Article Systemic Mesenchymal Stem Cell Treatment Mitigates Structural and Functional Retinal Ganglion Cell Degeneration in a Mouse Model of Multiple Sclerosis Oliver W. Gramlich1,2,AlexanderJ.Brown3,4, Cheyanne R. Godwin1,2, Michael S. Chimenti5, Lauren K. Boland6, James A. Ankrum6, and Randy H. Kardon1,2 1 Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA, USA 2 Center for the Prevention and Treatment of Visual Loss, Iowa City VA Health Care System, Iowa City, IA, USA 3 Department of Biomedical Research, National Jewish Health, Denver, CO, USA 4 Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 5 Iowa Institute of Human Genetics, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA 6 Roy J. Carver Department of Biomedical Engineering College, The University of Iowa, Iowa City, IA, USA Correspondence: Oliver W. Purpose: The purpose of this study was to determine mesenchymal stem cell (MSC) Gramlich, Department of therapy efficacy on rescuing the visual system in the experimental autoimmune Ophthalmology and Visual Sciences, encephalomyelitis (EAE) model of multiple sclerosis (MS) and to provide new mecha- The University of Iowa, 200 Hawkins nistic insights. Dr, Iowa City, IA 52242, USA. e-mail: Methods: [email protected] EAE was induced in female C57BL6 mice by immunization with myelin oligo- dendrocyte glycoprotein (MOG)35–55, complete Freund’s adjuvant, and pertussis toxin. Received: March 23, 2020 The findings were compared to sham-immunized mice. Half of the EAE mice received Accepted: May 26, 2020 intraperitoneally delivered stem cells (EAE + MSC). Clinical progression was monitored Published: July 10, 2020 according to a five-point EAE scoring scheme. Pattern electroretinogram (PERG) and Keywords: optic neuritis; EAE; retinal nerve fiber layer (RNFL) thickness were measured 32 days after induction. Retinas mesenchymal stem cells; were harvested to determine retinal ganglion cell (RGC) density and prepared for RNA- pattern-ERG; retinal ganglion cells sequencing. Citation: GramlichOW,BrownAJ, Results: EAE animals that received MSC treatment seven days after EAE induction Godwin CR, Chimenti MS, Boland LK, showed significantly lower motor-sensory impairment, improvement in the PERG ampli- Ankrum JA, Kardon RH. Systemic tude, and preserved RNFL. Analysis of RNA-sequencing data demonstrated statistically mesenchymal stem cell treatment significant differences in gene expression in the retina of MSC-treated EAE mice. Differ- mitigates structural and functional entially expressed genes were enriched for pathways involved in endoplasmic reticulum retinal ganglion cell degeneration in stress, endothelial cell differentiation, HIF-1 signaling, and cholesterol transport inthe a mouse model of multiple sclerosis. MSC-treated EAE group. Trans Vis Sci Tech. 2020;9(8):16, Conclusions: https://doi.org/10.1167/tvst.9.8.16 Systemic MSC treatment positively affects RGC function and survival in EAE mice. Better cholesterol handling by increased expression of Abca1, the cholesterol efflux regulatory protein, paired with the resolution of HIF-1 signaling activation might explain the improvements seen in PERG of EAE animals after MSC treatment. Translational Relevance: Using MSC therapy in a mouse model of MS, we discovered previously unappreciated biochemical pathways associated with RGC neuroprotection, which have the potential to be pharmacologically targeted as a new treatment regimen. cells, which causes visual impairment, fatigue, motor Introduction disabilities, pain, and cognitive deficits. Approximately 2.5 million people, primarily young adults, are afflicted Multiple sclerosis (MS) is a chronic neurodegen- with MS. The average age at disease onset is 30 years, erative disease, primarily mediated by autoreactive T and half of the patients require a wheelchair within Copyright 2020 The Authors tvst.arvojournals.org | ISSN: 2164-2591 1 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from tvst.arvojournals.org on 09/24/2021 Mesenchymal Stem Cell Therapy in Experimental Optic Neuritis TVST | July 2020 | Vol. 9 | No. 8 | Article 16 | 2 1–3 25 years of diagnosis. Vision loss due to acute optic dendrocyte glycoprotein (MOG)35–55 induced experi- neuritis and subsequent retinal ganglion cell (RGC) mental autoimmune encephalomyelitis (EAE) model. loss is the major initial feature in approximately 20 We sought to provide data showing that systemic MSC to 45% of cases with MS.4 There is a very high treatment significantly reduces motor-sensory impair- likelihood that patients diagnosed with MS by other ment in EAE mice. Furthermore, we used pattern initial symptoms will have at least one episode of MS- electroretinography (PERG) recordings to indicate related optic neuritis in the future. Many patients with rescue of the PERG amplitude in MSC-treated mice, acute optic neuritis completely recover their vision, and measurement of RNFL to indicate preservation but approximately 40% suffer from permanent vision of axons. Bioinformatics of the retina, including optic loss. Notably, faster recovery of vision is correlated nerve tissue, was used to characterize gene expres- with onset of treatment and the earlier the treatment sion to discover processes related to RGC death (e.g. starts, the better the prognosis is for visual outcome.5,6 HIF-1 pathway activation and endoplasmic reticu- Patients with MS with no history of optic neuritis show lum [ER] unfolded protein response), in MSC-treated evidence of RGC loss and thinning of the retinal nerve EAE mice when compared to untreated EAE mice. fiber layer (RNFL), determined by optical coherence Moreover, we identified differentially regulated gene tomography (OCT) imaging.7 Furthermore, impaired clusters associated with endothelial cell differentiation, contrast sensitivity in patients with MS correlates with visual system development, and response to retinoic severity of central nervous system (CNS) lesion size,8,9 acid that also influence regulation of cholesterol trans- demonstrating that the visual pathway is an important port in EAE mice having received systemic MSC treat- indicator of disease activity, even in patients with MS ment. These data indicate that MSC treatment in EAE- who have no visual symptoms and no history of optic mediated optic neuritis involves previously unappre- neuritis. ciated pathways associated with neuroprotection and Approaches using mesenchymal stem cells (MSCs) repair of visual system impairments that may be future in animal models and clinical studies have shown targets for therapeutics. considerable promise in reducing the severity of autoimmune-mediated disorders by decreasing effector T-cell populations and promoting T-regulatory cells. Methods There are currently 11 ongoing and 13 completed clinical trials worldwide using different sources of Animals and EAE Induction MSC (bone marrow-derived, umbilical cord-derived, etc.) and multiple routes of administration for treat- All animal experiments were approved by the Iowa ments (clinicaltrials.gov). One of the first clinical safety City VAHealth Care System Institutional Animal Care studies using MSCs in patients with secondary progres- and Use Committee (IACUC) and were conducted in sive MS indicated preservation of RNFL thickness accordance with the ARVO Statement for the Use of and improvement of visual function after MSC admin- Animals in Ophthalmic and Vision Research. Female istration.10 It is known that MSC has immunomod- two month old C57BL6/J (B6) mice were purchased ulatory properties that can directly suppress T-cell from Jackson Laboratories (Bar Harbor, ME) and activation11 and likely serve to reduce CNS damage. housed on a 12-hour light-dark cycle with food ad Moreover, recent studies have shown that MSC secrete libitum. EAE was induced in 40 mice, as described a large variety of trophic factors, such as platelet- by Bittner et al.14 by immunization with 200 μl derived growth factor, hepatocyte growth factor, and solution containing 200 μgMOG35–55 (Sigma Aldrich, insulin-like growth factor to promote retinal neuro- St. Louis, MO) emulsified with complete Freund’s protection.11,12 MSCs are also considered capable of adjuvant (Sigma) containing 2 mg/mL mycobacterium triggering remyelination,13 but mechanisms are not tuberculosis (BD Difco, Franklin Lakes, NJ) subcu- well understood. Discovery of biochemical pathways taneously at two sites along the back. A group through which MSCs improve visual function and of 20 control mice received a sham immunization facilitate RGC neuroprotection after an inflamma- with an equal volume of phosphate-buffered saline tory demyelinating event could provide new thera- (PBS) instead of MOG mixed with complete Freund’s peutic targets that can be augmented more specifi- adjuvant (control [CTRL]). MOG and CTRL mice cally through small molecules, drugs, diet, or gene- were injected with 400 ng pertussis toxin in 200 μl PBS therapeutic approaches. intraperitoneally at day 0 (day of immunization) and The purpose of our study is to overcome this knowl- again on day two. Clinical progression was monitored edge gap by validating systemic MSC therapy efficiency daily according to a five-point EAE scoring system by on rescuing the visual system using the myelin oligo- the following criteria: 0 = no symptoms, 0.5 = partial Downloaded from tvst.arvojournals.org on 09/24/2021 Mesenchymal