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J Neurochem. Author manuscript; available in PMC 2017 October 01. Published in final edited form as: J Neurochem. 2016 October ; 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. Smith1, Baerbel Rohrer1,3,5,*, Lee Wheless4, Supriti Samantaray2, Swapan K. Ray5, Jun Inoue7, Mitsuyoshi Azuma7, and Naren L. Banik2,3,6,*
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1Department
of Neurosciences, Medical University of South Carolina, Charleston, South Carolina
2Department
of Neurology and Neurosurgery, Medical University of South Carolina, Charleston, South Carolina
3Department
of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina
4Department
of Medicine–Division of Biostatistics and Epidemiology, Medical University of South Carolina, Charleston, South Carolina
5Department
of Pathology, Microbiology and Immunology, University of South Carolina
Service, Ralph H. Johnson VA Medical Center, Charleston, SC 29401; 3Alexion Pharmaceuticals, 352 Knotter Drive, Cheshire CT 06410
6Research
7Senju
Pharmaceutical Co Ltd, Kobe, Japan
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Abstract
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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-
Corresponding Authors: Naren L. Banik, 96 Jonathan Lucas St, MSC606, Charleston, SC 29425; [email protected], Baerbel Rohrer, 167 Ashley Ave, SEI614, Charleston, SC 29425; [email protected]. 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
Smith et al.
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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.
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INTRODUCTION
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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
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
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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).
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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 mcalpain, 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.
Author Manuscript
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
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
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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 selfmotion 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).
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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
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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
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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).
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
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Measurement of optokinetic response thresholds
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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
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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
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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
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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
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Enucleated eyes were collected on Day 26 and fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS, pH 7.4) for 4 hour. After fixation, the eyes were hemisected in dorsoventral 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
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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 = J Neurochem. Author manuscript; available in PMC 2017 October 01.
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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).
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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.
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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
J Neurochem. Author manuscript; available in PMC 2017 October 01. Published in final edited form as: J Neurochem. 2016 October ; 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. Smith1, Baerbel Rohrer1,3,5,*, Lee Wheless4, Supriti Samantaray2, Swapan K. Ray5, Jun Inoue7, Mitsuyoshi Azuma7, and Naren L. Banik2,3,6,*
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1Department
of Neurosciences, Medical University of South Carolina, Charleston, South Carolina
2Department
of Neurology and Neurosurgery, Medical University of South Carolina, Charleston, South Carolina
3Department
of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina
4Department
of Medicine–Division of Biostatistics and Epidemiology, Medical University of South Carolina, Charleston, South Carolina
5Department
of Pathology, Microbiology and Immunology, University of South Carolina
Service, Ralph H. Johnson VA Medical Center, Charleston, SC 29401; 3Alexion Pharmaceuticals, 352 Knotter Drive, Cheshire CT 06410
6Research
7Senju
Pharmaceutical Co Ltd, Kobe, Japan
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Abstract
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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-
Corresponding Authors: Naren L. Banik, 96 Jonathan Lucas St, MSC606, Charleston, SC 29425; [email protected], Baerbel Rohrer, 167 Ashley Ave, SEI614, Charleston, SC 29425; [email protected]. 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
Smith et al.
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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.
Author Manuscript Author Manuscript
INTRODUCTION
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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
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
Page 3
Author Manuscript
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).
Author Manuscript Author Manuscript
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 mcalpain, 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.
Author Manuscript
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
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
Page 4
Author Manuscript
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 selfmotion 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).
Author Manuscript
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
Author Manuscript
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
Author Manuscript
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).
J Neurochem. Author manuscript; available in PMC 2017 October 01.
Smith et al.
Page 5
Measurement of optokinetic response thresholds
Author Manuscript Author Manuscript
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
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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
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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
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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
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Enucleated eyes were collected on Day 26 and fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS, pH 7.4) for 4 hour. After fixation, the eyes were hemisected in dorsoventral 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
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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 = J Neurochem. Author manuscript; available in PMC 2017 October 01.
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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).
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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.
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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
