The Neuroprotective Role of Neuroglobin in Glaucoma: A Detailed Analysis

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Glaucoma is a neurodegenerative disease that is a leading cause of blindness worldwide, characterized by the progressive loss of retinal ganglion cells (RGCs) and glaucomatous optic neuropathy (GON). The only modifiable risk factor for glaucoma is intraocular pressure (IOP), and current management strategies focus on controlling IOP to slow disease progression. However, the search for neuroprotective agents that can be used alongside IOP-lowering treatments has garnered significant interest in recent years.

Neuroglobin (Ngb), a small hexacoordinated globin highly expressed in the brain and retina, emerged as a potential neuroprotective agent after its discovery in 2000. Numerous in vitro studies have demonstrated Ngb’s neuroprotective properties in neurons and astrocytes in both the brain and ocular regions. Moreover, transgenic rodent models have shown that Ngb overexpression can protect against stroke, retinal ischemia, and optic nerve disorders. The exogenous delivery of Ngb protein or gene therapy in rodent models of retinal hypoxia, glaucoma, and cerebral ischemia has also confirmed its neuroprotective effects.

Activated astrocytes and microglial cells are known to contribute to optic nerve damage in glaucoma through neuroinflammatory mechanisms, including the release of pro-inflammatory cytokines such as interleukin (IL)-6. Additionally, complement 3 (C3) activated astrocytes have been implicated in mediating RGC damage during neuroinflammation. Previous studies have demonstrated that exogenous IVT-Ngb can reduce RGC apoptosis by inhibiting the production of pro-inflammatory cytokines and microglial activation.

Given the phylogenetic similarities between primates and humans, primate models are invaluable for clinical translation studies. The primate microbead model of experimental glaucoma (EG) is particularly useful for investigating progressive changes in the optic nerve head (ONH) secondary to IOP elevation. Optical coherence tomography (OCT) parameters such as anterior lamina cribrosa depth (LCD), minimum rim width (MRW) thickness, and peripapillary retinal nerve fiber layer (RNFL) thickness are essential for monitoring ONH damage in EG. This study aimed to investigate the neuroprotective effects of IVT-Ngb against IOP-mediated structural changes, microgliosis, and astrogliosis in the optic nerve, as well as the ocular safety profile of IVT-Ngb using serial OCT measurements in a primate model of EG.

Investigation and Findings

This study focused on the effects of exogenous IVT-Ngb in a primate model of EG, examining IOP-mediated structural changes through OCT measurements and the responses of astroglia and microglia in the optic nerve. The ocular safety profile of IVT-Ngb was also assessed. Utilizing a bilateral EG model, we aimed to minimize inter-animal variability and ensure the IOP insult was consistent across both eyes. Baseline and serial OCT measurements of MRW, LCD, and RNFL were compared before and after IVT-Ngb treatment.

After treatment, IVT-Ngb-treated eyes exhibited significantly less structural damage in all three OCT parameters compared to sham-treated EG eyes. This trend of reduced structural changes continued up to the end of the study, suggesting Ngb’s neuroprotective role. Ex vivo optic nerve histology confirmed these in vivo OCT changes, further supporting Ngb’s efficacy in protecting against structural damage.

In the brain and optic nerve, astrocytes and microglial cells play crucial roles in neuronal homeostasis. Astrocytes around the ONH can become reactive in response to IOP elevation, a process known as reactive astrogliosis. Reactive astrocytes can be divided into neuroprotective A2 and neurotoxic A1 subtypes. A1 astrocytes express C3 and cleaved-caspase 3, contributing to microglial and astrocyte activation and neuroinflammatory responses.

In this study, Ngb overexpression was observed in GFAP-labelled astrocytes, aligning with previous reports of Ngb expression in astrocytes and neurons. In untreated EG eyes, there was a significant increase in reactive A1 neurotoxic astrocytes expressing C3 and cleaved-caspase 3, along with greater structural damage as evidenced by OCT. In contrast, IVT-Ngb-treated eyes showed higher numbers of Ngb-positive astrocytes with reduced C3 and cleaved-caspase 3 expression, indicating a shift towards the neuroprotective A2 phenotype. This was accompanied by fewer reactive microglia and overall preservation of astrocytes, suggesting Ngb’s role in mitigating EG-induced structural changes.

Methodology and Implications

The study employed a small sample size of six primates, comparable to other primate studies, to ensure ethical and cost constraints were met. Despite the sample size, the findings were significant and warrant further investigation with larger cohorts. Transient inflammation was observed post-IVT injection, treated with topical corticosteroids to minimize its impact on outcomes. No endophthalmitis cases were reported, highlighting the importance of strict sterility in IVT therapy preparation.

The proposed mechanism by which IVT-Ngb penetrates the retina involves its negative charge facilitating diffusion through the vitreous to the retina and ONH. The small size of Ngb (17 kDa) allows it to penetrate the internal limiting membrane of the retina, reaching RGCs and ONH astrocytes. Mechanical IOP and ischemic factors at the ONH activate pro-inflammatory astrocytes, leading to oxidative stress and RGC loss. Ngb treatment of cultured astrocytes reduces reactive oxygen species production and IL-6 release, decreasing mitochondrial oxidative stress and caspase 3 activation. In this study, IVT-Ngb increased optic nerve Ngb expression in astrocytes, reducing C3 and caspase 3 expression and preserving the neuroprotective astrocyte phenotype.

Conclusion

This study demonstrates that IVT-Ngb offers significant neuroprotection against IOP-mediated structural damage in a primate model of EG. The findings suggest that Ngb therapy may mitigate neuroinflammatory responses and preserve RGCs by promoting a neuroprotective astrocyte phenotype. Further studies with larger sample sizes and extensive molecular investigations are needed to confirm these findings and elucidate the precise mechanisms of Ngb’s neuroprotective effects. The potential for Ngb as a therapeutic agent in glaucoma management is promising, offering a novel approach to complement existing IOP-lowering treatments and improve outcomes for patients with glaucoma.


reference link : https://www.mdpi.com/2218-273X/13/6/961

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