted in a masked manner and compared between the treatment groups.ResultsSignificant protection against loss of RGCs and optic nerve axons was found following oral administration of macitentan in rats with elevated IOP. In addition, a protective trend for RGC function, as measured by pattern ERG analysis, was evident following macitentan treatment.ConclusionsMacitentan treatment had a neuroprotective effect on RGCs and their axons, independent of its IOP-lowering effect, suggesting that macitentan may complement existing treatments to prevent neurodegeneration during ocular hypertension. The findings presented have implications for the use of macitentan as an oral formulation to promote neuroprotection in glaucoma patients.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.