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By Barbara A. Mysona, Jing Zhao, Oceane De Greef, August Beisel, Parth A. Patel, Lindsay Berman, Sylvia B. Smith, and Kathryn Bollinger

Excerpt from the article published in Experimental Eye Research,  15 November 2022, 109308, ISSN 0014-4835, DOI: https://doi.org/10.1016/j.exer.2022.109308.

Abstract

Purpose: Glaucoma is a worldwide leading cause of irreversible blindness. Standard treatments lower intraocular pressure (IOP). Novel treatments to prevent optic nerve (ON) degeneration are needed. Here, we investigate the hypothesis that sigma-1 receptor (S1R) agonist (+)-pentazocine (PTZ) is neuroprotective in a Brown Norway (BN) rat, microbead model of glaucoma.

Methods: BN rats (9–11 weeks, male and female) were treated by intraperitoneal injection, 3 times per week with (+)-PTZ (2 mg/kg) or vehicle (VEH) alone. Treatment started 1 week prior to intraocular injection of polystyrene microbeads to elevate IOP. IOP was measured 2–3 times per week. Five weeks post microbead injection, rats were euthanized. ONs were removed, then fixed and processed for 63x oil, light microscope imaging of toluidine blue stained ON cross sections. To facilitate comparison of ON morphology from VEH and (+)-PTZ treated rats with similar ocular hypertensive insults, rats were assigned to low (IOP ≤15.8 mmHg), moderate (15.8 < IOP <28.0 mmHg), and high (IOP ≥28.0 mmHg) groups based on average IOP in the microbead injected eye. Axon numbers, axon density, axonal and glial areas, axon loss, and axon size distributions of naïve, bead, and contralateral ONs were assessed using QuPath program for automated image analysis.

Results: (+)-PTZ treatment of BN rats protected ONs from damage caused by moderate IOP elevation. Treatment with (+)-PTZ significantly reduced axon loss and glial areas, and increased axon density and axonal areas compared to ONs from VEH treated rats with moderate IOP. (+)-PTZ-mediated neuroprotection was independent of IOP lowering effects. At average IOP ≥28.0 mmHg, (+)-PTZ treatment did not provide measurable neuroprotection. ONs from contralateral eyes exhibited subtle, complex changes in response to conditions in the bead eyes.

Conclusions: S1R agonist (+)-PTZ shows promise as a neuroprotective treatment for glaucoma. Future studies to understand the complex molecular mechanisms by which (+)-PTZ provides this neuroprotection are needed.

Introduction

Glaucoma is a worldwide leading cause of irreversible blindness (Blindness, 2021). This multifactorial disease results in optic nerve (ON) degeneration characterized by loss of retinal ganglion cells (RGCs) and their axons. RGCs are the main output neurons of the retina (Weinreb et al., 2014; Friedman et al., 2004). RGC axons exit the retina at the back of the eye to form the ON which carries visual signals from the retina to the brain (Erskine and Herrera, 2014). Although the exact cause of glaucoma is often unknown, the most common risk factor is elevated intraocular pressure (IOP) (Sommer et al., 1991). Currently, lowering IOP is the main treatment for glaucoma (Kass et al., 2002; Heijl et al., 2002). Novel treatments that delay or prevent ON degeneration, independent of lowering IOP, are desperately needed. One promising class of neuroprotective compounds for glaucoma treatment are sigma-1 receptor (S1R) agonists.

S1R is a 223 amino acid, multifunctional, ligand-operated protein expressed in a wide variety of tissues, including the CNS (Schmidt et al., 2016, 2018; Ryskamp et al., 2019; Su, 1991; Gundlach et al., 1986; Alonso et al., 2000). In the retina and ON, S1R is expressed in neurons, glial, endothelial, and retinal epithelial cells (Ola et al., 2001; Bucolo et al., 1999; Jiang et al., 2006). Intracellularly, this transmembrane protein is localized primarily in the endoplasmic reticulum (ER) as well as in nuclear membranes (Jiang et al., 2006; Mavlyutov et al., 2015). S1R is known to act as a molecular chaperone that mediates ER-mitochondrial signaling by stabilizing mitochondria-associated membranes (MAM), regulating calcium flux, and modulating the ER stress response (Hayashi and Su, 2007; Hayashi and Fujimoto, 2010; Watanabe et al., 2016; Ha et al., 2011, 2014). S1R is also involved in post-translational processing of the neurotrophin, BDNF (Kikuchi-Utsumi and Nakaki, 2008; Fujimoto et al., 2012; Mysona et al., 2018), and in facilitating antioxidant responses (Pal et al., 2012; Wang et al., 2015). S1R activation typically mediates pro-survival and anti-apoptotic signaling pathways (Ryskamp et al., 2019).

Treatment with S1R agonists improves experimental outcomes in rodent models for a variety of neurodegenerative disorders (Ryskamp et al., 2019). In mouse and rat stroke models, the S1R agonists (+)-pentazocine (PTZ), PRE084, and fluvoxamine reduced infarct sizes (Nguyen et al., 2015; Vagnerova et al., 2006; Allahtavakoli and Jarrott, 2011; Sato et al., 2014). Treatment with the S1R agonist PRE084 has also been shown to be beneficial in mouse models of Parkinson’s disease and amyotrophic lateral sclerosis (ALS) (Mancuso et al., 2012; Francardo et al., 2014) while treatment with the S1R agonist pridopidine improved outcomes in a mouse model of Huntington’s disease (Ryskamp et al., 2017). In the mouse retina, treatment with the S1R agonist (+)-PTZ reduced RGC loss in the diabetic retina and in an NMDA model of excitotoxicity (Smith et al., 2008; Zhao et al., 2016). S1R was also shown to protect RGCs in an ON crush model of glaucoma (Li et al., 2021), and (+)-PTZ treatment preserved cone photoreceptor function in a mouse model of retinitis pigmentosa (Wang et al., 2016b). Few studies have examined the effectiveness of S1R agonists in slowing or preventing glaucomatous neurodegeneration. Recently, promising work by Geva and colleagues showed that the S1R agonist pridopidine reduced RGC loss in BN rats exposed to ocular hypertension induced by the hypertonic saline model of glaucoma (Geva et al., 2021).

Here, we investigate the extent to which the prototypical sigma-1 receptor (S1R) agonist (+)-PTZ provides neuroprotection in a BN rat, microbead model of glaucoma. We hypothesized that treatment with (+)-PTZ would protect ONs from the axon loss and gliosis that are hallmarks of ON degeneration in glaucoma. The QuPath program (Bankhead et al., 2017) was used to evaluate ON morphology from 63x oil light microscope images of toluidine blue stained ON cross sections (Mysona et al., 2020). QuPath automated image analysis allowed us to perform in depth characterization of ON morphology. Variables measured included axon numbers, axon density, axon and glial areas, and axon size distributions. The inclusion of contralateral ONs and ONs from VEH and (+)-PTZ treated naïve rats provided valuable insights into the effects of ocular hypertension on the contralateral ON and the effects of (+)-PTZ treatment independent of ocular hypertension.

Section snippets

Animals

Fifty-five BN rats, 9–11 weeks of age (Charles River Laboratories, Wilmington, MA) were randomly assigned to VEH treated (17 female/12 male) and (+)-PTZ treated (11 female/15 male) groups (Fig. 1A). Rats were maintained at Augusta University animal facility in accord with the National Research Council’s guide for the care and use of laboratory animals. All work was performed under animal protocols approved by Augusta University institutional animal care and use committee (IACUC) and experiments 

Results

This work tested the hypothesis that the sigma-1 receptor (S1R) agonist (+)-PTZ is neuroprotective in a rat microbead model of glaucoma. Nine to eleven week-old male and female BN rats were treated with VEH (29 rats) or (+)-PTZ (26 rats) (Fig. 1A). (+)-PTZ and VEH treatment (3x/week, 2 mg/kg) began 1 week prior to microbead induced IOP elevation and continued for an additional 5 weeks (Fig. 1B). Releasing aqueous humor before bead injection and using air to force microbeads into the

Discussion

In this study, we demonstrated that treatment with the prototypical S1R agonist (+)-PTZ provided neuroprotection in a BN rat microbead model of glaucoma. Three key findings emerged. First, (+)-PTZ treatment provided neuroprotection at moderate IOP elevations. Second, (+)-PTZ treatment did not result in measurable neuroprotection in the high IOP insult group. Third, contralateral eyes showed signs of undergoing complex and subtle changes in response to varying conditions in ocular hypertensive

Conclusions

In conclusion, the robust ability of (+)-PTZ treatment to preserve ON morphology provides support for the use of S1R agonists as neuroprotective treatments for glaucoma, especially in cases of moderate IOP-induced insult. Investigations into neuroprotective capabilities of multiple types of S1R agonists in different glaucoma models are important. Additional investigations to understand the mechanisms by which S1R activation exerts neuroprotection are greatly needed.