Pre-treatment with vinpocetine protects against retinal ischemia

Accepted Manuscript Pre-treatment with vinpocetine protects against retinal ischemia Lisa Nivison-Smith, Pauline Khoo, Monica L. Acosta, Michael Kalloniatis PII:

S0014-4835(16)30489-4

DOI:

10.1016/j.exer.2016.11.018

Reference:

YEXER 7066

To appear in:

Experimental Eye Research

Received Date: 25 August 2016 Revised Date:

17 October 2016

Accepted Date: 22 November 2016

Please cite this article as: Nivison-Smith, L., Khoo, P., Acosta, M.L., Kalloniatis, M., Pre-treatment with vinpocetine protects against retinal ischemia, Experimental Eye Research (2016), doi: 10.1016/ j.exer.2016.11.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Pre-treatment with Vinpocetine protects against retinal ischemia Lisa Nivison-Smith1,2, Pauline Khoo2, Monica L Acosta3,4, and Michael Kalloniatis1,2,3,4

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Centre for Eye Health; 2 School of Optometry and Vision Science, University of New South

National Eye Centre, University of Auckland, New Zealand.

Correspondence:

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Dr Lisa Nivison-Smith

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Wales, Sydney, 2052, Australia; 3 School of Optometry and Vision Science and 4New Zealand

Phone: Int +61 2 81150791 Fax: Int +61 2 81150799 E-mail: [email protected]

Number of pages: 29

Word Count Abstract: 172 words

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Manuscript: 4,480 words

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Number of figures: 9

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School of Optometry and Vision Science, UNSW Australia, Sydney, 2052, NSW, Australia.

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Running title: Vinpocetine pre-treatment protects against retinal ischemia

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Abstract Vinpocetine has been shown to have beneficial effects for tissues of the central nervous system subjected to ischemia and other related metabolic insults. We recently showed

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vinpocetine promotes glucose availability, prevents unregulated cation channel permeability and regulates glial reactivity when present during retinal ischemia. Less is known however about the ability of vinpocetine to protect against future ischemic insults. This study

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explores the effect of vinpocetine when used as a pre-treatment in an ex vivo model for retinal ischemia using cation channel permeability of agmatine (AGB) combined with

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immunohistochemistry as a measure for cell functionality. We found that vinpocetine pretreatment reduced cation channel permeability and apoptotic marker immunoreactivity in the GCL and increased parvalbumin immunoreactivity of inner retinal neurons in the inner nuclear layer following ischemic insult. Vinpocetine pre-treatment also reduced Müller cell

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reactivity following ischemic insults of up to 120 minutes compared to untreated controls. Many of vinpocetine’s effects however were transient in nature suggesting the drug can

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applications.

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protect retinal neurons against future ischemic damage but may have limited long-term

Key words: vinpocetine; retinal ischemia; cation channel; AGB

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Introduction Retinal ischemia is involved in the pathogenesis of major vision-threatening diseases such as diabetic retinopathy, retinal vein thrombosis and acute angle closure glaucoma (Osborne et al., 2004). Ischemia deprives the retina of immediate access to oxygen and metabolic

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substrates leading to acute cellular dysfunction. Even with reperfusion, retinal damage

continues with death of retinal neurons including ganglion cells, amacrine cells and bipolar cells (Dijk and Kamphuis, 2004a; Lafuente et al., 2002; Osborne et al., 2004; Schmid et al.,

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2014; Sun et al., 2007).

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Neuroprotective strategies against retinal ischemia focus on modulating the disease mechanisms which are thought to lead to cell death including glutamate excitotoxicity, free radical production, cationic imbalance, oxidative stress and mitochondrial dysfunction (Osborne et al., 2004; Szabadfi et al., 2010). Molecules such as antioxidants, growth factors,

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glutamate receptor agonists and cation channel blockers have all shown to be effective at attenuating parts of the ischemic cascade (Dilsiz et al., 2006; Osborne et al., 2004; Szabadfi et al., 2010). Pre-treatment methods such as ischemic or hypothermic pre-conditioning also

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appear to activate endogenous pathways which alter gene regulation and lead to improved

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protection against future ischemic insults (Roth et al., 2006; Traustason et al., 2007). However, there is no safe and established clinical treatment for retinal ischemia (Osborne et al., 2004).

Vinpocetine is a natural herbal supplement from the Vinca Minor plant that has been shown to have a beneficial effect in a range of models for ischemia. We have shown that vinpocetine promotes glucose availability and prevents unregulated cation channel permeability in in vitro and in vivo rodent models of retinal ischemia (Nivison-Smith et al.,

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2014; Nivison-Smith et al., 2015). Vinpocetine also has neuroprotective effects in in vitro ischemia models generated using brain-derived glia (Vas and Gulyas, 2005) and in vivo rodent models of cerebral ischemia (Jincai et al., 2014; Rischke and Krieglstein, 1990, 1991; Sauer et al., 1988; Wang et al., 2014). Clinical trials involving patients with ischemic diseases

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such as stroke, chronic ischemic cardiovascular disease and vascular dementia, show improved cerebral blood flow, glucose metabolism and cognitive performance with

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vinpocetine treatment (Bonoczk et al., 2002; Feher et al., 2009; Gulyas et al., 2001; McDaniel et al., 2003; Nagy et al., 1998; Szakall et al., 1998; Szilagyi et al., 2005).

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Vinpocetine treatment has also been assessed in age-related macular degeneration and shown to improve visual function (Avetisov et al., 2007).

The assessment of vinpocetine’s actions in the retina is limited to its application during or after an ischemic insult (Nivison-Smith et al., 2014; Nivison-Smith et al., 2015). However,

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pre-treatment of animal models with vinpocetine prior to cerebral ischemia have found increased blood flow and improved metabolic profile of the affected site compared to non

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pre-treated animals (Rischke and Krieglstein, 1990, 1991; Sauer et al., 1988). Similar effects occur in animals pre-treated with vinpocetine before exposure to NMDA induced

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excitotoxicity damage (Nyakas et al., 2009). Pre-treatment of cultured neurons with vinpocetine provides neuroprotection against glutamate excitotoxicity and reduces reactive oxygen species production and lipid peroxidation following exposure to oxidising agents (Santos et al., 2000; Tarnok et al., 2008). The purpose of this study was to determine the effects of vinpocetine pre-treatment in retinal ischemia. Using an established ex vivo model for retinal ischemia, we assessed the role of vinpocetine pre-treatment in modifying cation channel permeability and calcium

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binding protein immunoreactivity as these processes are typically altered in models of ischemia (Dijk and Kamphuis, 2004a, b; Gunn et al., 2011; Kim et al., 2010; Kwon et al., 2005; Osborne et al., 2004; Osborne et al., 1995; Osborne and Larsen, 1996; Osborne et al., 2002). While vinpocetine treatment post-insult results in improved retinal homeostasis

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(Nivison-Smith et al., 2014; Nivison-Smith et al., 2015), we analyse here whether pre-

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treatment can potentially reduce the severity of the ischemic insult.

Tissue preparation and incubation

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Materials and Methods

All experimental protocols were approved by the University of New South Wales Animal Ethics Committee and were in adherence with the statement for The Use of Animals in

(ARVO).

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Ophthalmic and Vision Research, Association for Research in Vision and Ophthalmology

Six week-old Sprague Dawley rats obtained from Animal Research Centre (Western

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Australia, Australia) were deeply anesthetised using ketamine/medetomidine (75mg/kg/0.5mg/kg), the eyes enucleated (whilst the animals were alive but anaesthetised)

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and the retina dissected and placed on a 0.8µm pore Metricel membrane filter paper (Gelman Sciences, Ann Arbor, MI, USA). The retinal pigment epithelium, choroid and sclera were removed and retina pieces placed in incubation medium in an in-house, ex vivo incubation system (Nivison-Smith et al., 2014). Retina pieces (n = 6 for each condition) were incubated in normal physiological brain buffer containing 125 mM NaCl, 2.5mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 10 mM dextrose, 2 mM CaCl2 and 1 mM MgCl2 (Edwards et al., 1989), bubbled with a gas mixture of 95% O2/5% CO2 at 37˚C. For samples pre-treated with 5

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vinpocetine (14-ethoxycarbonyl-(3α, 16α-ethyl)-14,15-eburnamine; Sigma-Aldrich, St. Louis, MI, USA), retinal pieces were placed in normal physiological buffer with 100µM vinpocetine added and an equimolar reduction in NaCl removed to maintain buffer osmolarity. The vinpocetine concentration used was previously effective against ex vivo ischemia (Nivison-

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Smith et al., 2014; Nivison-Smith et al., 2015). Pre-treatment incubations were performed for 5, 15, 30 or 60 minutes with buffer replenished for incubations over 30 minutes to

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ensure glucose and ionic concentrations remained constant. After vinpocetine pretreatment, retinal samples were transferred to a new chamber containing normal

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physiological buffer without vinpocetine (control conditions) or a chamber with physiological buffer without dextrose and bubbled with a gas mixture of 95% N2/5% CO2 (ischemic conditions) for 35 minutes followed by assessment for cation channel permeability.

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For experiments determining vinpocetine’s effects as a function of duration of the ischemic insult, all tissues were pre-treated with vinpocetine for 60 minutes as described above.

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Retinal samples were then transferred to new chambers and incubated under control or ischemic conditions for 15, 55 or 115 minutes before cation channel assessment.

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Assessment of cation channel permeability Cation channel permeability was assessed through entry of 1-amino-4-guanidobutane (AGB). AGB is an organic cation which permeates through open, non-selective cation channels and has been used to track the cation channel permeability of neurons in a range of tissues including retina in rodent, rabbit and post-mortem human samples, olfactory bulb and frog end-plate (Chua et al., 2009; de Souza et al., 2012a, b; Kalloniatis et al., 2015; Marc, 1999; Marc et al., 2007; Mobley et al., 2008; Nivison-Smith et al., 2014; Nivison-Smith et al., 6

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2013a; Nivison-Smith et al., 2013b; Sun et al., 2007; Yoshikami, 1981; Zhu et al., 2013). Following incubation in control or ischemic conditions, retinal tissues were transferred to a new chamber containing the same incubation buffer with the addition of 25mM AGB for 5 minutes (Nivison-Smith et al., 2014). Total incubation time is quoted as time spent in

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normal/ischemic buffer following vinpocetine pre-treatment (15 – 115 minutes) plus 5 mins incubation time with AGB.

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Immunohistochemistry

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Tissue fixation and immunolabelling was performed after incubations as previously described (i.e. Nivison-Smith et al., 2014; Nivison-Smith et al., 2013a; Nivison-Smith et al., 2015; Nivison-Smith et al., 2013b). Briefly, tissue samples were fixed in 4% (w/v) paraformaldehyde, 0.01% (w/v) glutaraldehyde in 0.1M phosphate buffer for 30 minutes,

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cryoprotected in 30% (w/v) sucrose overnight and cryosectioned in the retinal vertical plane (10 µm) using a Leica Cryostat CM1501S (Leica Microsystems Pty Ltd, Nussloch, Germany). Prior to immunolabelling, retinal sections were blocked against non-specific antibody

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binding (6% (v/v) goat serum, 1% (w/v) bovine serum albumin, 0.1% (v/v) Triton-X, 0.1M

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phosphate buffer, 1 hour at room temperature) then incubated overnight with primary antibodies diluted in an antibody buffer containing 3% (v/v) goat serum, 1% (w/v) bovine serum albumin, 0.1% (v/v) Triton-X at 4˚C (Table 1). Cell markers, calretinin and parvalbumin, were specifically chosen as they label large, functionally important populations of retinal neurons such as AII amacrine cells and cholinergic amacrine cells (Gabriel and Witkovsky, 1998; Voigt, 1986; Wassle et al., 1993) which have been previously shown to have immunoreactivity modified by ischemia (Dijk and Kamphuis, 2004a, b; Gunn et al., 2011; Kim et al., 2010; Kwon et al., 2005; Osborne et al., 1995; Osborne and Larsen, 7

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1996; Osborne et al., 2002) and are amendable to AGB tracking to detect changes in cation channel entry (Chua et al., 2009; Nivison-Smith et al., 2014; Nivison-Smith et al., 2013b; Sun et al., 2007). Caspase 2 was used as a marker of apoptosis in cells of the ganglion cell layer (Singh et al., 2001). Labelling was detected using anti-mouse AlexaFluor 594 or anti-rabbit

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AlexaFluor 488/ AlexaFluor 594 secondary antibodies (Life Technologies, Carlsbad, CA)

diluted 1:500 in antibody buffer for 2 h at room temperature. Sections were counter-stained

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with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, Life Technologies, Carlsbad, CA).

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Image quantification

All samples were imaged using the FluoView FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan). Scans were collected with z-axis sequence with a step size of 1.16

sample.

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μm per frame with 2048 x 800 pixels. A minimum of three sequences were taken per

Quantification was performed on single optical images. Cell counts for cell markers

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calretinin and parvalbumin were performed as previously reported (Chua et al., 2009;

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Nivison-Smith et al., 2014; Nivison-Smith et al., 2013b; Sun et al., 2007) and cells quantified over a minimum 1,400 µm of retina length in all groups (n = 296 – 830 cells in inner nuclear layer; INL; per treatment group; 113 - 327 cells in ganglion cell layer; GCL; per treatment group). The cation channel permeability of these cells was then quantified as co-localisation of AGB labelling with cell marker labelling and expressed as a percentage of the total marker-labelled cells. All data is presented as the mean ± standard error of the mean (SEM). Representative images were modified for brightness and contrast using Adobe Photoshop software (version 12.1, Adobe Systems, Mountain View, CA). No modifications were made 8

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to microscope images used for cell counts. Caspase 2 immunoreactivity was quantified in the GCL only in the same manner as cell markers and plotted as number of immunoreactive cells per retinal length in mm. Glial fibrillary acidic protein (GFAP) immunoreactivity was quantified as described in Nivison-Smith et al. (2015). Briefly, images were converted to

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binary images using ImageJ (National Institutes of Health) and thresholded to remove

background fluorescence. GFAP was quantified as the percentage of GFAP positive pixels

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over the total number of pixels in the area of the inner plexiform layer.

Statistical Analysis

Statistical analysis for pre-treatment timecourse (i.e. Figures 2 and 4) were assessed using a two-way ANOVA using SPSS (Version 23; IBM corporation, Chicago, USA) run for all outcomes, with group (i.e. control or ischemia), time (i.e. minutes of pre-treatment) and the

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group*time interaction in the model. For analysis of duration of pre-treatment protection, a three-way ANOVA was conducted with group (control or ischemia), treatment (with

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vinpocetine or without vinpocetine) and time (minutes of incubation) in SAS 9.4 (SAS Institute Inc., Cary, NC, USA, 2012) with specific hypothesis testing using each variable. For

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caspase-2 labelling, groups were compared using a Student’s t-test for unpaired groups with unequal variance in GraphPad Prism (v6, GraphPad Software, Inc., La Jolla, CA, USA). Significance was considered as p < 0.05.

Results Pre-treatment with vinpocetine protects calretinin positive retinal neurons

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Retinal pieces incubated under normal physiological ex vivo conditions displayed typical calretinin immunoreactivity (Figure 1A). Calretinin positive cells were found within the inner nuclear layer (INL; 60.4 ± 0.92 cells/mm) and to a lesser extent the GCL (36.6 ± 0.72

low cation channel permeability (Figure 1B-C).

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cells/mm). Cell within both these layers had low levels of co-localisation with AGB indicating

Under ischemic conditions, the number of calretinin cells decreased to 38.7 ± 1.42 cells/mm

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in the INL and 14.4 ± 0.90 cells/mm in the GCL (Figure 1D). Co-localisation of calretinin

positive cells with AGB however significantly increased indicating greater cation channel

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permeability of these cells (Figure 1E-F). All results were consistent to previous studies using this ex vivo incubation system (Nivison-Smith et al., 2014; Nivison-Smith et al., 2015). Pre-treatment with vinpocetine (representative images in Figure 1G-R) had no significant

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effect on the number of calretinin positive cells in the INL in the control retina or ischemic retina (two-way ANOVA, p = 0.09 for time, p = 0.578 for interaction; Figure 2A). Vinpocetine pre-treatment time also had no effect on cation channel permeability of calretinin labelled

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cells based on the co-localisation of calretinin and AGB (two-way ANOVA, p = 0.178 for time,

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p = 0.24 for interaction; Figure 2C). In the GCL, vinpocetine pre-treatment had no effect on the control retina (Figure 2B, D) but significantly affected the ischemic retina, increasing the number of calretinin labelled cells in the GCL (two-way ANOVA p < 0.001 for time; Figure 2B) and decreasing cation channel permeability of these cells based on AGB co-localisation (two-way ANOVA, p < 0.001, Figure 2D). Following 60 minutes of pre-treatment, vinpocetine reduced calretinin/AGB colocalisation to levels similar to the control retina (p = 0.81, Figure 2D). Vinpocetine pre-

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treatment however did notmaintain calretinin immunoreactivity of the GCL to that of the normal retina (p<0.001; Figure 2B). Pre-treatment with vinpocetine protects parvalbumin positive retinal neurons

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For parvalbumin labelled cells, cells were predominately found in the INL of the control retina (42.1 ±2.31 cells/mm) rather than the GCL (16.5 ±0.62 cells/mm; Figure 3A) and had low cation channel permeability based on low co-localisation with AGB as seen with

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calretinin labelled cells (Figure 3B-C). In the ischemic retina, less parvalbumin positive cells were observed in the INL but no change was observed in the GCL (Figure 3D). Both cell

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groups showed more co-localisation with AGB in the ischemic retina (Figure 3E-F). Vinpocetine pre-treatment significantly increased the number of parvalbumin positive cells in the INL of the control and ischemic retina in a time dependent manner (two-way ANOVA:

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p < 0.001 for time, Figure 4A) but had no effect on cation channel permeability of these cells (two-way ANOVA, p = 0.525 for time, p = 0.835 for interaction; Figure 4C). In contrast, vinpocetine pre-treatment had no effect on the number of parvalbumin cells in the GCL of

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the control or ischemic retina (two-way ANOVA: p = 0.097 for time, Figure 4B) but significantly decreased cation channel permeability of these cells in the ischemic retina

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(two-way ANOVA, p < 0.001 for time and interaction; Figure 4D). Pre-treatment of ischemic retinae for 30 or 60 minutes lead to co-localisation values which were not significantly different to the control retina (Figure 4D).

Vinpocetine’s pre-treatment effects as a function of ischemic insult duration

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Following confirmation that vinpocetine pre-treatment can protect against ischemic insult, we evaluated the effect of vinpocetine’s pre-treatment as a function of the duration of ischemic insult. Retinal samples were pre-treated with vinpocetine for 60 minutes then exposed to normal physiological buffer or ischemic insults for 20, 60 and 120 minutes

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(Figures 5 – 6).

The number of calretinin positive cells in the INL of the control and ischemic retina

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significantly decreased throughout the incubation but to a greater extent in the latter

(three-way ANOVA: p <0.001; Figure 5I). Pre-treatment with vinpocetine (dotted lines) had

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no significant effect on these cells in either the control or ischemic retina (p = 0.83 for control, p = 0.99 for treatment*time interaction; Figure 5I). In the GCL, the number of calretinin positive cells also significantly decreased with ischemia duration (three-way ANOVA: p = 0.05 for time for untreated ischemia; Figure 5J). Pre-treatment with vinpocetine

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increased the number of calretinin cells in the GCL in the ischemic retina following 20 minutes of insult but this effect was lost at the 60 and 120 minute time points (three-way

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ANOVA: p = 0.82 for treatment*time interaction Figure 5J). For cation channel permeability, calretinin positive cells in the INL of the control retina

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demonstrated a slight but significant increase in AGB co-localisation over time (p < 0.05 for untreated control). This was significantly less than the ischemic retina, where co-localisation significantly increased with insult duration (p < 0.001 for time for ischemia; Figure 5K). Vinpocetine pre-treatment had no effect on co-localisation in the INL of the control or ischemic retina (p = 0.76 for control, p = 0.99 for ischemia for treatment*time; Figure 5K). In the GCL, cation channel permeability of calretinin cells was also low at all time points in the control retina (p = 0.29) but significantly increased in the ischemic retina with incubation 12

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duration (p < 0.001; Figure 5L). Vinpocetine pre-treatment of the control retina resulted in a significant increase in AGB-calretinin co-localisation over time (p < 0.01 for time) but overall no significant interaction difference between the untreated and treated control retina (three-way ANOVA: p = 0.98 for treatment*time interaction for ischemia). In the ischemic

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retina, co-localisation was lowered at 20 minutes in vinpocetine treated samples then

returned to levels not significantly different from the untreated retina by 120 minutes of

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incubation (three-way ANOVA: p = 0.93 for treatment*time interaction for ischemia; Figure 5L).

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For parvalbumin positive cells, the number of cells in the INL did not change following incubation under normal physiological conditions (three-way ANOVA, p = 0.89 for untreated control with time) but significantly decreased in the INL with ischemic insult of up to 120 minutes (p < 0.01; Figure 6I-J). Pre-treatment with vinpocetine appeared to increase the

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number of parvalbumin cells in the INL following 20 minutes of ischemia but overall there was no significant difference with vinpocetine treatment (three-way ANOVA: p = 0.79 for

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treatment*time interaction for ischemia; Figure 6I). In the GCL, treatment and insult duration had no effect on number of parvalbumin labelled cells in the ischemic retina

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(Figure 6J).

Cation channel permeability of parvalbumin cells in the INL was similar to calretinin labelled cells: co-localisation remained low at all time points in the control retina but increased significantly in the ischemic retina in a time dependent manner (three-way ANOVA: p = 0.31 for time for untreated control vs p <0.001 for time for untreated ischemia; Figure 6K). Vinpocetine pre-treatment had no effect on these cells of the INL in the control retina or

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ischemic retina (three-way ANOVA: p = 0.99 for control, p = 0.99 for ischemia for treatment*time interaction; Figure 6K). In the GCL, parvalbumin and AGB co-localisation significantly increased over time for all

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groups although co-localisation in the control retina significantly was less than that of the ischemic retina (three-way ANOVA: p < 0.01 for group; Figure 6L). Vinpocetine pre-

treatment significantly increased co-localisation of parvalbumin cells in the GCL across the

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time course in the control retina (p < 0.001) but there was not significant interaction difference between untreated and treated control retina over time (p = 0.77 for

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treatment*time). In the ischemic retina, vinpocetine pre-treatment decreased cation channel permeability following 20 minutes of insult but showed no significant effect across the whole 120 minutes time course (three-way ANOVA: p = 0.88 for treatment*time; Figure

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6L).

Finally, we assessed whether vinpocetine pre-treatment was associated with changes in RGC cell death, a hallmark of retinal ischemia (Figure 7). Based on the apoptotic marker caspase

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2 , there was a significantly greater number of caspase 2 positive cells in the untreated ischemic retina following a 120 minute insult compared to treated or untreated control

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retinae as expected (p < 0.05, Student’s t-test). Pre-treatment with vinpocetine significantly lowered the number of caspase 2 positive cells in the ischemic retinae (p < 0.001, Student’s t-test) to levels not significantly different to control retinae (p = 0.1; Figure 7B). Effect of vinpocetine on Müller cells Vinpocetine has been previously shown to modify GFAP immunoreactivity in Müller cells when present during an ischemic insult (Nivison-Smith et al., 2015). We assessed GFAP

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immunoreactivity following pre-treatment with vinpocetine to determine if it could still protect against glia hyperreactivity. GFAP immunoreactivity was low in control retina with or without vinpocetine pre-treatment, restricted mostly to the GCL (Figure 8A, C). In the untreated ischemic retina, GFAP immunoreactivity was greater than the control retina and

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observed to expand from the GCL to the IPL (Figure 8B). In the ischemic retina pre-treated with vinpocetine, a reduced level of GFAP immunoreactivity was observed in the IPL. Time

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course assessment indicated vinpocetine pre-treatment followed by up to 120 minutes of ischemia significantly decreased GFAP immunoreactivity in the IPL compared to untreated

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ischemic retina. Pre-treated ischaemia retinae still had significantly more GFAP immunoreactivity than control retinae (p <0.05; Figure 8E).

Discussion

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This is the first study to find that vinpocetine pre-treatment can protect the retina against ischemic insult as evidenced by reduced cation channel permeability, preservation of calretinin and parvalbumin immunoreactivity of inner retinal neurons, decreased apoptotic

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marker expression and reduced glial cell gliosis. Against a 40 minute ischemic insult, vinpocetine was protective when provided for at least 15 minutes prior to the insult. Many

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of these effects however were lost when the duration of the ischemic insult exceeded 60 minutes except caspase 2 in the GCL and GFAP immunoreactivity in the IPL which remained reduced for up to 120 minutes of ischemic insult in vinpocetine pre-treated retina. Vinpocetine pre-treatment is effective against retinal ischemia Against a 40 minute ischemic insult, vinpocetine could protect retinal tissue with only 15 minutes of pre-treatment. This is within the pre-treatment time frames shown to be effective in other in vitro studies using brain neurons subjected to ischemia or 15

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pharmacologically induced glutamate excitotoxicity (Lakics et al., 1995; Rischke and Krieglstein, 1990, 1991; Sauer et al., 1988; Tarnok et al., 2008; Zelles et al., 2001). Whilst no study has specifically determined the uptake of vinpocetine by cells in vitro, studies in cynomolgous monkeys show vinpocetine rapidly crosses the blood/brain barrier and is

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taken up by brain tissue when administered intravenously (within 2 minutes; Gulyas et al. (1999)). Vas et al. (2002) also demonstrated that vinpocetine appears in blood serum only

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20 minutes following oral administration in humans. Vinpocetine is also neuroprotective when provided during an ischemic or pharmacological insult (Erdo et al., 1990; Sitges et al.,

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2006; Sitges et al., 2005; Vas and Gulyas, 2005; Wang et al., 2014), suggesting its actions under these conditions are unlikely to involve long-term mechanisms such as gene expression or neurogenesis.

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Vinpocetine pre-treatment actions are not identical to those of post-treatment We found vinpocetine’s pre-treatment effects on cation channel permeability and calretinin/parvalbumin immunoreactivity were quite similar to those observed when the

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drug was administered during an ischemic insult in the INL but not necessarily the GCL (Figure 9; Nivison-Smith et al., 2014). For example, vinpocetine administered during an

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ischemic insult typically reduced loss of calretinin and parvalbumin immunoreactivity (Nivison-Smith et al., 2014) but in this study when vinpocetine was administered as a pretreatment, this effect was much less pronounced (Figure 9). Calretinin and parvalbumin are calcium binding proteins which may potentially provide neuroprotection through buffering of intracellular Ca2+ (Osborne, 1999; Osborne et al., 2004; Osborne et al., 1995; Osborne and Larsen, 1996; Sitges et al., 2005). Considering changes in intracellular Ca2+ occur during ischemia rather than before, vinpocetine actions relating to these calcium binding proteins

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may only be activated in the presence of this cation imbalance. It is currently not clear how vinpocetine interacts with these proteins, however it is unlikely the drug directly binds to these proteins, as Hagiwara et al. (1984) found vinpocetine did not directly interact with

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other calcium binding proteins, notably calmodulin. In terms of cation entry, vinpocetine pre-treatment appears to be more effective at

reducing unregulated cation entry into calretinin and parvalbumin labelled retinal neurons

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in the GCL than vinpocetine administered during ischemia (Figure 9; Nivison-Smith et al., 2014). The localisation of vinpocetine’s effects to this retinal layer is important as ganglion

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cells have been shown to be highly susceptible to ischemic cell death (Chidlow and Osborne, 2003; Goto et al., 2002; Kergoat et al., 2006; Lafuente et al., 2002; Mukaida et al., 2004). However, as we did not confirm the identity of immunoreactive cells in the GCL, some of these cells could also be displaced amacrine cells. This is particularly critical for calretinin

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labelled cells as cholinergic amacrine cells, one of the largest populations of calretinin labelled amacrine cells in the rodent retina and are commonly displaced in the GCL (Gabriel

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and Witkovsky, 1998; Voigt, 1986). Vinpocetine could modulate cation channel permeability of cholinergic amacrine cells through its actions on the Nav1.8 ion channel or NMDA

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glutamate receptors, both localised to cholinergic amacrine cells (Erdo et al., 1990; Kiss et al., 1991; Nivison-Smith et al., 2014; Nyakas et al., 2009; O'Brien et al., 2008; Zhou et al., 2003). A limitation of this study was that we only examined the actions of vinpocetine on two cell markers and therefore do not know if vinpocetine exhibits additional effects in other retinal areas. Further investigation using other cell markers such as cholinergic acetylcholine transferase (a specific cell marker for cholinergic amacrine cells) is currently underway.

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Interestingly, pre-treatment with vinpocetine significantly increased cation channel permeability of cells in the GCL layer of the control retina despite conversely decreasing cation channel permeability of these cells in the ischemic retina. These actions are consistent with those observed previously (Nivison-Smith et al., 2014; Nivison-Smith et al.,

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2015) and suggest vinpocetine may have different mechanisms of protecting against

ischemia when administered to control tissue. The increase in cation channel permeability

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in the normal retina by vinpocetine – essentially reflecting an “ischemic-like” action by a neuroprotective compound may be similar to actions associated with ischemic pre-

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conditioning – a phenomenon whereby a short, prior ischemic insult leads to neuroprotection against future insults. Indeed, ischemic pre-conditioning has been shown to be associated with activating endogenous defence mechanisms via adenosine (Sakamoto et al., 2004) and vinpocetine is known to inhibit the re-uptake of adenosine by neurons

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(Krieglstein and Rischke, 1991) thereby potentially priming normal retinal neurons against ischemia. Whilst further investigation is warranted on the matter to ensure vinpocetine does not have detrimental effects on normal tissue, its current, widespread use as a

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supplement withlimited side effects (Balestreri et al., 1987), suggest the changes in normal

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cation permeability are unlikely to have detrimental consequences. In addition, labelling with apoptotic markers, namely caspase 2 indicated no increase in cell death of the ganglion cell layer following vinpocetine pre-treatment of the normal retina. Pre-treatment with vinpocetine was only effective short-term In our model, vinpocetine’s effect on cation channel permeability and cell markers for retinal neurons was limited to ischemic insults of less than 60 minutes duration. This could be related to vinpocetine’s short half-life, reported as 1-2h in serum (Bonoczk et al., 2000)

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and/or the length of the ischemic insult which caused more cellular damage than can be counteracted with vinpocetine. Interestingly, vinpocetine’s effect on glial cell reactivity and neuronal cell death extended beyond this timeframe with reduced caspase 2 and GFAP immunoreactivity still evident up to 120 minutes post pre-treatment suggesting not all of

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vinpocetine’s effects may be short term. In addition, clinical studies show single daily doses of vinpocetine improves cognitive outcomes for patients with chronic cerebrovascular

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dysfunction (Balestreri et al., 1987), dementia (Hindmarch et al., 1991) and degenerative central nervous system disorders (Manconi et al., 1986) suggesting the drug may have

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applicability in clinical scenarios when provided beyond a single pre-treatment dose.Cochrane reviews on vinpocetine’s effects for acute ischemic stroke and dementia however have concluded current clinical trials are insufficient to draw solid conclusions and therefore additional studies incorporating additional pre-treatment time points and

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alternative ischemic insult models may be needed to determine whether vinpocetine’s short-term actions are effective in preventing long-term damage from retinal ischemia

Conclusion

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(Bereczki and Fekete, 2008; Szatmari and Whitehouse, 2003).

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This study demonstrates for the first time that pre-treatment of retinal tissue can protect it from impending ischemic insults. We found that a maximal pre-treatment effect was obtained with only 15 minutes and that pre-treatment for 60 minutes resulted in protection of inner retinal neurons from ischemic damage but many of these effects were lost for insults over 1 hour in duration. Vinpocetine pre-treatment reduced cation channel permeability in the GCL to a greater extent than vinpocetine treatment administered during an ischemic insult. Vinpocetine also effectively reduced immunoreactivity of GFAP in the IPL 19

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suggesting its actions may also affect Müller cells. These results suggest administration of vinpocetine prior to a metabolic insult may protect the inner retinal neurons from ischemic

Conflicts of interest

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All the authors certify that there is no conflict of interest.

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damage.

Acknowledgements

This work was supported, in part, by research grants from the National Health and Medical Research Council of Australia (#1033224), the University of New South Wales Early Career

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Research Grant 2015 and 2016 (#PS35430) and the New Zealand Optometric Vision Research Foundation (NZOVRF #3625663). Guide Dogs NSW/ACT is also a partner on the NHMRC grant and provided support for LN-S. The authors would also like to thank Natalie

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analysis.

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Lim, Andrea Pham for technical support and Nancy Briggs for assistance on statistical

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Figure Legends Figure 1: Representative images of rat retinae that were (A-F) untreated, (G-L) pre-treated with vinpocetine for 15 minutes or (M-R) pre-treated with vinpocetine for 60 minutes

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following incubation in normal physiological buffer (denoted control, A-C, G-I, M-O) or ischemic buffer (D-F, J-L, P-R). All retinae were labelled with calretinin (red) and AGB (green) and co-localisation ratio was used to determine AGB entry into calretinin immunoreactive

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cells. Scale bar is 20μm. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer;

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GCL, ganglion cell layer.

Figure 2: Labelling density and cation channel permeability of calretinin cells as a function of vinpocetine pre-treatment duration. Cells were pre-treated with vinpocetine for 0 – 60 mins then subjected to 40 mins of normal physiological conditions (black lines) or ischemic

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conditions (red lines). The total number of calretinin immunoreactive cells per mm length of retina (n=6 for each condition) in the (A) INL and (B) GCL was determined and used to determine the percentage of calretinin cells co-localised with AGB in the (C) INL and (D) GCL.

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Asterisks indicate significant difference in interaction effects between the two groups based

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on a two-way ANOVA. *** p < 0.001. Figure 3: Representative images of rat retinae that were (A-F) untreated, (G-L) pre-treated with vinpocetine for 15 minutes or (M-R) pre-treated with vinpocetine for 60 minutes following incubation in normal physiological conditions (denoted control, A-C, G-I, M-O) or ischemic conditions (D-F, J-L, P-R). All retinae were labelled with parvalbumin (red) and AGB (green) and co-localisation used to determine AGB entry into parvalbumin immunoreactive cells. Scale bar is 20μm. Abbreviations as in Figure 1.

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Figure 4: Labelling density and cation channel permeability of parvalbumin cells as a function of vinpocetine pre-treatment duration. Cells were pre-treated with vinpocetine for 0 – 60 mins then subjected to 40 mins of normal physiological conditions (black lines) or ischemic conditions (red lines). The number of calretinin immunoreactive cells per mm

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length of retina (n=6 for each condition) in the (A) INL and (B) GCL was determined and used to determine the percentage of parvalbumin cells co-localised with AGB in the (C) INL and

groups based on a two-way ANOVA. *** p < 0.001.

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(D) GCL. Asterisks indicate significant difference in interaction effects between the two

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Figure 5: Vinpocetine’s (vinp) pre-treatment effects on calretinin immunoreactive cells as a function of ischemia duration. Retinae were pre-treated with vinpocetine or normal physiological buffer for 60 mins then incubated in normal or ischemic buffer for 20, 60 or 120 mins. Representative images demonstrate (A – B) untreated or (C - D) vinpocetine pre-

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treated control retina following a (A, C) 20 min and (B, D) 60 min incubation and (E – F) untreated or (G - H) vinpocetine pre-treated ischemic retina following a (E, G) 20 min and (F,

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H) 60 min incubation. The number of calretinin positive cells per mm length of retina (n=6 for each condition) in the (I) INL and (J) GCL was determined and the percentage of these

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calretinin cells co-localised with AGB was quantified for the (K) INL and (L) GCL. Scale bar is 20μm. All other abbreviations as in Figure 1. Figure 6: Vinpocetine’s (vinp) pre-treatment effects on parvalbumin immunoreactive cells as a function of ischemia duration. Retinae were pre-treated with vinpocetine or normal physiological buffer for 60 mins then incubated in normal or ischemic buffer for 20, 60 or 120 mins. Representative images demonstrate (A – B) untreated or (C - D) vinpocetine pretreated control retina following a (A, C) 20 min and (B, D) 60 min incubation and (E – F)

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untreated or (G - H) vinpocetine pre-treated ischemic retina following a (E, G) 20 min and (F, H) 60 min incubation. The number of parvalbumin positive cells per mm length of retina (n=6 for each condition) in the (I) INL and (J) GCL was determined and the percentage of

Scale bar is 20μm. All other abbreviations as in Figure 1.

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these parvalbumin cells co-localised with AGB was quantified for the (K) INL and (L) GCL.

Figure 7: Vinpocetine’s (vinp) pre-treatment effects on caspase 2 immunoreactivity. (A)

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Representative images demonstrating caspase 2 immunoreactivity (green) of untreated and vinpocetine treated retinae followed by incubation in normal physiologically (control) or

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ischemic conditions for 120 minutes. (B) Caspase 2 immunoreactivity quantified as number of immunopositive cells in the ganglion cell layer per mm of retinal length (n=6 for each condition). Scale bar is 20μm. ** p < 0.01. All other abbreviations as in Figure 1.

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Figure 8: Effect of vinpocetine pre-treatment on GFAP immunoreactivity. Representative images demonstrate GFAP immunoreactivity (green) of untreated retinae incubated in (A) normal physiological buffer or (B) ischemic buffer for 120 minutes. Other images represent

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vinpocetine pre-treated retinae (C) exposed to 120 minute incubation in normal physiological buffer or (D) 120 minutes of ischemia. GFAP immunoreactivity for each

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condition was then quantified as (E) the percentage of immunopositive pixels within the total area of the IPL (n=6 for each condition). Scale bar is 20μm. All other abbreviations as in Figure 1.

Figure 9: Summary of vinpocetine’s actions in retinal ischemia. The actions of vinpocetine to bring cell marker loss (top bars) or unregulated cation entry (based on AGB co-localisation, bottom bars) from ischemic levels to normal levels is compared when the drug is administered during a retinal insult (denoted by post-treatment arrows; extracted from 23

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(Nivison-Smith et al., 2014) or as a pre-treatment prior to retinal ischemia (denoted by pretreatment arrows; extracted from this study). Note vinpocetine’s actions are essentially identical in the INL regardless of time of administration but varied in the GCL and vinpocetine pre-treatment is more effective then post-treatment for reducing unregulated

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cation entry but not reducing cell marker loss. All comparisons are made for identical

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ischemia durations (40 mins).

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Zhu, Y., Misra, S., Nivison-Smith, L., Acosta, M.L., Fletcher, E.L., Kalloniatis, M., 2013. Mapping cation entry in photoreceptors and inner retinal neurons during early degeneration in the P23H-3 rat retina. Vis Neurosci 30, 65-75.

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ACCEPTED MANUSCRIPT Table 1: Details of the antibodies used in this study. Abbreviations – Rb, rabbit; Ms, mouse. Manufacturer, Cat No

Host

Dilution

Agmatine (AGB)

Agmatine conjugated to bovine serum albumin

Millipore; AB1568

Rb; polyclonal

1:100

Calretinin (CR)

Rat calretinin, amino acids 38-151

BD Transduction

Ms; monoclonal

1:1000

Glial fibrillary acidic protein (GFAP)

GFAP from pig spinal cord

Sigma Aldrich; G3893

Ms; monoclonal

1:1000

Parvalbumin (PV)

Frog muscle parvalbumin

Sigma-Aldrich; P3088

Ms; monoclonal

1:500

RI PT

Immunogen

AC C

EP

TE D

M AN U

SC

Antigen

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Highlights Vinpocetine pretreatment reduces neuron cation channel permeability during ischemia

•

Vinpocetine pretreatment can reduce glial cell reactivity in retinal ischemia

•

Vinpocetine pretreatment effects on cell marker inmmunoreactivity differs to posttreatment

•

Vinpocetine pretreatment effects on ischemic retinal neurons is transient

AC C

EP

TE D

M AN U

SC

RI PT

•