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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: J Neurochem. 2016 Sep 27;139(2):270–284. doi: 10.1111/jnc.13770

Calpain Inhibition Reduces Structural and Functional Impairment of Retinal Ganglion Cells in Experimental Optic Neuritis

Amena W Smith 1, Baerbel Rohrer 1,3,5,*, Lee Wheless 4, Supriti Samantaray 2, Swapan K Ray 5, Jun Inoue 7, Mitsuyoshi Azuma 7, Naren L Banik 2,3,6,*
PMCID: PMC5056830  NIHMSID: NIHMS810209  PMID: 27513991

Abstract

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis (MS). ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells (RGCs) and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrifice (Day 26). Visual function was determined by electroretinogram (ERG) recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias towards unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern ERG amplitudes and paralysis, with attenuation of RGC death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with MS. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON.

Graphical Abstract

As in multiple sclerosis patients, optic neuritis and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE). Daily oral treatment with calpain inhibitor SNJ1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a) and their axons (myelin oligodendrocyte specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in optic neuritis.

graphic file with name nihms810209u1.jpg

INTRODUCTION

Manifestations of optic neuritis (ON), inflammation of the optic nerve, include decreased sensation of light brightness, muted color vision, and reduced visual acuity, which are the initial signs of multiple sclerosis (MS) in 15–20% of patients (Arnold 2005). The visual dysfunction that accompanies ON is a common cause of disability in MS (Mowry et al. 2009). There is recent evidence that significant macular volume loss, particularly in the ganglion cell layer and inner plexiform layer, occurs in MS patients, even in those who do not report ON (Walter et al. 2012). Moreover, Optical Coherence Tomography (OCT) studies have demonstrated a direct relationship between disease severity in MS (e.g. controls versus relapsing remitting versus secondary progressive) or MS-associated ON (Clinically Isolated Syndrome versus 1st ON versus Recurrent ON) and the thickness of the ganglion cell layer (Saidha et al. 2011, Fernandes et al. 2012). Saidha et al, reported a decrease in combined ganglion cell layer and inner plexiform layer thickness among patients with secondary progressive MS compared with healthy controls. Importantly, thickness of the ganglion cell and inner plexiform layers is correlated with ability of patients to read Snellen charts with low-contrast letters (Saidha et al. 2011). Deficits in these areas are associated with worse quality of life (Sakai et al. 2011). Although intravenous methylprednisolone hastens visual recovery and reduces the risk of MS after a clinically isolated syndrome, it does not improve chronic visual outcomes (Osborne & Volpe 2009).

In this study, the experimental autoimmune encephalomyelitis (EAE) model was used to study the onset and progression of ON as well as the efficacy of intervention with a putative neuroprotective compound. Serial MRI studies of a prospective cohort of EAE mice revealed that disruption of the blood–optic nerve barrier began as early as 3 days post-immunization and resolved spontaneously at 2 weeks (Guy 2008). This suggests that ON may be an early event during EAE progression. As observed in MS patients, EAE-ON animals also demonstrate decreased visual evoked potential (VEP) and electroretinogram (ERG) recordings, implicating visual dysfunction due to pathophysiological insult to the optic nerve and retina (Pietrobon et al. 1990, Banik et al. 1994, Petratos et al. 2010). Furthermore, in a mouse model of MS, the pattern ERG (pERG) amplitude was decreased early relative to gait abnormality or paralysis score and before CNS demyelination was evident by MRI (Enriquez-Algeciras et al. 2011). Perivascular cuffing of immune cells, splitting of the myelin lamellae, and cleavage of myelin and cytoskeletal proteins, such as α-spectrin and neurofilament protein (NFP), have also been shown in optic nerves of EAE-ON animals (Banik et al. 1985, Shields & Banik 1998). The aforementioned myelin and cytoskeletal proteins are substrates of the activated calcium (Ca2+)-dependent neutral protease calpain (Ray et al. 2002, Greenwood et al. 1993, Zimmerman & Schlaepfer 1982). Calpain activation is also integral to immunological events including T cell activation (Gerondakis et al. 1998) and chemotaxis (Butler et al. 2009), signal transduction (Hendry & John 2004), and apoptosis (Wu et al. 2007). Increased expression and activity of μ-calpain and m-calpain, requiring μM and mM Ca2+ concentrations for activation, respectively, has been detected in EAE-ON optic nerves prior to paralysis onset (Shields & Banik 1998). We recently demonstrated that calpain inhibition prevents apoptosis of RGCs in the retina of EAE rats by downregulating expression of pro-apoptotic proteins and the pro-inflammatory molecule nuclear factor-kappa B (NF-κB) (Smith et al. 2011b). Thus, calpain inhibition may represent a novel strategy for the preservation of visual function in ON.

Few studies have investigated function of the visual pathway throughout the EAE disease course. Since the blood-optic nerve barrier is compromised early, parameters of vision in ON mice should be routinely evaluated to further our understanding of the disease process and determine the efficacy of novel treatments.

As aforementioned, measurement of visual acuity (VA) is widely used to quickly assess MS patients’ visual ability. VA is clarity of vision, especially form vision (e.g. letters, checks, bars), which depends on three components: optics, the health and resolving power of the retina, and finally the interpretative faculty of the visual cortex. To quantify VA is to measure the ability of a subject to identify black symbols on a white background at a standardized distance as the size of the symbols is varied, also known as the spatial frequency of the forms. An optomotor behavioral method that records the reflexive optokinetic response (OKR) of rodents watching a changing pattern stimulus consisting of black/white vertical bars has been developed to determine the highest spatial frequency a mouse responds to during a testing session (Douglas et al. 2005). The OKR is a class of compensatory eye movements that function to stabilize images on the retina during self-motion and/or motion of the visual surround. It is controlled primarily through direct projections from the retina to the accessory optic system (Douglas et al. 2005, Nakajima et al. 2011, Jeon et al. 2012, Lee et al. 2012), and corresponding pathways are found in humans and lab rodents (Koh et al. 2012, Surh et al. 2001).

The present study determined that EAE mice manifest losses in OKR sensitivity throughout the progression of ON. Staining of EAE retinas revealed significant RGC apoptosis. Daily oral treatment with the calpain inhibitor SNJ 1945, initiated after the onset of OKR threshold decline, improved OKR sensitivity, inner retinal function, and paralysis, with associated attenuation of histopathology.

MATERIALS AND METHODS

EAE-ON Induction

An EAE model was selected which would produce a high mean disease severity score, for evaluation of the therapeutic efficacy of a calpain inhibitor (Papenfuss et al. 2004). Accordingly, male B10.PL mice (6–8 weeks) were obtained from the Jackson Laboratory and provided water and food pellets ad libitum. Mice were immunized subcutaneously in each flank with a 100 μL emulsion containing 200 μg of guinea pig myelin basic protein (MBP) in sterile saline emulsified with an equal volume of complete Freund’s adjuvant (CFA) supplemented with heat-inactivated Mycobacterium tuberculosis (10 mg/ml). Pertussis toxin (100 ng) was administered intraperitoneally at the time of immunization on days 0 and 48 h later. Control mice were injected with saline/CFA only. Paralysis scores of experimental animals were assigned by a masked investigator as follows: 0, no clinical disease; 0.5, piloerection; 1, tail weakness; 1.5, tail paralysis; 2, hindlimb weakness; 3, hindlimb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; 5, moribund or death. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the MUSC Animal Care and Use Committee.

Administration of Calpain Inhibitor

Mice received b.i.d. oral dosing of the calpain inhibitor SNJ 1945 (50 mg/kg) or 0.5% carboxymethylcellulose vehicle from Day 9 post-induction until sacrifice on Day 26. SNJ 1945 is a dipeptidyl-ketoamide 5 derived from SJA 6017 that contains an amphipathic moiety. This compound has a much higher penetration into the retina after oral administration than SJA 6017. SNJ 1945 also exhibits an excellent oral bioavailability and a long half-life (4.3 hours). It has high enzyme inhibitory activity for m-calpain (IC50: 0.062 μM) and m-calpain (IC50: 0.045 μM).

Measurement of optokinetic response thresholds

The OptoMotry® Virtual Optomotor System (VOS, Cerebral Mechanics, New York, NY) was utilized to record quick, daily assessment of rodent visual acuity via recording of optokinetic responses (OKR) (Prusky et al. 2004). The moving, full-field stimulus (a large, rotating virtual drum) invokes slow eye and head movements in the direction of rotation. With prolonged rotation, the compensatory slow eye movements are interrupted by quick repositioning fast phases, or saccades in the opposite direction. The eye movements form the OKR while the head movements are called tracking. Use of a virtual cylinder allows the stimulus pattern to be easily changed. Also, by monitoring the position of a freely moving animal inside the system with a camera, the virtual drum can be kept centered on the head. Recognition of specific spatial frequencies (cycles/degree) are indicated by a tracking movement of the rodent’s head. The experimenter records a “yes” or “no” reaction to the change, which increases or decreases the stimulus frequency until a spatial frequency threshold is attained separately for each eye (Douglas et al. 2005). Spatial frequency thresholds of EAE-ON and control mice were recorded at 100% contrast and a constant drift speed from the day prior to induction of EAE until Day 25.

Pattern ERG (pERG) recordings

The pERG directly accesses activity of RGCs (Porciatti 2007, Porciatti et al. 2007). On Day 25 post-EAE induction mice were anesthetized with intraperitoneal injections of a mixture of Ketamine (80 mg/kg) and Xylazine (10 mg/kg) and were kept at a constant body temperature of 37.0°C using a feedback-controlled heating pad. The recording electrode is a thin (0.25 mm diameter) gold wire configured to a semicircular loop of 2 mm radius, which is carefully placed on the corneal surface ensuring that the pupil is encircled without obstructing vision. The reference electrode is a small stainless steel needle (12 mm (1/2 inch) × 29 gauge, LKC Technologies, Gaithersburg, MD) inserted into the skin just superior/dorsal to the nose. Sterile saline drops were administered to the eye surfaces every 30 minutes to maintain the cornea and lens in optimally hydrated condition. Pattern stimuli consisted of vertical bars presented at a 0.1 c/d spatial frequency at 100% contrast and a 1Hz pattern reversal frequency. Stimuli were displayed on a TV monitor whose center was aligned with the visual axis and presented from a short distance (15 cm) to stimulate a large retinal area centered on the optic disc. Substantial averaging (150 sweeps) was used to isolate the response from background noise. The first, most positive peak amplitude (P1) and the immediately following negative trough (N2) were measured from each left and right eye recording, evaluating both amplitudes and implicit times.

Luxol Fast Blue staining of optic nerves

Mouse optic nerves were dissected on day 26 and Luxol Fast Blue (LFB) staining was performed as reported previously (Tyor et al. 2002). Briefly, slides were stained for 4 hours at 60°C in 0.1% LFB (Solvent Blue 38; Sigma-Aldrich, St. Louis, MO) in acidified 95% ethanol. Differentiation and counterstaining were performed with 0.01% Li2CO3 and incubation in nuclear fast red [0.1% in 5% Al2(SO4)3] for 5 min. Stained sections were viewed on an Olympus microscope (BH-2; Olympus, Melville, NY) at 200x magnification. Images were captured with a Magna Fire SP CCD camera (Optronics, Goleta, CA). A blinded investigator ranked the demyelination score within optic nerve. Total number of nuclei stained with nuclear fast red were counted within a 300 μm2 region of each of 3 optic nerve images per mouse using ImageJ software (NIH, Bethesda, MD), and the mean nuclei count was calculated for each treatment group. Data was expressed as nuclei/tissue section.

Immunohistochemistry

Enucleated eyes were collected on Day 26 and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 4 hour. After fixation, the eyes were hemisected in dorso-ventral orientation through the optic nerve and the superior and inferior oblique muscles, to ensure the same orientation for all eyes. The tissue was then saturated in 30% sucrose, embedded in OCT, and cryosectioned (14 μm). Optic nerves were cryosectioned (7 μm) and fixed for 15 minutes in 95% ethanol. After washing in PBS, slides containing tissue sections were blocked for 1 hour with a 2% solution of the same blocking serum that was used to dilute the secondary antibody. The PBS solution was then replaced by a primary antibody solution containing an anti-rabbit polyclonal m-calpain or μ-calpain antibody raised in our lab (Banik et al. 1983), or antibodies raised against NeuN (neuronal nuclear marker, 1:500, Millipore, Billerica, MA), Brain-specific homeobox/POU domain protein 3A (Brn3a, RGC marker, 1:200, Santa Cruz Biotechnologies, Santa Cruz, CA), myelin oligodendrocyte specific protein (MOSP, oligodendrocyte marker (1:500, Millipore), dephosphorylated neurofilament protein (deNFP, axonal degradation marker, 1:1000, Covance, Princeton, NJ), neurofilament protein (NFP, axonal marker, 1:1000, Covance) or glial fibrillary acidic protein (GFAP, astrocyte marker, 1:500, Millipore) overnight at 4°C. After washing, the sections were incubated for 30 min in the dark in blocking solution containing the appropriate secondary antibody conjugated with FITC or Texas Red (1:100, Vector Laboratories, Burlingame, CA). The slides were mounted with 1 drop of Vectashield Mounting Medium with or without DAPI (Vector Laboratories) and coverslipped. The sections were viewed under a fluorescence microscope at 200x magnification (Carl Zeiss Meditec, Dublin, CA).

Image analysis

For quantification of antibody binding in the optic nerve, the total area of each of 3 optic nerve images per mouse was selected, and the mean pixel density quantified using ImageJ software (NIH, Bethesda, MD) as described previously (Sribnick et al. 2005). For RGC counts, Brn3a-positive cell bodies were counted within four fields (approximately 400 μm in length) in two identical areas of each central retina and two areas of each peripheral retina (one eye each from 3–5 animals, 4 sections per eye), using ImageJ software (NIH). The fields were placed at a maximum of 500 μm from the optic disk. Brn3a-positive cells were counted in each field, excluding cells that were also TUNEL positive, the values were averaged and the standard error of the means (SEM) was calculated. The percentage difference from the control-vehicle counts, set at 100%, was calculated for each treatment group.

Statistical Analyses

Optokinetic response profiles were investigated over time using a linear mixed effects model for repeated measures (Bolker et al. 2009). The overall model has the form acuity = treatment day treatment*day. The interaction term of treatment*day was included to determine if there is a different effect of the treatment over time. Moreover, the direction of the grating rotation (right or left) was added to the main effect model to determine if there was an association between the right or left eye and the acuity score. A random intercept was set for each individual with an unstructured covariance, which allows the program to find the best fitting model. An autoregressive regressive structure was used for the repeated measures. The significance of the random effects in the model was assessed with conditional F-tests. Assumptions underlying the models were visually checked with diagnostic plots of residuals. T-test contrasts for EAE-Vehicle vs. Control-Vehicle or EAE-SNJ 50 mg/kg vs. Control-Vehicle were tested at each time point for the affected eye to determine the first day on which a significant difference in the acuity score emerged. The chi-square test was utilized to determine differences in the proportion of days that mice in each group manifested poor OKN sensitivity (value ≤ STDEV + STERROR of the control cumulative daily mean). The linear mixed model analyses and t-tests were performed using SAS 9.1 (SAS Institute, Cary, NC) and the chi-square tests were performed using PASW statistical software (IBM Corporation, Somers, NY).

The statistical tests of EAE paralysis were chosen based on standard recommendations for analyzing data from EAE studies (Fleming et al. 2005). Clinical scores were analyzed with the nonparametric Friedman test for ordinal data, followed by post-hoc analyses using Wilcoxon Signed-Rank Tests with Bonferroni corrections applied. The chi-square test was used to determine differences in the proportion of mice in each group that exhibited paralysis.

For the PERG and histological analyses, one-way ANOVAs followed by Tukey’s HSD tests were used to calculate P-values between treatment groups, unless the data were nonparametric. In this case, the Kruskall-Wallis test was run followed by Mann-Whitney U pairwise comparisons. The above tests of parametric and non-parametric means were performed using PASW statistical software (IBM Corporation). A significant difference was defined as a P-value <0.05.

RESULTS

Visual sign onset is early and follows a relapsing/remitting pattern

When left and right eye OKR visual acuity thresholds were compared for individual mice over the twenty-five days tested (Fig. 1), a significant decline in either the left or right eye measurements for a given mouse was noted, while the other eye typically maintained a threshold that was not different from the baseline value. No difference was found between the overall right and left eye mean acuity values, so a bias did not exist in tracking directionality (Table 1). Due to the strong trend for eyes to be unilaterally affected, the analyses were subdivided to compare thresholds of eyes manifesting more (affected, Fig. 1b) versus less (fellow, Fig. 1c) severe OKR impairment. Compared to the pattern of acuity among control mice, there was a decline in acuity over time for affected eyes of EAE-vehicle mice (P =0.001), but no difference for fellow eyes (P =0.483) (Table 1). The first significant loss of acuity occurred on day 7 (Fig. 1b). This event occurred early in the disease course relative to the onset of paralysis on approximately day 14 (Papenfuss et al. 2004). Sharp declines in OKR threshold loss were followed by recovery of acuity until roughly day 14. Beyond this point there was virtually no further response recovery. Indeed, by day 25, the mean acuity of affected EAE eyes had decreased to 53% of the control acuity (0.220 c/d versus 0.410 c/d). However, essentially no difference was found between the OKR thresholds of control and EAE fellow eyes by day 25 (Fig. 1c, Table 1). Moreover, no significant difference in acuity was found between EAE fellow eyes on the first and last days of the testing period (0.446 c/d versus 0.425 c/d, Fig. 1c).

Figure 1. Alterations in visual acuity occur early and follow a relapsing/remitting pattern.

Figure 1

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Mice were monitored daily from the day prior to induction until Day 25 for threshold OKRs to a virtual optokinetic drum that consisted of a pattern of vertical black and white bars. The pattern stimulus varied in spatial frequency as well as the direction of rotation (100% contrast). (a) Combined (mean of left and right eyes) spatial frequency thresholds are shown for the group of control vehicle-treated and EAE-vehicle treated mice. (b) The first sharp decline in OKR sensitivity occurs early in the affected EAE eye (Day 7). The initial decline is followed by a trend of gradual decline with intermittent marginal recovery. (c) The mean threshold of the eyes less affected with EAE-ON (fellow) is contrasted with the control group mean. *P <0.05 versus Control-Vehicle; N = 7 Control, 8 EAE.

Table 1. Effect of calpain inhibition upon cumulative mean visual acuity in EAE-ON.

The mean acuity values (cycles/degree) for OKR testing over the 25-day disease course are presented for respective eyes. The percentage of days on which the group mean OKR value was low (≤ 0.371 c/d) relative to the control mean is also presented. The day of first event represents the first day on which the mean OKR sensitivity was significantly lower than that of control.

Mean Visual Acuity Low OKR Threshold Days Day of First Event
Combined Affected Other Left Right Combined Affected Other
Control-Vehicle 0.421 ± .009 n/a n/a 0.421 ± .006 0.422 ± .006 n/a n/a n/a n/a
EAE-Vehicle 0.362 ± .006 0.319 ± .008** 0.402 ± .007 0.367 ± .009 0.357 ± .009 50% 79% 21% 7
EAE-50mg/kg SNJ 1945 0.401 ± .012 0.382 ± .016* 0.422 ± .013 0.402 ± .014 0.400 ± .014 20% 43% 14% 21
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*

P =0.021 versus EAE-Vehicle;

**

P =0.025 versus Control-Vehicle.

N =7 Control, 8 EAE.

Daily oral treatment with calpain inhibitor improves spatial frequency sensitivity

Spatial frequency thresholds of EAE vehicle-treated mice were compared with those of mice treated with 50 mg/kg SNJ 1945 after the onset of acuity decline (Day 9 start). SNJ 1945 treatment improves affected eye OKR thresholds and delays the onset of the onset of acuity loss from Day 7 to Day 21 post-EAE induction (Fig. 2, Table 1). The mean OKR sensitivity of the fellow eyes was similar to that of EAE-vehicle fellow eyes (Table 1).

Figure 2. Daily oral treatment with calpain inhibitor improves spatial frequency sensitivity.

Figure 2

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Mice were dosed orally twice daily with vehicle (0.5% carboxymethylcellulose in water) or 50 mg/kg SNJ 1945 from Day 9 until Day 25. (a) The mean OKR sensitivity of eyes more severely affected (affected) with EAE-ON is plotted over time for mice treated with 50 mg/kg SNJ 1945 and EAE-vehicle treated mice. Treatment improves acuity, particularly in the last several days of the disease course. (b) Mean OKR sensitivity of the eyes less affected with EAE-ON (fellow) is preserved regardless of the treatment group. Overall P =0.024 versus EAE-Vehicle; N =7 Control, 8 EAE, 5 EAE SNJ.

A goal of current disease modifying therapy (DMT) for MS is to reduce the frequency of relapses experienced by patients diagnosed with a relapsing-remitting form of the disease. Documentation of an exacerbation begins with subjective report, as signs and symptoms that had been quiescent re-emerge. As described in Figure 1, the progression of OKR threshold loss follows a relapsing-remitting pattern relative to control thresholds. This finding raised the question of whether daily treatment with calpain inhibitor can reduce the frequency of days on which EAE mice exhibit a low OKR threshold. A low threshold was defined as the control mean threshold minus the sum of the control mean standard deviation and the SEM (low threshold = 0.371 c/d). The number of days on which the mean OKR threshold was less than or equal to this value were counted for each treatment group, and the results are presented as the percentage of days mice exhibited low spatial frequency thresholds during the disease course (Table 1). Treatment with 50 mg/kg SNJ 1945 reduced the frequency of low OKR threshold days from 50% to 20% when combined eye thresholds were measured (χ2 =2.489, P =0.115) from 79% to 43% when more severely affected eyes were measured (χ2 =3.743, P =0.053) and from 21% to 14% when less affected eyes were compared (χ2 =0.373, P =0.541) (overall χ2 =21.778, P =0.001).

Daily oral treatment with calpain inhibitor improves clinical scores of paralysis

The most utilized assessment of disease activity in EAE is the paralysis score (Papenfuss et al. 2004). The daily disease scores of control-vehicle, EAE-Vehicle, and EAE SNJ 1945-treated mice were recorded from Days 0 to 25. The timing of paralysis onset, monophasic disease peak, and associated characteristics (Fig. 3, Table 2) of B10.PL EAE vehicle-treated mice was consistent with what has been described in the literature (Papenfuss et al. 2004). Specifically, of the 8 EAE mice, 2 never developed paralysis symptoms but were still included in the analysis as intention to treat. Of the 6 mice that developed symptoms, their peak paralysis severity occurred between Day 20–22 and individual peak paralysis scores did not all occur on the same day, so the mean of the scores does not reflect individual peak paralysis severity. Individual peak scores ranged from 2 to 3.5. Our observed peak disease severity and the finding that most but not all mice develop symptoms are consistent with the literature for this model (Spach & Hayes 2005, Grant et al. 2003). Overall, there was a statistically significant difference in the paralysis scores between Control-Vehicle, EAE-Vehicle, and EAE-SNJ 1945-treated mice, χ 2 =36.487, P <0.001. Furthermore, there was a difference between the Control and EAE-Vehicle groups (Z = −5.597, P <0.001), the EAE-Vehicle and EAE-SNJ 1945-treated groups (Z = −3.748, P <0.001) and the Control and EAE SNJ 1945-treated groups (Z = −2.456, P =0.014). Compared with EAE-Vehicle, the mean paralysis score was significantly lower upon treatment with SNJ 1945 (mean ± SD; 0.06 ± 0.27 versus 0.41 ± 0.82). In summary, mice treated with SNJ 1945 experienced significantly milder manifestations of the disease (Table 2), although the monophasic peak of paralytic signs was not delayed relative to EAE-vehicle mice (Fig. 3a).

Figure 3. Daily oral treatment with calpain inhibitor improves clinical scores of paralysis.

Figure 3

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After immunization, mice were observed over 25 days for the development of clinical signs of paralysis according to this scale: 0, no clinical disease; 0.5, piloerection; 1, tail weakness; 1.5, tail paralysis; 2, hindlimb weakness; 3, hindlimb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; 5, moribund or death. (a) Clinical scores (0<5) are compared for control-vehicle, EAE-Vehicle, and EAE SNJ 1945-treated mice. (b–c) The peak severity of visual acuity loss coincides in time with the peak severity of paralysis during EAE progression. N =7 Control, 8 EAE, 5 EAE SNJ.

Table 2. Effect of calpain inhibition upon paralysis and OKR in EAE-ON.

Treatment with 50 mg/kg SNJ 1945 from Day 9 post-induction until Day 25 reduces the incidence of paralysis and the maximum clinical score (0<5). There was not a significant association between the severity of paralysis and the severity of acuity loss within individual animals with EAE, regardless of treatment group. N =7 Control, 8 EAE, 5 EAE SNJ.

Paralysis Incidence Day of Paralysis Onset Maximum Clinical Score Relationship of Paralysis vs. Affected Eye OKR Sensitivity Loss (Adjusted R2)
Control-Vehicle 0% n/a n/a n/a
EAE-Vehicle 75% 17 ± 0.4 4 0.007
EAE-50mg/kg SNJ 1945 20% 17 2 0.268
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Furthermore, plotting of the daily mean OKR thresholds of the affected eyes and daily clinical scores on dual axes revealed that the timing of OKR threshold loss coincides with the timing of peak paralysis severity in EAE mice (Fig. 3b). The mice appeared to recover more quickly from paralysis than from visual acuity loss. A final inquiry was whether or not a relationship exists between the severity of paralysis and the severity of OKR threshold loss for an individual animal. Since a common disease peak emerged for both parameters (Day 20), the within-subject strength of correlation between the paralysis score and affected eye acuity was tested at this time point; significant relationships did not exist for untreated (P =0.345) or treated mice (P =0.204) (Table 2).

Pattern ERG amplitudes are decreased in chronic EAE retina and preserved with calpain inhibitor

Overall, these findings indicate that the OKR is progressively impaired during the course of EAE-ON, and daily oral administration of calpain inhibitor relieves the decline of OKR thresholds. It was next postulated that the inner retina might be subjected to loss of function due to thinning of the retinal nerve fiber layer and subsequent death of retinal ganglion cells (RGCs), two events which have been well characterized in clinical (Burkholder et al. 2009) and experimental (Gramlich et al. 2011) ON studies. Furthermore, it was hypothesized that calpain inhibition was effective due to anti-inflammatory and/or neuroprotective mechanisms. PERG testing, a gold standard of inner retinal function measurement, was utilized to determine whether calpain inhibition ultimately provided protection to RGCs late in the course of EAE-ON. On Day 25, the control mouse pERG consisted of a prominent positive component that peaked at a mean ± SEM of 55 ± 5 msec after each reversal (P1) at 100% contrast. P1 was followed by a slower negative component (N2) whose trough was 85 ± 3 msec after each reversal at 100% contrast. The waves were similar in appearance to the P50 and N95 waves of the human pERG, named for the peak and trough latencies, respectively. Among EAE mouse eyes, the P1 latency was delayed to an average of 79 ± 6 msec (P <0.05) and was maintained at a mean of 45 ± 4 msec for SNJ-treated mice. The N2 latency was delayed to 106 ± 5 msec among EAE mouse eyes (P <0.05), whereas the N2 latency in treated mice was 79 ± 6 msec. On Day 25, the mean first, most positive peak (P1) amplitude was diminished by 2-fold in affected eyes of EAE-vehicle treated mice (Fig. 4a; 4.28 ± 1.59 μV versus 2.22 ± 1.65 μV, P =0.041), whereas the mean P1 amplitude recorded from EAE SNJ 1945-treated mice was comparable to that of control (4.39 ± 1.39 μV versus 4.28 ± 1.59 μV, P =0.992) and significantly increased relative to vehicle-treated mice (4.39 ± 1.39 μV versus 2.22 ± 1.65 μV, P =0.018). The full pERG amplitude (P1-N2) reflected a similar trend (Fig. 4b), and although the decline in P1-N2 amplitude recorded from EAE mice was not significant (3.76 ± 1.04 μV versus 2.66 ± 1.18 μV, P =0.482), affected eyes of mice treated with SNJ 1945 exhibited a response amplitude that was increased relative to vehicle-treated mice (4.94 ± 2.42 μV versus 2.66 ± 1.18 μV, P =0.028). Taken together, these observations may imply that components of the P1 response may be more susceptible to dysfunction or atrophy in EAE-ON.

Figure 4. PERG amplitudes are decreased in chronic EAE retina and preserved with calpain inhibitor.

Figure 4

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Response amplitudes of control EAE mice were measured on Day 25 during presentation of a stimulus of 4 black and 4 white vertical bars at 100% contrast. When SNJ 1945 treatment was begun after onset of visual acuity decline (Day 9), the P1 amplitude (a) and P1-N2 amplitude (b) was comparable to the control amplitude with calpain inhibition. *P < 0.05 versus Control-Vehicle, #P <0.05 versus EAE-Vehicle. N =7 Control, 8 EAE, 5 EAE SNJ.

Calpain inhibition improved RGC survival in the EAE-ON retina

Upregulation of calpain activity was previously demonstrated in the retina of acute EAE-ON rats during disease peak, concurrent with cleavage of pro-apoptotic proteins that are known substrates of calpain, such as caspase-3 and Bid (Smith et al. 2011a). In the current study, 76kD m-calpain expression was detected in retinal neurons (NeuN), primarily located in the innermost ganglion cell layer, but with sparse detection in the inner nuclear layer and in displaced neurons of the inner plexiform layer (Fig. 5a). Having confirmed that EAE-ON mice exhibited signs of impaired inner retinal function, RGCs (Brn3a) were stained and counted in the ganglion cell layer of control-vehicle, EAE-vehicle, and EAE SNJ 1945-treated mice that were sacrificed on Day 26 (Fig. 5b). Brn3a is an excellent marker for rodent RGCs, and it has been shown to label as many RGCs as retrograde Fluorogold tracing. Moreover, Brn3a is still present in injured but not dead cells (Nadal-Nicolas et al. 2009). As predicted, there was a 34.6% ± 4.4% loss of RGCs in EAE affected retinas (P =0.0002) compared with an 8.4% ± 5.3% loss (P =0.001) in treated retinas, indicating that calpain inhibition increased survival of RGCs (Fig. 5b).

Figure 5. Calpain inhibition improved RGC survival in the EAE-ON retina.

Figure 5

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Active calpain is expressed primarily in retinal neurons of the ganglion cell layer and daily administration of SNJ 1945 (day 9 start) increased survival of RGCs relative to the EAE vehicle-treated group in retinas dissected on day 26. (a) Retinal sections were stained with anti-calpain and anti-NeuN antibodies. (b) Retinal sections were stained with anti-Brn3a antibody to detect living RGCs, and RGCs counted in EAE-Vehicle and EAE-SNJ 1945-treated retinas were quantified relative to Control-Vehicle-treated retinas. *P < 0.05 versus Control-Vehicle, #P <0.05 versus EAE Vehicle. N =7 Control, 8 EAE, 5 EAE SNJ.

Calpain inhibition attenuated inflammatory demyelinating histopathology in EAE-ON optic nerves

To continue the investigation of EAE-ON pathobiology and specific effects of calpain inhibition on the disease process, histological parameters of inflammation, demyelination, and axonal damage in the optic nerves collected at sacrifice (Day 26) were studied. To assess the level of inflammatory cell infiltration and demyelination, an LFB stain with an eosin counterstain was performed (Fig. 6a). Quantification of stained nuclei revealed a 2.36-fold increase in the mean nuclei/area counted in EAE-Vehicle optic nerves versus control (P =0.049), whereas the mean nuclei/area increased by only 57% in SNJ 1945-treated optic nerves (P =0.180). Furthermore, there was a trend towards a decreased demyelination score for calpain inhibitor-treated optic nerves (1.2 ± 0.18 versus 0.7 ± 0.18, P =0.104), which indicates preservation of myelin. Moreover, staining for anti-mature oligodendrocyte (OL) specific protein (MOSP) in EAE optic nerves revealed an altered pattern of MOSP (Fig. 6b), which translated as a decrease in the overall intensity of MOSP in vehicle-treated optic nerves (P =0.021). On the other hand, MOSP expression in SNJ 1945-treated optic nerves appeared to be more highly concentrated in OL cell bodies (Fig. 6b), and MOSP intensity was increased relative to vehicle-treated nerves (P =0.021). Since calpain inhibition appeared to modestly limit myelin degradation, anti-m-calpain was co-stained with MOSP in EAE vehicle-treated optic nerves, under the premise that calpain activation due to Ca2+ influx in injured OLs may contribute to myelin breakdown and/or OL cell death (Ray et al. 2002). Substantial active (76 kD) m-calpain expression was localized to the processes of mature OLs (Fig. 6c).

Figure 6. Calpain inhibition attenuated inflammatory demyelinating histopathology in EAE-ON optic nerves and altered MOSP expression in EAE optic nerves.

Figure 6

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Calpain inhibition attenuated inflammatory cell infiltration and modestly limited demyelination of EAE-ON optic nerves obtained at sacrifice (Day 26). Optic nerve sections were stained with LFB and an eosin counter-stain (a), and nuclei/tissue section were quantified for control-vehicle, EAE-vehicle, and EAE-SNJ 1945 treated optic nerves. In each treatment group, a demyelination score was assigned to 3 areas of optic nerve per mouse using the following scale: 0=no demyelination 1=mild demyelination 2=moderate demyelination 3=severe demyelination. The mean demyelination score and SEM are presented for EAE vehicle-treated and EAE-SNJ 1945-treated groups. (b) Optic nerves were obtained on Day 26 and the intensity/area of anti-mature oligodendrocyte specific protein (MOSP) antibody staining was quantified in 3 areas of optic nerve per mouse and compared between treatment groups. (c) Optic nerves were co-stained with anti-m-calpain and anti-MOSP antibody. *P <0.05 vs. Control-Vehicle, #P <0.05 vs. EAE-Vehicle. N =7 Control, 8 EAE, 5 EAE SNJ.

Calpain inhibition attenuated degradation of axonal neurofilament protein and limited reactive astrocytosis in EAE-ON optic nerves

A third major component of EAE-ON pathology is axonal degeneration, featuring truncation of axons with terminal axonal swelling (Trapp et al. 1998). Since neurofilament protein (NFP) is a substrate of calpain(Banik et al. 1983), it was hypothesized that daily calpain inhibition might prevent degradation of NFP, which could contribute to axonal integrity and thus account, at least in part, for the observed improvements in OKR spatial frequency thresholds (Fig. 2). Among EAE vehicle-treated optic nerves collected on Day 26, there was a decrease in intact NFP expression (P =0.012) with a corresponding trend towards an increase in deNFP expression (P =0.091) relative to control (Figs. 7a,b), whereas SNJ 1945-treated optic nerves displayed increased NFP (P =0.024) and decreased deNFP expression (P =0.004) relative to vehicle-treated nerves (Figs. 7a,b). Acute injury of myelinated optic nerves leads to a quick, transient retraction of astrocytic processes from the damaged axons with a corresponding change in astrocyte morphology (Sun et al. 2010). Over the next few days-weeks, astrocytes reorganize to repair axonal damage sites, but upon chronic insult, glial fibrillary acidic protein (GFAP) is upregulated with formation of glial scars, which may serve as a barrier to regeneration (Johnston-Wilson et al. 2000). Since there was evidence of damage to axonal neurofilament, the optic nerves were next stained for the presence of GFAP (Fig. 7c). The intensity of GFAP expression was increased in EAE vehicle-treated optic nerve tissue (P =0.002) (Figs. 7c,d). Moreover, a distinct change was observed in the pattern of GFAP expression in disease tissue, with dense, punctate staining in lieu of extensive staining of glial processes (Fig. 7c). SNJ 1945-treated nerves exhibited increased intensity of GFAP expression (P <0.001) and a pattern similar to that of control (Figs. 7c,d).

Figure 7. Calpain inhibition attenuated degradation of axonal neurofilament protein and limited reactive astrocytosis in EAE-ON optic nerves.

Figure 7

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Calpain inhibition prevented degradation of axonal neurofilament protein and attenuated GFAP expression in EAE-ON optic nerves obtained on Day 26. (a) Optic nerve sections were stained with anti-deNFP and anti-NFP antibodies and the staining intensity was quantified (b) in 3 areas of optic nerve per mouse for control-vehicle, EAE-vehicle, and EAE-SNJ 1945 treated optic nerves. (c) Optic nerves were stained with anti-GFAP antibody and staining intensity/area was quantified (d) in 3 areas of optic nerve per mouse and compared between treatment groups. *P <0.05 versus Control-Vehicle, #P <0.05 versus EAE-Vehicle. N =7 Control, 8 EAE, 5 EAE SNJ.

DISCUSSION

The findings in this MS model are in agreement with clinical reports that visual field impairment and RGC loss are consistent and clinically meaningful features of MS-associated ON. Onset of visual signs in EAE-ON occurs early in the disease course relative to the onset of paralysis, and OKR spatial frequency threshold loss follows a relapsing/remitting pattern. These findings parallel the clinical picture in which manifestations of visual system damage tends to occur prior to paresthesia and paralysis in the extremities. Interestingly we noticed a strong bias towards unilateral OKR impairment among EAE-ON eyes. This phenomenon has a clear clinical correlate, as the majority of documented ON cases are unilateral (Osborne & Volpe 2009).

Visual impairment and paralysis were attenuated with calpain inhibitor, importantly, when treatment was started on Day 9, after the onset of spatial frequency threshold decline. In a model of EAE-ON in female SJL/J mice immunized with proteolipid protein peptide, inflammatory cell infiltration of optic nerves was detected by histology as early as 9 days after immunization, with demyelination, loss of RGC axons, and finally loss of RGC soma occurring in sequence through Day 14 (Shindler et al. 2008). However, the results herein suggest that OKR response sensitivity is impaired by immune cell infiltration of the optic nerves much earlier. Furthermore, calpain inhibition did not completely abrogate the increased presence of inflammatory cells relative to control, nor did it significantly prevent demyelination (Fig. 6a), but there was a significant sparing of mature oligodendrocytes (MOSP) by Day 25 (Fig. 6b). The increased detection of MOSP in treated optic nerves suggests that SNJ 1945 prevents oligodendrocyte cell death, and these oligodendrocytes have the potential to form new myelin (Mu & Dyer 1994).

An EAE model that produced mild-moderate paralysis severity was chosen to ensure survival of the mice so recovery could be monitored. Regarding the association between paralysis and visual signs, the onset of visual signs in EAE-ON occurs early in the disease course relative to the onset of paralysis. These findings parallel the clinical picture in which manifestation of visual system damage tends to occur prior to paralysis. This may be accounted for at least in part by differences in gray matter volume of the optic nerve vs. spinal cord (Edgar et al. 2008). Since there is a direct relationship between the sizes of individual neurons and their axons in the anterior visual system (Sadun 1986), this finding lends support to the hypothesis that smaller diameter axons may be preferentially vulnerable to injury in MS and its models (Ganter et al. 1999). Furthermore, ON occurs at some point during the MS disease course in 38–66% of patients (Beck et al. 1992, Arnold 2005, Frohman et al. 2005, Foroozan et al. 2002); and, prospective studies have estimated that 38% to 75% of patients with a first episode of ON were eventually diagnosed with MS (Beck et al. 2003). This EAE model appears to follow the same pattern in which some mice with visual symptoms don’t experience spinal cord symptoms, or at least not to a severe degree. Although optic neuritis signs began and persisted well in advance of paralysis onset, there was a steady increase in the severity of OKR sensitivity losses and paralytic scores until peak severity of both coincided on Day 21. Thus, there was a gross association of paralysis and visual symptoms. Interestingly, in this model paralysis immediately begins to recover after Day 21 (Fig. 3) while OKR threshold sensitivity remains poor relative to control through the duration of testing, presumably due to loss of a significant portion of RGCs (Fig. 4). This may also be an artifact of differing sensitivity between these tests.

Calpain inhibition improved RGC survival in the EAE-ON retina, even when administered after the onset of OKR sensitivity loss. Activated calpain was strongly expressed in retinal neurons of the ganglion cell layer in EAE-ON, concurrent with a 33% loss of Brn3a positive RGCs at Day 25. Treatment of EAE-ON with corticosteroids after optic neuritis onset (days 10–14) was shown to attenuate EAE progression, but suppression of RGC loss on day 14 was lost by Day 18 (Dutt et al. 2010). However, our results demonstrated that daily, oral administration of SNJ 1945 from Day 9 of disease was sufficient to reduce loss of Brn3a positive cells to 10% by Day 25. Whether SNJ 1945 can preserve RGCs long-term in this model or after withdrawal of daily therapy has yet to be determined. Active calpain was also expressed in occasional neurons displaced to the inner plexiform layer. These cells likely represent displaced amacrine cells, which are responsible for input to retinal ganglion cells. Typically, they form synapses with RGCs (and bipolar cells) in the inner plexiform layer. If amacrine cells are subject to cell death in this model, this would likely contribute to impaired retinal function in the modes tested (pERG, optokinetic responses) (Yoshida et al. 2001). It has been reported that the combination of retinal ganglion cell layer plus inner plexiform layer thinning is significantly correlated with both visual function and vision-specific quality of life in MS and may serve as a marker of disease activity (Walter et al. 2012). Thus, any influence on the synaptic connections in the IPL, such as death of RGCs or amacrine cells may be functionally important.

As anticipated due to the loss of RGCs, pERG amplitudes were decreased in chronic EAE retina and preserved with calpain inhibitor. The P1 response is generated by coordination between ON pathway bipolar cells and RGCs in the innermost retina, and the contributions of these neurons is mostly spiking; the N2 response is generated by OFF bipolar cells and RGCs more proximal to the IPL, and it reflects spiking activity from these neurons as well as nonspiking contributions from both OFF and ON pathway neurons (Miura et al. 2009). In the current study, the decreased P1 response amplitude and the increased latencies of both the P1 and N2 components indicate that RGC responses driven by both the ON and the OFF inner retinal pathways are impaired. These data suggest that RGCs are most susceptible to damage and cell death in optic neuritis, and may support the hypothesis that RGC death is a retrograde event following primary retrobulbar optic neuritis.

Recent MS literature describes reductions in both the retinal nerve fiber layer and ganglion cell layer thickness for MS or ON patients (Costello et al. 2010). As hypothesized based on this literature, both RGC loss and changes to the optic nerve, particularly RGC axons in the optic nerve, were found to be crucial aspects of ON pathology in this model. In this study, SNJ 1945 treatment initiated from Day 9 of disease was sufficient to attenuate degradation of RGC axonal neurofilament protein and limit reactive astrocytosis in EAE-ON optic nerves. Calpains can cleave all three neurofilament subunits in vitro (NFL, NFM, NFH) (Greenwood et al. 1993), and recently, major in vitro and in vivo sites of calpain cleavage of bovine NFM have been identified that are well conserved relative to human NFM (Shaw et al. 2004). Furthermore, NFP is lost in animal models of acute axonal injury (Serbest et al. 2007). NFP is also considered an MS biomarker, and increased levels of phosphorylated NFP in MS CSF are reflective of axonal damage (Gunnarsson et al. 2011) and have been strongly correlated with CSF levels of MBP, gadolinium ELV, and T2-weighted MRI abnormalities and clinical disability in MS (Lim et al. 2005).

A significant increase in GFAP immunoreactivity was detected in EAE vehicle-treated optic nerves as well as a striking change of astrocyte morphology that featured dense, punctate staining of soma with protracted dendritic processes. We are not the first to report that astrocytes assume a hypertrophic morphology during reactive gliosis in EAE (Guo et al. 2011). Calcium influx with calpain activation has been associated with a rapid increase in GFAP immunoreactivity in astrocytes (Lee et al. 2000. GFAP is not only an established substrate of calpain, but an MS disease biomarker (Gunnarsson, 2011 #66). We found that calpain inhibition attenuated GFAP immunoreactivity and preserved astrocyte morphology in EAE optic nerves. The reduced GFAP immunoreactivity was possibly either a direct result of calpain inhibition or the indirect result of a less pro-inflammatory milieu. The CSF presence of GFAP has been identified as biomarker of MS disease progression and disability (Axelsson et al. 2011). Moreover, the mean annual increase of GFAP was 6.5 ng/L for controls, 8.1 ng/L for RRMS patients, and 18.9 ng/L for SPMS patients, and the GFAP level upon initial examination had predictive value for neurological disability 8–10 years later (based on the Kurtzke Expanded Disability Status Scale [EDSS]). Interestingly, GFAP levels are not reduced by the disease modifying therapy natalizumab (Gunnarsson et al. 2011), which may imply that a strictly anti-inflammatory therapy is not sufficient to taper astrocyte reactivity during an MS exacerbation. In the present study, the reduction of astrocyte reactivity in EAE, as produced with SNJ 1945 treatment (Fig. 7), suggests that combination therapy of an anti-inflammatory agent and a calpain inhibitor may effectively taper astrocytosis.

In conclusion, immunized EAE mice were found to manifest early and severe losses in spatial frequency threshold sensitivity, especially in affected eyes. Structural damage resulting in decreased OKR thresholds appears to occur in both optic nerves and retinas. Daily oral treatment with the calpain inhibitor SNJ 1945, initiated after the onset of OKR threshold decline, improves OKR sensitivity, inner retinal function and paralysis, with associated attenuation of histopathology. Future studies are necessary to characterize the specific inflammatory mechanisms at work in this disease model and distinguish the T cell responses of vehicle and SNJ 1945-treated mice.

Supplementary Material

Supp Table S1

Acknowledgments

This work was supported in part by NINDS grants NS41088, NS56176, and NS65456, NEI grant EY019320, and Department for Veterans Affairs Merit Awards RX000444 and 1IO1BX002349.

List of abbreviations

Brn3a

brain-specific homeobox/POU domain protein 3A

CFA

complete Freund’s adjuvant

CSF

cerebral spinal fluid

deNFP

dephosphorylated neurofilament protein

DMT

disease modifying therapy

EAE

experimental autoimmune encephalomyelitis

ERG

electroretinogram

GFAP

glial fibrillary acidic protein

LFB

Luxol Fast Blue

MBP

myelin basic protein

MOSP

myelin oligodendrocyte specific protein

MRI

magnetic resonance imaging

MS

multiple sclerosis

NF-κB

nuclear factor-kappa B

NFH

heavy molecular weight neurofilament

NFL

light molecular weight neurofilament

NFM

medium molecular weight neurofilament

NFP

neurofilament protein

OCT

optical coherence tomography

OKR

optokinetic response

OL

oligodendrocyte

ON

optic neuritis

PBS

phosphate-buffered saline

PERG

pattern electroretinogram

RGC

retinal ganglion cell

SEM

standard error of the mean

VA

visual acuity

VEP

visual evoked potentials

Footnotes

Competing Financial Interests

JI and MA are employees of Senju Pharmaceutical Co Ltd, Kobe, Japan.

All other authors have no financial conflicts of interest.

SNJ 1945 was obtained by NLB under a Material Transfer Agreement between MUSC and Senju Pharmaceutical Co Ltd, Kobe, Japan.

Conflicts of interest: None

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