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Retinal Ganglion Cell Background
The relatively thin size of RGC axons is important for energy consumption as well as action potential generation, but there are also consequences for axons being so thin (Calkins, 2012; Crish et al., 2010). These aforementioned calcium changes are mediated by either calcium influx through voltage-gated plasma membrane calcium channels or release from intracellular stores (Gleichmann & Mattson, 2011; Mallick et al., 2016).
Glial cells of the Retina
Glial cells also protect the blood-retinal barrier, as evidenced by their activation in the peripapillary chorio-retinal response to stress or injury (Barres, 2008; Vecino et al., 2016). Whether the role of astrogliosis is beneficial or detrimental is up for debate (Varela et al., 1997; Morgan, 2000).
Glaucoma Background
Genetics of Glaucoma
Types of Glaucoma
There are two main types of glaucoma - primary open-angle glaucoma and primary angle-closure glaucoma. Primary open-angle glaucoma is often a diagnosis of exclusion because, in these patients, there are no obvious ocular or systemic causes for it.
Intraocular Pressure and its Relationship to Glaucoma
The majority of research into the mechanical damage that occurs in patients with normal tension glaucoma focuses on a structure in the eye known as the lamina cribrosa. This mesh-like structure is extremely important in the route between the axons in the eye and the axons that exit via the optic nerve.
Current Standards of Treatment for Glaucoma
These treatments have fewer systemic side effects, but they are more inconvenient for patients because they have to be administered several times a day and therefore have very low patient compliance (Kimura et al., 2017a; Rusciano et al., 2017) . Primary angle-closure glaucoma is considered a medical emergency, so patients should have these procedures performed immediately in combination with other IOP-lowering treatments (Khazaeni & Khazaeni, 2022; Weinreb et al., 2014a).
Clinical v. Experimental Glaucoma
These mice show a gradual increase in IOP as they age that corresponds to axonal loss in the optic nerve (Harada et al., 2019; Quillen et al., 2020). In the photocoagulation model, a diode laser (wavelength of 532 nm) is aimed at the trabecular meshwork and episcleral vessels (Yun et al., 2014).
Microbead Occlusion Model of Glaucoma
Previous optic nerve cross-sectional studies in both rats and mice showed a decrease in axon density, a decrease in axon number, and an increase in the number of degenerative axons 4 weeks after IOP elevation compared with saline-injected controls. (Calkins et al., 2018; Lambert et al., 2020; Sappington et al., 2010). Furthermore, this model has been used to measure visual function in rats and mice at multiple time points after induced ocular hypertension and both species show reduced amplitudes of their visual evoked potential compared to saline-injected controls (Bond et al., 2016; Hines-Beard et al., 2016a).
The Relationship Between Oxidative Stress and Glaucoma
We have previously shown an endogenous increase in these antioxidant proteins at 2 weeks after IOP elevation (S. Naguib et al., 2021). The endogenous retinal antioxidant response is mediated by the PI3K/Akt pathway (Naguib et al., 2021).
Antioxidant Defenses Against Oxidative Stress
The Relationship Between Antioxidants and Glaucoma
Some glaucoma patients in one particular study treated with vitamin C saw a detectable decrease in IOP (Hui et al., 2020). In a phase III clinical trial in Australia, oral vitamin B3 had impressive preliminary findings in patients with primary open-angle glaucoma (Hui et al., 2020).
NRF2, KEAP1 and the Antioxidant Response Element
The most important regulatory domain of the protein is Neh2, which is located in the N-terminus, and is involved in maintaining the protein's stability (Huang et al., 2015; Vriend & Reiter, 2015). NRF2's antioxidant activity is dependent on its dissociation from KEAP1 so that it can accumulate in the cytosol and translocate into the nucleus to bind to the ARE (Nguyen et al. Raghunath et al., 2018).
NRF2 and Glaucoma
In the well-characterized microbead occlusion model (MOM) of glaucoma, we have previously shown that Nrf2 is phosphorylated by the PI3K/Akt pathway at 2 weeks after IOP elevation (S. Naguib et al., 2021). Previous data showed correlations between decreases in VEP N1 amplitude and axonal degeneration at 5 weeks after IOP elevation in MOM (Bond et al., 2016; S.
Neuroprotection and Glaucoma
EPO as a Neuroprotective for Glaucoma
Furthermore, in a model of glaucomatous degeneration, systemic EPO treatment protected RGCs (Wang et al., 2012; Zhong et al., 2007). Furthermore, EPO treatment protected photoreceptors in a rat photodamage model (Rex et al., 2009; Sullivan et al., 2011).
EPO-R76E
The first use of this mutated form of EPO was tested in mouse model of photoreceptor death (Sullivan et al., 2011). We have also previously published using EPO-R76E formulated into microparticles containing PLGA in a model of blast-indirect traumatic optic neuropathy (DeJulius et al., 2021).
Conclusions and Hypotheses
We have previously published that RGC function declines as early as 2 weeks after IOP elevation in MOM, as measured by the photopic negative response (PhNR) of the ERG (Naguib et al., 2021). Quantification of the number of degenerative axons in optic nerves at 2 weeks after IOP elevation, *p<0.05.
Retinal Oxidative Stress Activates the NRF2/ARE Pathway: An Early Endogenous
Introduction
Although this is relatively effective, many patients continue to experience vision loss despite these treatments (Goldberg, 2003; Killer . & Pircher, 2018; Weinreb et al., 2014a). Similar findings of increased susceptibility to RGC death in Nrf2 KO mice were found in an ischemia-reperfusion model ( Xu et al., 2015 ).
Materials and Methods
For comparison, the 2 week wild-type data from Figure 2.2 is re-shown in Figure 2.6 to compare with Nrf2 KO mice. For comparison, the 2 week wild-type data in Figure 2.2 was re-displayed in Figure 2.6 to compare with the data from Nrf2 KO mice.
Results
Compared to wild-type counterparts, there was a significant increase in DHE fluorescence in saline-injected Nrf2 KO mice, p=0.0001 (Figure 2.6A, B). Quantification of the number of total optic nerve axons showed fewer axons in microbead-injected Nrf2 KO mice compared to saline-injected Nrf2 KO controls, p=0.0054 (Figure 2.6I).
Discussion
Here we show that although there are no differences in b-wave amplitudes 2 weeks after IOP. PhNR amplitude was reduced in microbead-injected mice as early as 2 weeks after IOP elevation compared with saline-injected controls.
Neuronal NRF2 Modulates Glaucoma Onset and Severity
Introduction
Antioxidants are therapeutic in several glaucoma models in preventing RGC degeneration and protecting visual function (Hines-Beard et al., 2016a; Inman et al., 2013b; Kansanen et al., 2013). Nrf2 KO mice have been used in several glaucoma models to show further increased susceptibility to RGC degeneration and death, suggesting that Nrf2 plays an important role in glaucomatous pathology and RGCs in MOM (Nakagami, 2016; Z. Xu et al. al., 2015).
Materials and Methods
We injected 2 μl of 15 μm diameter FluoSpheres polystyrene microbeads into the anterior chamber of anesthetized rats (Thermo Fisher, Waltham, MA) as described previously (Bond et al., 2016; D. J. Calkins et al., 2018; Sa. pping; 2010). Just before imaging, mice were anesthetized with ketamine/xylazine and eyes were dilated with 1% tropicamide.
Results
Overall, we found that knocking down Nrf2 in either RGCs or astrocytes was sufficient to affect the PhNR amplitude at 2 weeks post-IOP elevation. We also assessed antioxidants at the protein level using western blot analysis at both 2 and 5 weeks post-IOP elevation (Figure 3.6G, H).
Discussion
Here, we sought to determine whether phosphorylation of NRF2 in the presence of PLGA.EPO-R76E would occur by the same mechanism. Importantly, Gargini et al. showed that optic nerve damage caused deficits in the ERG amplitudes that correlated with the timing of endogenous.
Erythropoietin-Mediated Activation of NRF2 via MAPK Protects RGCs and Astrocytes
Introduction
One of these is through the transcription factor, NRF2, which our laboratory and others have implicated as a potential therapeutic target for glaucoma (Fujita et al., 2017; In that study, we showed that a single intravitreal injection of of these microparticles one day after injury protected against optic nerve degeneration and visual function deficits (DeJulius et al., 2021).
Materials and Methods
We raised IOP bilaterally using anterior chamber occlusion with 2 µl injections of 15 µm diameter FluoSpheres polystyrene microbeads (Thermo Fisher, Waltham, MA) as previously described (Bond et al., 2016; D. J. Calkins et al. , 2018; S; Naguib et al., 2021; Sappington et al., 2010). We measured IOP immediately before microbead injection and biweekly thereafter using the Icare TonoLab rebound tonometer (Colonial Medical Supply, Franconia, NH) as previously described (D. J. Calkins et al., 2018; Hines-Beard et al., 2016a). .
Results
In this study, 2 weeks after IOP elevation, the PhNR amplitudes of mice treated with PLGA.EPO-R76E were 50% greater than their blank PLGA counterparts. Because the ratio of phosphorylated to total MAPK was greatest in mice that received PLGA.EPO-R76E 2 weeks after IOP elevation, we sought to determine whether NRF2.
Discussion
As previously reported, bITON caused a reduction in ERG b-wave amplitude (bmax) compared to sham mice (Fig. 7.1G; Naguib et al., 2019). We detected increases in both IL-1α and IL-1β in the retina after bITON, as previously reported (Fig. 7.2A and B; Bernardo-Colón et al., 2018).
Discussion
Limitations and Future Directions
We previously reported a role for the inflammasome pathway, and particularly levels of IL-1α and IL-1β in the retina in this model (Bernardo-Colón et al., 2018). Certainly, the synaptic terminals are disconnected from their downstream targets at 1-month post-bITON, which contributes to the decrease in the ERG b-max (Naguib et al., 2019).
Appendix 1: Galantamine Protects Against Synaptic, Axonal and Visual Deficits in
Methods
Subcutaneous platinum needle electrodes were placed on the snout and back of the head at the location of the visual cortex. Twenty microliters of supernatant samples were injected using a Water 2707 autosampler onto a Phenomenex Kintex C18 HPLC column (100 x 4.60 mm, 2.6um).
Results…
Control sham and galantamine-treated bITON nerves exhibit more axons than the control bITON nerve (Figure 6.3A-C). There was no statistically significant difference in fluorescence levels between galantamine sham mice and galantamine-treated biTON mice (Figure 6.4F).
Discussion
The levels of the anti-inflammatory cytokines, IL-1α and IL-1β, were increased in mice that received an intravitreal injection. A previous study showed that intravitreal injection of saline induced temporary vacuole formation in photoreceptor outer segments and RPE, which resolved 3 weeks after injury (Hombrebueno et al., 2014).
Appendix 2: Intravitreal Injection Worsens Outcomes in a Mouse Model of Indirect
Manuscript
The increase was disproportionately even greater in the bITON saline group compared to the non-injected bITON group. However, both IL-1α and IL-1β levels were increased in the optic nerve of saline-injected mice compared to the uninjected mice (Fig. 7.2E and F).
