Optic Nerve and Retinal Ganglion Cell Protection, Rejuvenation, and Regeneration as Glaucoma Treatment Strategiess

Once destroyed, neurons and their axons in the mammalian central nervous system, including retinal ganglion cells (RGCs) and their axons in the eye and neurons in the thalamic and cortical brain regions involved in visual perception, cannot automatically be replaced. Intrinsic inhibitory chemicals and structural components, suppressive transcription factors, scar formation, and the sheer long distances the RGC axons have to travel to the brain prevent or reduce regenerative capacity in the visual system damaged by aging and various diseases such as glaucoma. However, non-clinical and some clinical uses of transcorneal electrical stimulation, redlight therapy, gene-therapy, and cell replacement, among other novel technologies and techniques, appear promising to help overcome some of these hurdles. Early results indicate that indeed neuronal rejuvenation; potential regeneration and ultimate replacement of the lost RGCs and their axons, such as in glaucoma; and the reestablishment of the retina-optic nerve−brain connections may be possible. Improvement and/or partial restoration of eyesight due to ocular and neurological disease-induced visual impairment in humans may thus be possible in the near future. These aspects will be discussed in this chapter

MicroRNA profile of extracellular vesicles released by Müller glial cells

IntroductionAs with any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions, and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells.MethodsWe conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture and used bioinformatics tools to evaluate their potential role in the neuroprotective signalling attributed to these cells.ResultsSequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b, and the let-7 family. A total of 51 miRNAs were found to be differentially enriched in EV compared to the whole cells from which EV originated. Bioinformatics analyses also indicated that preferential enrichment of species was demonstrated to regulate genes involved in cell proliferation and survival, including PTEN, the master inhibitor of the PI3K/AKT pathway.DiscussionThe results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks is likely to represent a significant proportion of the neuroprotective effect observed after the transplantation of these cells into animal models of retinal ganglion cell (RGC) depletion. Future studies will seek to evaluate the modulation of putative genes as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons

Human Trabecular Meshwork Cell Volume Decrease by NO-Independent Soluble Guanylate Cyclase Activators YC-1 and BAY-58-2667 Involves the BK Ca Ion Channel

PURPOSE. There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved. METHODS. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3Ј,5Ј-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BK Ca channel were used to characterize their involvement in the YC-1-and BAY-58-2667-induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay. RESULTS. YC-1 (10 nM-200 M) and BAY-58-2667 (10 nM-100 M) each elicited a biphasic effect on TM cell volume. YC-1 (1 M) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1-induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BK Ca channel inhibitor IBTX. The BAY-58-2667-induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BK Ca channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667-induced decreases in cell volume. CONCLUSIONS. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K ϩ efflux. (Invest Ophthalmol Vis Sci. 2009;50:3353-3359) DOI:10.1167/iovs.08-3127 A queous humor exits the eye through the trabecular meshwork (TM) and Schlemm canal. Activation of sGC by NOdependent donors increases the rate at which aqueous humor flows through the TM and Schlemm canal. These changes in outflow facility occur concomitantly with sGC-induced decreases in TM cell volume. sGC comprises an ␣-subunit and a smaller heme-containing ␤-subunit, 1,2 both of which constitute the active enzyme. Heterodimers are activated by NO binding to the heme moiety, whereas homodimers exhibit little or no synthetic activity, even in the presence of the ligand. Binding of NO to sGC results in the formation of 3Ј,5Ј-cyclic guanosine monophosphate (cGMP) from guanosine 5Ј-triphosphate (GTP). Increased cGMP activates protein kinase G (PKG), NO acting through the sGC, cGMP, and PKG pathways decreased TM cell volume in a time course that correlated with the NO-induced increases in outflow facility in perfused eye anterior segments. 5 Therefore, the need to identify other activators of sGC that regulate TM cell function is of vital interest. YC-1 [3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzyl indazole], 6 a benzyl indazole derivative, and BAY-58-2667 7 are NO-independent activators of sGC. As with NO activation of sGC, YC-1, and BAY-58-2667, activation of sGC also results in increases in cGMP and PKG phosphorylation events. Alterations of the contractile states and volume of the TM cells would regulate aqueous humor outflow. 8 -17 Changes in cell volume are influenced by the activities of the Na-K-2Cl cotransporter, MATERIALS AND METHODS Cell Culture Eyes from human donors with no history of ocular disease or surgery were obtained from Lions Eye Institute (Tampa, FL) within 24 to 30 hours of death. Primary human TM cell lines (numbers represent the ages of the donors: HTM26, HTM71, HTM36, HTM80, and HTM86) were developed. For our experimental protocols cells from early passages (passages 3-5) were used. Human TM explants were obtained from whole eyes that were stored in a moist environment at 4°C or from corneal scleral rims stored in ophthalmic solution (Optisol; Dexol; Chiron Ophthalmics, Irvine, CA) at 4°C. TM cells were isolated after collagenase digestion of TM explants. 22 Collagenase-treated cells were grown in low-glucose (1g/L) Dulbecco modified Eagle medium (DMEM; Mediatech, Herndon ,VA) in the presence of 10% fetal bovine serum (Mediatech), 100 U/mL penicillin, and 100 g/mL streptomycin (Mediatech) and then passaged into six-well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator at 37°C in 5% CO 2 . We validated human TM cells by their morphology From th

Preclinical pharmacology, ocular tolerability and ocular hypotensive efficacy of a novel non-peptide bradykinin mimetic small molecule

We sought to characterize the ocular pharmacology, tolerability and intraocular pressure (IOP)-lowering efficacy of FR-190997, a non-peptidic bradykinin (BK) B2-receptor agonist. FR-190997 possessed a relatively high receptor binding affinity (Ki = 27 nM) and a high in vitro potency (EC50 = 18.3 ± 4.4 nM) for inositol-1-phosphate generation via human cloned B2-receptors expressed in host cells with mimimal activity at B1-receptors. It also mobilized intracellular Ca2+ in isolated human trabecular meshwork (h-TM), ciliary muscle (h-CM), and in immortalized non-pigmented ciliary epithelial (h-iNPE) cells (EC50s = 167–384 nM; Emax = 32–86% of BK-induced response). HOE-140, a selective B2-receptor antagonist, potently blocked the latter effects of FR-190997 (e.g. IC50 = 7.3 ± 0.6 nM in h-CM cells). FR-190997 also stimulated the release of prostaglandins (PGs) from h-TM and h-CM cells (EC50s = 60–84 nM; Emax = 29–44% relative to max. BK-induced effects). FR-190997 (0.3–300 μg t.o.) did not activate cat corneal polymodal nociceptors and did not cause ocular discomfort in Dutch-Belted rabbits, but it was not well tolerated in New Zealand albino rabbits and Hartley guinea pigs. A single topical ocular (t.o.) dose of 1% FR-190997 in Dutch-Belted rabbits and mixed breed cats did not lower IOP. However, FR-190997 efficaciously lowered IOP of conscious ocular hypertensive cynomolgus monkey eyes (e.g. 34.5 ± 7.5% decrease; 6 h post-dose of 30 μg t.o.; n = 8). Thus, FR-190997 is an unexampled efficacious ocular hypotensive B2-receptor non-peptide BK agonist that activates multiple signaling pathways to cause IOP reduction.Peer reviewe

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Recently Approved Drugs for Lowering and Controlling Intraocular Pressure to Reduce Vision Loss in Ocular Hypertensive and Glaucoma Patients

Serious vision loss occurs in patients affected by chronically raised intraocular pressure (IOP), a characteristic of many forms of glaucoma where damage to the optic nerve components causes progressive degeneration of retinal and brain neurons involved in visual perception. While many risk factors abound and have been validated for this glaucomatous optic neuropathy (GON), the major one is ocular hypertension (OHT), which results from the accumulation of excess aqueous humor (AQH) fluid in the anterior chamber of the eye. Millions around the world suffer from this asymptomatic and progressive degenerative eye disease. Since clinical evidence has revealed a strong correlation between the reduction in elevated IOP/OHT and GON progression, many drugs, devices, and surgical techniques have been developed to lower and control IOP. The constant quest for new pharmaceuticals and other modalities with superior therapeutic indices has recently yielded health authority-approved novel drugs with unique pharmacological signatures and mechanism(s) of action and AQH drainage microdevices for effectively and durably treating OHT. A unique nitric oxide-donating conjugate of latanoprost, an FP-receptor prostaglandin (PG; latanoprostene bunod), new rho kinase inhibitors (ripasudil; netarsudil), a novel non-PG EP2-receptor-selective agonist (omidenepag isopropyl), and a form of FP-receptor PG in a slow-release intracameral implant (Durysta) represent the additions to the pharmaceutical toolchest to mitigate the ravages of OHT. Despite these advances, early diagnosis of OHT and glaucoma still lags behind and would benefit from further concerted effort and attention

Glaucomatous optic neuropathy treatment options: the promise of novel therapeutics, techniques and tools to help preserve vision

Peripheral vision loss followed by “tunnel vision” and eventual irreversible blindness is the fate of patients afflicted by various forms of glaucoma including primary open-angle glaucoma (POAG) and normotensive glaucoma (NTG). These complex and heterogeneous diseases are characterized by extensive death of retinal ganglion cells (RGCs) accompanied by retraction and severance of their axonal connections to the brain and thus damage to and thinning of the optic nerve. Since patients suffering from this glaucomatous optic neuropathy (GON) first notice visual impairment when they have lost > 40% of their RGCs, early diagnosis is the key to retard the progression of glaucoma. Elevated intraocular pressure (IOP), low cerebrospinal and/or low intracranial fluid pressure, advancing age, and ethnicity are major risk factors associated with POAG. However, retinal vascular abnormalities and a high sensitivity of RGCs and optic nerve head components to neurotoxic, inflammatory, oxidative and mechanical insults also contribute to vision loss in POAG/GON. Current treatment modalities for POAG and NTG involve lowering IOP using topical ocular drugs, combination drug products, and surgical interventions. Two recently approved multi-pharmacophoric drugs (e.g., rho kinase inhibitor, Netarsudil; a drug conjugate, Latanoprostene Bunod) and novel aqueous humor drainage devices (iStent and CyPass) are also gaining acceptance for treating POAG/ NTG. Neuroprotective and regenerative agents, coupled with electroceutical, mechanical support systems, stem cell transplantation and gene therapy are emerging therapeutics on the horizon to help combat GON. The latter techniques and approaches hope to rejuvenate RGCs and repair the optic nerve structures, thereby providing a gain of function of the visual system for the glaucoma patients