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. 2020 Jul 10;9(8):16.
doi: 10.1167/tvst.9.8.16. eCollection 2020 Jul.

Systemic Mesenchymal Stem Cell Treatment Mitigates Structural and Functional Retinal Ganglion Cell Degeneration in a Mouse Model of Multiple Sclerosis

Oliver W Gramlich  1   2 , Alexander J Brown  3   4 , Cheyanne R Godwin  1   2 , Michael S Chimenti  5 , Lauren K Boland  6 , James A Ankrum  6 , Randy H Kardon  1   2
Affiliations

Systemic Mesenchymal Stem Cell Treatment Mitigates Structural and Functional Retinal Ganglion Cell Degeneration in a Mouse Model of Multiple Sclerosis

Oliver W Gramlich et al. Transl Vis Sci Technol. .

Abstract

Purpose: The purpose of this study was to determine mesenchymal stem cell (MSC) therapy efficacy on rescuing the visual system in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) and to provide new mechanistic insights.

Methods: EAE was induced in female C57BL6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55, complete Freund's adjuvant, and pertussis toxin. The findings were compared to sham-immunized mice. Half of the EAE mice received intraperitoneally delivered stem cells (EAE + MSC). Clinical progression was monitored according to a five-point EAE scoring scheme. Pattern electroretinogram (PERG) and retinal nerve fiber layer (RNFL) thickness were measured 32 days after induction. Retinas were harvested to determine retinal ganglion cell (RGC) density and prepared for RNA-sequencing.

Results: EAE animals that received MSC treatment seven days after EAE induction showed significantly lower motor-sensory impairment, improvement in the PERG amplitude, and preserved RNFL. Analysis of RNA-sequencing data demonstrated statistically significant differences in gene expression in the retina of MSC-treated EAE mice. Differentially expressed genes were enriched for pathways involved in endoplasmic reticulum stress, endothelial cell differentiation, HIF-1 signaling, and cholesterol transport in the MSC-treated EAE group.

Conclusions: 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.

Keywords: EAE; mesenchymal stem cells; optic neuritis; pattern-ERG; retinal ganglion cells.

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Conflict of interest statement

Disclosure: O.W. Gramlich, None; A.J. Brown, None; C.R. Godwin, None; M.S. Chimenti, None; L.K. Boland, None; J.A. Ankrum, None; R.H. Kardon, None

Figures

Figure 1.
Figure 1.
Systemic MSC administration reduces clinical EAE scoring. (A) Untreated MOG35-55-induced EAE mice (black: EAE) display a moderate EAE disease course by means of a five-scale EAE scoring system whereas sham-immunized control mice (blue: CTRL) show no signs of motor-sensory impairment over time. EAE mice having received as single systemic injection of 106 human MSC (green: EAE + MSC) at day seven after EAE induction (arrow) demonstrate a milder disease course when compared to untreated EAE mice. Mean EAE scores per day with SEM are given and * indicates P < 0.05. (B) Area under curve (AUC) analysis obtained from MSC-treated and untreated EAE mice demonstrate significantly lower overall disease severity (mean ± SEM) in MSC-treated EAE mice. Weakening of the tail or any other signs of motor-sensory impairment were not observed in sham-immunized controls.
Figure 2.
Figure 2.
MSC treatment in EAE mice preserves Pattern-ERG amplitude. (A) The latency (L) was measured as the time from presentation of the stimulus to the P1 peak and the PERG amplitude (A) was determined as the distance from the P1 peak to the N2 trough. (B) Thirty-two days after induction, sham-immunized controls and MSC-treated EAE mice show almost identical PERG amplitudes, whereas untreated EAE mice demonstrate a significantly declined amplitude. This result indicates a robust MSC-related rescue effect on RGC function.
Figure 3.
Figure 3.
MSC treatment mitigates RNFL thinning in EAE mice. (A) Thickness of the RNFL (dotted yellow lines) was determined in 4 quadrants at 400 µm distance from the center of the optic nerve head (OHN). (B) Treatment of EAE mice with MSC at day 7 leads to preservation of the RNFL thickness when compared to untreated EAE mice, measured 32 days after EAE induction. (C) Representative OCT images from EAE mice and MSC-treated EAE mice show differences in RNFL thickness. Note overall thicker RNFL, with areas of RNFL hypertrophy (between asterisks) and enlarged blood vessels (arrows) in MSC-treated EAE mice.
Figure 4.
Figure 4.
MSC treatment alleviates RGC loss in EAE mice. (A) Representative images of Brn3a immunolabeling (red) display differences in RGC density in the peripheral retina of controls, EAE mice, and MSC-treated EAE mice. (B) Analysis of the average RGC density reveals a significant loss of Brn3a+ cells in untreated EAE mice and a remarkably higher RGC density in MSC-treated mice. (C) Regional differences in the RGC density between controls and EAE mice is most prominent in the mid-periphery. A notably upward trend in RGC numbers of EAE mice with MSC treatment is observed in the peripheral and central region, although this is not statistically significant.
Figure 5.
Figure 5.
MSC treatment significantly influences gene expression in the retina and optic nerve of EAE mice. (A) In the volcano plot, all 259 significantly differentially expressed (DE) genes are represented in terms of their measured expression change (x-axis) and the significance of the change (y-axis). The significance is represented in terms of the negative log (base 10) of the P value. The dotted lines represent the thresholds used to select the differentially expressed genes: 0.5 for expression change and 0.1 for significance. The bar plots show the measured gene expression of processes that are found to be significantly differently (P value in headlines) regulated in MSC-treated EAE mice. The top five processes are related to (B) HIF-1 signaling, (C) endoplasmic reticulum unfolded protein response, (D) visual system development, (E) cellular response to retinoic acid, and (F) positive regulation of endothelial cell proliferation. The upregulated genes (positive log fold change) are shown in red, whereas the downregulated genes are blue.
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