Dietary protection against the visual and motor deficits induced by experimental autoimmune encephalomyelitis

Introduction Five to eight percent of the world population currently suffers from at least one autoimmune disorder. Despite multiple immune modulatory therapies for autoimmune demyelinating diseases of the central nervous system, these treatments can be limiting for subsets of patients due to adverse effects and expense. To circumvent these barriers, we investigated a nutritional intervention in mice undergoing experimental autoimmune encephalomyelitis (EAE), a model of autoimmune-mediated demyelination that induces visual and motor pathologies similar to those experienced by people with multiple sclerosis (MS). Methods EAE was induced in female and male mice and the impact of limiting dietary carbohydrates by feeding a ketogenic diet (KD) enriched in medium chain triglycerides (MCTs), alpha-linolenic acid (an omega-3 fatty acid), and fiber was evaluated in both a preventive regimen (prior to immunization with MOG antigen) and an interventional regimen (following the onset of symptoms). Motor scores were assigned daily and visual acuity was measured using optokinetic tracking. Immunohistochemical analyses of optic nerves were done to assess inflammatory infiltrates and myelination status. Fatty acid and cytokine profiling from blood were performed to evaluate systemic inflammatory status. Results The KD was efficacious when fed as a preventive regimen as well as when initiated as an interventional regimen following symptom onset. The KD minimally impacted body weight during the experimental time course, increased circulating ketones, prevented motor and ocular deficits, preserved myelination of the optic nerve, and reduced infiltration of immune cells to optic nerves. The KD also increased anti-inflammatory-associated omega-3 fatty acids in the plasma and reduced select cytokines in the circulation associated with EAE-mediated pathological inflammation. Discussion In light of ongoing clinical trials using dietary strategies to treat people with MS, these findings support that a KD enriched in MCTs, omega-3 fatty acids, and fiber promotes a systemic anti-inflammatory milieu and ameliorates autoimmune-induced demyelinating visual and motor deficits.


Introduction
Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe disabilities. Deficits include losses of mobility, balance and coordination, blindness, depression, fatigue, memory loss, and decreased quality of life. MS is more common in females, with onset typically occurring in the third and fourth decades of life [reviewed in (1)(2)(3)].
The increased incidence of MS and other autoimmune disorders in recent decades coincides with global increases in obesity, hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, and type 2 diabetes (4). Mounting evidence points to the excessive consumption of ultra-processed foods containing highly-processed carbohydrates and pro-inflammatory fats as drivers of metabolic syndrome (5-7), poor health outcomes (8,9), and all-cause mortality (10). These ultra-processed, hyperpalatable, calorically-dense foodstuffs promote hyperphagia (11), and can exacerbate autoimmunity by disrupting microbiomehost symbiosis and promoting systemic inflammation (12). Evidence from clinical trials and animal studies support that diet profoundly impacts MS severity and disease trajectory (13)(14)(15), consistent with the observation that insulin resistance and adiposity correlate with more severe disability scores for people with MS (16, 17).
To determine the impact of reducing carbohydrate-laden foods in the diet, we investigated the therapeutic efficacy of a ketogenic diet (KD) in the mouse MOG 35−55 -experimental autoimmune encephalomyelitis (MOG 35−55 -EAE) model, hereafter referred to as "EAE." This model of autoimmune-mediated demyelination induces visual and motor pathologies similar to those experienced by people with MS. Despite the caveats and limitations of preclinical models (e.g., the lack of genetic diversity inherent to humans), EAE rodent studies have proven valuable with respect to the development of FDA-approved therapeutics for MS (18).
The studies presented here were done to determine whether the KD developed by D'Agostino and colleagues (19) that is enriched in fiber and contains medium chain triglycerides [MCTs; caprylic acid (C8) and capric acid (C10)] along with flaxseed oil and canola oil as the sources of fat can preserve motor and visual function in both male and female C57BL/6J mice undergoing EAE. Previous studies of nutritional interventions using this EAE model have demonstrated that caloric restriction attenuates EAE motor deficits and correlates with reduced levels of IL-6 and leptin (20). Likewise, fast-mimicking diets (21,22), intermittent fasting (23, 24), and KDs (22,25,26) reduce EAE motor disabilities, and a KD improved long-term potentiation, spatial learning, and memory (26).
Nutritional ketosis has been used since the 1920s to treat children with drug-refractory seizures (27, 28), and KDs have gained popularity for weight loss and as an anti-diabetic strategy because the diet suppresses hunger and reduces energy intake (29-31). These and other findings [reviewed in (32,33)] have led to multiple clinical trials (e.g., NCT01538355, NCT03718247, NCT01915433, and NCT05007483) including "Nutritional Approaches in Multiple Sclerosis" (NAMS; NCT03508414), a randomized controlled clinical trial in Germany for people with active MS to compare a KD vs. a fasting protocol vs. a fat-modified standard diet over an 18-month period (34).
KDs can differ in composition but share at least two properties. The diets are comprised primarily of fats with moderate protein content and low amounts of carbohydrates, typically in the range of 0-70 g daily for humans, excluding indigestible fiber. Secondly, KDs induce the liver to produce the ketone bodies β-hydroxybutyrate, acetoacetate, and acetone. People in nutritional ketosis have circulating ketones of 0.5-4 mM with blood glucose <150 mg/dl, irrespective of fasting or fed state. This nutritional ketosis is distinguished from the pathological ketoacidosis associated with uncontrolled diabetes that yields ketones exceeding 25 mM and blood glucose >240 mg/dl.
Here we report that a KD can robustly protect against EAE-mediated motor and vision loss concomitant with reducing immune cell infiltration and preserving myelination of the optic nerve in both female and male mice. Functional preservation and protection against neurodegeneration were robust when the KD was fed as a preventive regimen prior to immunization to induce EAE and likewise when implemented as an intervention after symptom onset, demonstrating the translational feasibility of this nutritional approach. The KD increased circulating levels of multiple omega 3 (ω3) fatty acids associated with endogenous resolution pathways of acute inflammation and reduced circulating factors associated with neutrophil-mediated inflammation and MS pathogenicity. Together, these findings show that an MCT-based KD enriched in fiber confers neuroprotection and can reverse the loss of motor and visual function caused by autoimmunemediated demyelination.

Mice
Male and female C57BL/6J mice were housed in microisolator cages (n = 4-5 per cage) under a 12-h light/dark cycle and fed ad libitum [Picolab R Rodent Diet (cat # 5053)]. All animal care and experimental procedures were performed in compliance with ARRIVE guidelines, an Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee (IACUC)-approved protocol, and complied with standards delineated by the Institute for Laboratory Animal Research. These studies adhered to The Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Research. All studies used C57BL/6J female and male mice. Breeders were purchased from Jackson Laboratories (stock # 000664). At the termination of experiments, mice were anesthetized to collect blood by cardiac puncture and subsequently sacrificed by CO 2 asphyxiation followed by cervical dislocation.  Figure 1A and Supplementary Figure 1A. Food and water were provided ad libitum and additional hydration/electrolytes were provided to animals showing signs of dehydration using intraperitoneal saline injections.

MOG-EAE
EAE was induced in 10-12 week old mice by subcutaneous flank injection of 150 µg of myelin oligodendrocyte glycoprotein peptide (residues 35-55; MOG 35−55 ; Genemed Synthesis, Inc., San Francisco, USA) emulsified in incomplete Freund's adjuvant (Thermo Fisher Scientific; DF0639606) supplemented with 5 mg/ml heat-inactivated Mycobacterium tuberculosis (Thermo Fisher Scientific; DF3114338). Mice were injected intraperitoneally with 250 ng Bordetella pertussis toxin (List Biological Laboratories, Inc. #181) the day of and 2 days following MOG 35−55 immunization. For the prevention experiments, mice were euthanized for post-mortem histology 21-22 days post-immunization (dpi). For the intervention experiments (Figure 7), all mice were maintained on standard chow until symptom onset at which time animals were switched to either the KD or CD and followed until 28 dpi. For the study presented in Supplementary Figures 7B, C, KD-fed mice were followed until 35 dpi. Manifestations of progressive ascending paralysis of classical EAE were assessed daily, using a more granular scoring system than we previously described (35-37): 0-no disease, 0.5-reduced tail tone, 1-loss of tail tone, 1.5-limp tail and ataxia, 2-hind limb paresis, 2.5-one hind limb paralyzed, 3complete hind limb paralysis, 3.5-complete hind limb paralysis and forelimb weakness, 4-hind limb paralysis and forelimb paresis, 5-moribund or dead. Mice were weighed daily to ensure weight loss did not exceed 25% of starting weight at the time of immunization.

Visual acuity assessment
Visual acuity threshold was measured daily by OKT response using Optometry software and apparatus (Cerebral Mechanics Inc., Alberta, Canada) as previously described (35)(36)(37). Briefly, mice are placed on a pedestal inside a box with a virtual cylinder consisting of vertical lines projected on four computer screens of the box surrounding the animal. The vertical lines rotate at varying frequencies, and tracking behavior is assessed in a stepwise manner as the thickness of the lines are reduced. Visual acuity is represented as the highest spatial frequency at which mice track the rotating cylinder. Optokinetic tracking is a temporal-to-nasal reflex, and therefore counter-clockwise and clockwise rotations exclusively test the right and left eye, respectively. When EAE-induced motor deficits rendered mice without the balance and stability to adequately perform OKT testing, a measurement was not recorded for the affected mouse on that day.

Retinal flatmount analysis
Retinal flatmounts were prepared and RGCs labeled and quantified as described (35-37) using anti-Brn3a (goat; Santa Cruz, Santa Cruz, CA; sc31984; 1:500), Alexa 546nm Fluor-conjugated donkey anti-goat IgG (Molecular Probes; 1:1,000), and Hoechst 33342 (2 µg/ml), all diluted in 3% BSA/10% donkey serum/PBS. Retinas were washed in PBS before mounting with Prolong Gold mounting medium (Life Technologies, Grand Island, NY) and examined with a Nikon 80i microscope with a 60X objective. Images were captured with a DXM1200C camera using NIS-Elements software (Nikon, Inc. Tokyo, Japan). Photomicrographs were captured from the four leaflets comprising the flatmount with representative images captured from the peripheral, medial, and central retina within each quadrant, yielding 12 pictures per retina. Images were identically contrast-enhanced. Brn3a-positive RGCs were counted manually using the FIJI Cell Counter Plugin.

Fatty acid analysis
Plasma fatty acid profiles were determined by extracting total lipids from 40 to 50 µl of plasma following the method of Bligh and Dyer (38) with slight modifications (39). The purified total lipid extracts were stored under nitrogen until use. To these lipid extracts, 50 nmol each of 15:0 and 17:0 internal standards were added and the total extracts subjected to acid hydrolysis/methanolysis to generate fatty acid methyl esters (FAMEs) (40). All reagents for fatty acid extraction and derivatization were of the highest quality available from Sigma-Aldrich. FAMEs were identified using an Agilent Technologies 7890A gas chromatograph with a 5975C inert XL mass spectrometer detector (Agilent Technologies, Lexington, MA) as described (40). The gas chromatograph-mass spectrometer was operated in the electron impact total ion monitoring mode. The injection volume was 1 µl and the inlet, held at 325 • C, was set to pulsed splitless mode. An Agilent Technologies HP-5MS column (30 m × 0.25 mm × 0.25 µm) was used with a helium carrier gas flow rate of 1.2 ml/min. The oven temperature began at 130 • C for 1.0 min, was ramped to 170 • C at 6.8 • C/min, and was then ramped to 215 • C at 2.9 • C/min. After holding at 215 • C for 15.0 min, the oven was ramped to 260 • C at 20 • C/min and held for 5.0 min. The oven was then ramped to 325 • C at 15 • C/min and held for 18.0 min. The mass spectrometer transfer line, ion source, and quadrupole temperatures were 325, 230, and 150 • C, respectively.
FAMEs were quantified using an Agilent Technologies 6890N gas chromatograph with flame ionization detector (GC-FID) (41). Sample concentrations were determined by comparison to internal standards 15:0 and 17:0. The injection volume was 1 µl and the inlet, held at 290 • C, was set to pulsed split mode (10:1 ratio). An Agilent Technologies DB-23 column (60 m × 0.32 mm × 0.25 µm) was used with a hydrogen carrier gas constant pressure of 13.1 psi. The oven temperature began at 130 • C for 0.8 min, was ramped to 170 • C at 8.2 • C/min, and was then ramped to 215 • C at 3.5 • C/min. After holding at 215 • C for 9.5 min, the oven was ramped to 230 • C at 50 • C/min, and was then held for 8 min. The oven was then ramped to 290 • C at 12.0 • C/min and was held for 12 min. The .
/fneur. . Mice were fed a KD or CD for weeks prior to MOG − immunization to acclimate and were maintained on their respective diets for the duration of the experiment. Motor scores and visual acuity were tracked daily, and blood draws (red drop symbol) for glucose and ketone levels were taken at days − , , and dpi. Tissues and plasma were harvested weeks post immunization at study termination. (C-E) Graphs of body weights (C), Frontiers in Neurology frontiersin.org . /fneur. .

FIGURE (Continued)
blood glucose levels (D), and blood ketones (E) as a function of days before and after MOG − immunization. For graphs, blue and green traces show female and male mice on KD, respectively. Red and purple traces show female and male mice on CD, respectively. N = -mice/sex. Whisker-bar standard deviations and p-values for di erences between curves in C were computed with mixed linear model implementation of lme function, nlme R package. Asterisks denote statistical significance: *p < . , **p < . , ****p < . ; ns, not statistically significant. Data compiled from to independent experiments. detector was held at 290 • C. Data for the diets is represented as µg of each fatty acid per mg of diet, and data from plasma is represented as nmol of fatty acid per mg of plasma protein.

Blood glucose and ketone measurements
Blood ketone levels (non-fasting) were measured with a Precision Xtra Blood Ketone Monitoring System and fasting blood glucose was measured after 6 h of food withdrawal using a True Metrix Blood Glucose Meter. Blood was drawn from the tail vein.

Cytokine analyses
Twenty-six cytokines and chemokines in the plasma of EAE mice 21 dpi were assayed using custom xMAP multiplex cytokine panels from Biotechne, Inc. according to the manufacturer's recommendations and processed in the OMRF Arthritis and Clinical Immunology Human Phenotyping Core. Six factors were below the detection limit of the assay in all samples (IL-1β, IL-2, IL-4, IL-13, IL-17A, and IL-27) and excluded from the table in Figure 6.

Immunohistochemistry of optic nerves
To assess oligodendrocytes (as a marker of myelination) as well as lymphocyte and macrophage infiltration, we incubated sequential de-paraffinized sections with anti-2 ′ ,3 ′ -cyclicnucleotide 3 ′ -phosphodiesterase (CNPase; mouse CNPase: 1:200 dilution; Biolegend, Inc.), anti-CD3 (rabbit; 1:250; Abcam) or anti-Iba1 (mouse; 1:200; Millipore, Inc.) antibodies after antigen retrieval in R-buffer B. Images were captured using a Nikon TE2000 fluorescent microscope. Quantification of staining was performed using FIJI Software. CNPase, Iba1, and CD3 staining on sections spanning the entire length of the optic nerve were quantified by a masked experimentalist using representative images from both ends and the middle of the optic nerve. The number of positive cells within a defined area were counted using the Cell Counter Plugin, and the number of positive-stained cells per 10,000 pixels was calculated for each optic nerve.

Statistical analysis
Confidence intervals and p-values for the statistical significance of each studied effect in the longitudinal or binocular data were determined by fitting the data to a linear mixed-effects model, using the lme function implemented in the nlme R package, as detailed in Larabee et al. (35), Axtell et al. (42), Laird and Ware (43), and Lindstrom and Bates (44). This function is an extended version of regular linear regression but can accommodate complex data collection design features, such as longitudinal measurements, nested layers, and within-group correlation. Standard testing methods, such as the Student's paired or unpaired t-test and the Mann-Whitney exact test, with Bonferroni correction for multiple testing, were employed when no embedding was involved. Graphpad Prism 9 was used to display the results. Testing for differences in marker levels among the five mice groups in Figure 4 was performed following log transformations for improving normality by unequal group variance ANOVA and Tukey's post hoc adjustment. Differences in cytokine and chemokine levels in Figure 6 were assessed with robust linear regression using the rlm function in MASS R package, and p-value computations relied on robust F-Testing (Wald) performed with f.robftest function from sfsmisc package. Principal component exploratory analysis (PCA) and biplot generation were carried out with specific functions from stats package in R. Multidimensional 95% confidence regions were added to the biplot using the draw.ellipse function from the plotrix package. Throughout the manuscript, the asterisks code denoting significance is: * p < 0.05, * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001; ns, not statistically significant.

Results
The current study tested the efficacy of a KD in the EAE mouse model of autoimmune-mediated demyelination. Many preclinical studies that have demonstrated therapeutic efficacy with a KD have used formulations containing lard, soybean oil, and hydrogenated fats [e.g., (22,45,46)]. In contrast, the KD used here is enriched in fiber and the sources of fat are MCTs, flaxseed oil, and canola oil ( Figure 1A). This diet was designed to be antiinflammatory by minimizing ω6 fatty acids and hydrogenated fats (19). A complete list of the diet ingredients is provided in Supplementary Figure 1A.
The experimental paradigm used to test the KD in a preventive regimen is shown in Figure 1B. Animals were fed either a KD or an ingredient-matched control diet (CD) for 2 weeks prior to MOG 35−55 peptide immunization to acclimate mice to the diet based on previous strategies testing dietary impacts on CNS autoimmunity in the EAE model [e.g., (47)]. Mice were maintained on their respective diets for the duration of the experiment during which motor scores, vision, and body weights were longitudinally tracked and blood was collected. Three weeks post-immunization, mice were euthanized for postmortem analyses. Notably, consumption of the KD for 5 weeks in the absence of EAE led to modest weight reduction whereas 5 weeks of the CD did not (Supplementary Figure 1B). Animals were randomized to KD or CD groups and had comparable weights within each sex at study initiation ( Figure 1C). From immunization to completion of experiments at 21dpi, among EAE mice fed the KD, females slightly gained weight whereas males were weight stabilized. In contrast, both sexes on the CD undergoing EAE lost body weight (Supplementary Figure 1C), consistent with sickness behavior (e.g., inflammatory anorexia) (48). Compared to the CD, the KD reduced fasting blood glucose levels after 2 weeks of feeding despite no differences at study onset or termination ( Figure 1D). Elevated non-fasting ketones are a defining molecular signature of a KD (49) and circulating, nonfasting ketones (β-hydroxybutyrate specifically) were increased in mice fed the KD for 2 weeks and persisted through the duration of the study ( Figure 1E). Mice on the CD also had moderately elevated circulating ketones 3 weeks after immunization, likely related to sickness behavior (e.g., inflammatory anorexia) (48). These data show that the KD lowered fasting blood glucose and induced nutritional ketosis but did not promote obesity during 5 week-long experiments.

KD prevents the onset of motor and visual deficits in EAE mice
Remarkably, mice fed the KD were spared the ascending paralysis and motor deficits induced by EAE (Figure 2A), with comparable efficacy observed for females and males (Figures 2B, C, respectively). Because optic neuritis and visual disturbances are major sequelae of autoimmune demyelinating disease in humans (50)(51)(52), we complemented the motor score measurements with visual acuity measurements using optokinetic tracking (OKT). OKTs were recorded at the initiation of the diets, prior to MOG 35−55 immunization, and then daily beginning ∼10 dpi, .
/fneur. . coincident with symptom onset in CD-fed animals ( Figure 1B). As we reported previously (36), mice subjected to MOG-EAE typically undergo episodic monocular vision loss, consistent with what is experienced by people with relapsing-remitting MS (RRMS) (53, 54). These deficits are readily detectable as diminished visual acuity in one eye (35, 36). The OKT measures each eye separately, so every mouse has a designated more affected (MA) and less affected (LA) eye. Coincident with the mitigation of motor deficits, the KD preserved vision in the MA eyes of EAE mice ( Figure 3A). Although visual deficits were minimal in the LA eyes of mice on either diet, the LA eyes of KDfed mice retained better vision than their CD-fed counterparts ( Figure 3B, inset). In alignment with the efficacy of the KD on motor function, this preservation of vision was independent of sex ( Supplementary Figures 2A, B).
The axons of retinal ganglion cells (RGCs) are bundled together into the optic nerve and episodes of optic neuritis induce RGC apoptosis (55-59). We therefore quantified RGC counts and found that the functional preservation of vision mediated by the KD was accompanied by a sparing of Brn3A + RGCs in the central and medial retina of MA eyes from EAE mice (Figures 3C, D). As we reported previously (36), RGCs in the peripheral retina are not typically lost during MOG-EAE ( Figure 3D), fitting with RGC loss in longstanding MS cases being most prominent in the central retina, nearest the optic nerve head (60). When analyzed as a function of diet and sex, the KD-mediated sparing of RGCs in the MA eyes was significant in the central retina for both females and males (Supplementary Figure 2C).

The KD preserves oligodendrocytes and restricts inflammatory infiltration of the optic nerve in EAE mice
To complement the OKT and RGC analyses, immunohistochemistry (IHC) was performed on paraffinembedded optic nerves from the MA eyes of the mice analyzed for visual function in Figure 3. For each marker, labeling was quantified from sections captured along the entire length of the optic nerve, and optic nerves from healthy (i.e., no EAE) mice were included for comparison. CNPase, an oligodendrocyte marker and proxy of myelination, was largely preserved on the optic nerves of KD-fed mice ( Figure 4A). CNPase labeling was quantified by comparing perinuclear staining, with the specificity of this staining corroborated by labeling optic nerves from healthy mice ( Figure 4A, left panel inset). Labeling for Iba1 and CD3 to mark macrophages/microglia and T cells, respectively, revealed that mice fed the KD had reduced Iba1 and CD3 labeling of the optic nerve compared to mice fed the CD ( Figures 4B, C, respectively).
When the data were further examined to determine the combined impacts of diet and sex, both females and males on the KD had significantly preserved CNPase labeling compared to their CD-fed counterparts (Supplementary Figure 3A). Likewise, KD-fed male optic nerves had decreased Iba1 and CD3 labeling compared to CD-fed males (Supplementary Figures 3B, C, respectively). Due to higher variability between samples, differences in Iba1 and CD3 labeling did not reach significance between the KD and CD female mice (Supplementary Figures 3B, C, respectively), despite the preservation of visual acuity for female mice on the KD (Supplementary Figure 2A). Notably, all groups had statistically significant increases in Iba1 labeling compared to optic nerves from healthy mice (Supplementary Figure 3B), despite no loss of visual acuity for the KD group ( Figure 3A). For CD3 labeling, females and males on the KD were not statistically different than healthy mice whereas EAE males and females on the CD showed significant increases in CD3 + infiltrates (Supplementary Figure 3C).
Principal components exploratory analysis ( Figure 4D) confirmed that the relatively higher CNPase labeling and lower Iba1 and CD3 labeling for mice fed the KD correlated with lower motor and visual acuity deficits. The first principal component reveals that the KD-fed mice are statistically comparable to healthy mice but significantly different from the CD-fed group, presenting significantly improved motor and visual acuity scores and higher CNPase levels, along with lower Iba1 and CD3 levels. The second component suggests marginal separation of the healthy mice group from KD-fed mice, in terms of Iba1 and CD3 levels.
We also observed several circulating FAs changed as a combined function of sex and diet. . 20:5n3 was also increased in KDfed females but did not reach significance because of high variability among 20:5n3 levels within female CD-fed mice (data not shown). Together, these data are consistent with the KD conferring protection from EAE pathologies at least in part by enriching the systemic milieu with ω3 fatty acids associated with SPM-mediated neuroprotection and decreasing fatty acids, such as 20:3n9, that are precursors for the biosynthesis of pro-inflammatory leukotrienes.
To determine which of the enriched circulating ω3 fatty acids in EAE mice consuming the KD were contributed directly by the diet, we analyzed the fatty acid content of each diet (Supplementary Figure 4C). Notably, compared to the CD, the additional flaxseed oil in the KD led to an enrichment by 4.3 µg fatty acid/mg sample (or 1.6-fold) in 18:3n3 (α-linolenic acid; ALA). 18:3n3 can be biosynthetically converted in vivo to the corresponding 20:4n3, 20:5n3, and 22:5n3 (63, 64). As 20:4n3, 20:5n3, and 22:5n3 were absent from the KD and CD diets, the elevated levels of these ω3 fatty acids in the plasma of EAE mice consuming the KD ( Figure 5B) appear to be derived from the endogenous conversion of 18:3n3.

Cytokines impacted by the KD in EAE mice
Complementary evidence supporting the anti-inflammatory milieu resulting from the KD came from an analysis of 26 different cytokines and chemokines in the plasma 21 dpi, of which 20 were detectable in some or all samples within the sensitivity of the multiplex assay ( Figure 6A). Of the cytokines detected, granulocyte-colony stimulating factor (G-CSF), C-X-C Motif Chemokine Ligand 2 (CXCL2), C-C motif chemokine ligand 11 (CCL11), and IL-6 were all significantly decreased 21 dpi in the plasma of EAE mice consuming the KD ( Figure 6B). Two sex-specific changes were also detected .
/fneur. .  accompanying p-values above the bars indicate statistical significance as calculated by unequal group variance ANOVA with Tukey's post hoc adjustment. Size bar in (C) corresponds to µm. (D) Principal components (PC) biplot to summarize, in d projection, the similarities among mice groups and their relationships to the five measured scores (i.e., motor scores, changes in OKT from baseline, CNPase, Iba , CD ). The dots on the graph are samples, colored according to their phenotype group, with the measured variables shown as arrows. Arrow coordinates on the two axes show each variable contributing loading on the first two principal components. Multidimensional % confidence regions for each mouse group, projected in d, are shown as ellipses. All data acquired from n ≥ mice/sex/diet. The graph to the right of the PC biplot makes explicit, for the five markers, the di erence between the two clusters of mice (Healthy + KD vs. CD) separated by the first principal component. Whisker bars depicting % confidence intervals are shown for each variable both in Healthy + KD mice and in CD mice with the p-values assessing the significance of their di erences marked on the graph. (Supplementary Figure 5). EAE females on the KD had elevated monocyte-colony stimulating factor (M-CSF) whereas males had reduced C-reactive protein (CRP). Curiously, CRP levels in KD female mice were comparable to the levels in KD-fed males but CRP was not elevated in the female CD-fed group (Supplementary Figure 5).

EAE mice fed a KD mount a T-cell response to MOG − immunization
To rule out that the protection conferred by the KD in the preventive regimen was not a consequence of the diet blunting an immune response to the MOG antigen, splenoctyes were isolated 21 dpi from EAE mice on each diet, stimulated ex vivo in culture with MOG 35−55 peptide, and subsequently with PMA, iononmycin, and monensin prior to antibody labeling to identify activated T cells (i.e., CD3 + , CD4 + , CD44 ++ ) and their respective intracellular levels of IL-17 and IFN-γ by flow cytometry (Supplementary Figure 6A). IL-17 and IFN-γ expression by MOG 35−55 -activated splenic T cells is a signature EAE response following immunization with MOG [e.g., (65,66)]. For comparison purposes, unstimulated sets of splenocytes from mice on either the KD or CD were similarly stained for intracellular IL-17 and IFN-γ. These analyses showed that the percent of live CD3 + CD4 + CD44 ++ T cells expressing IL-17 in the spleen was comparable between diets whereas a greater faction of stimulated splenocytes from KD-fed animals expressed IFN-γ vs. their CD-fed counterparts (Supplementary Figures 6A, B). Notably, the level of activated CD3 + CD4 + CD44 ++ T cells was similar between the KD and CD groups (Supplementary Figure 6C).
Of note, we observed a small of percentage of "breakthrough" mice consuming the KD that began to exhibit EAE symptoms ∼19-21 dpi (Supplementary Figure 7A). To determine whether all or most KD-fed mice would manifest EAE symptoms if the experimental time course was extended, we tracked EAE mice on the KD for an additional 2 weeks (i.e., 2 weeks pre-immunization plus 5 weeks post-immunization). Compared to the 90% of CDfed mice (100% males and 80% females) that showed symptoms   Table of cytokine and chemokine measurements from the plasma of EAE mice. Asterisks and bolding highlight factors that are statistically significantly di erent as a function of diet. "AVG" denotes average and "SD" denotes standard deviation. "ND" indicates that all samples analzyed were below the detection limit of the assay and "N/A" indicates that insu cient numbers of samples were within the detectable range of the assay to perform statistical analyses. (B) Graphical presentation of the four factors that di er in the plasma dpi as a function of diet. EAE mice consumed either the KD (green) or a CD (orange), -mice per diet per sex from ≥ independent experiments. Statistical significance for cytokines and chemokines was determined with robust linear regression using the rlm function in MASS R package. p-values relied on robust F-Testing (Wald) performed with f.robftest function from sfsmisc package. For graphs in (B), *p < . , ***p < . .
within 10-16 dpi (11-12 dpi for males and 10-16 dpi for females), extending the study out to 7 weeks total revealed that disease incidence among KD-fed mice was ∼ 33% (20% for females and 50% for males) with symptoms not detected until 22-33 dpi (22-33 dpi for males and 31-32 dpi for females; Supplementary Figure 7B). Furthermore, the severity of motor deficits among the subset of KD-fed animals that eventually developed symptoms was significantly reduced compared to their CD-fed counterparts (Supplementary Figure 7C). Together, these lines of evidence support that although most mice on the KD are protected from the overt pathologies induced by the EAE model, the underlying mechanism is not a failure to mount a T cell response to immunization with the MOG antigen. Collectively, the KD effectively prevented disease onset in most mice and mitigated disease severity in the subset of mice that manifested functional deficits.

The KD as an interventional regimen restores motor and visual function
Complementary studies in female and male mice were performed to determine the efficacy of the KD as an interventional regimen ( Figure 7A). All mice were maintained on standard chow until EAE symptom onset. At the first observable sign of motor and visual deficits, mice were switched to either the KD or the CD for the remainder of the study. Within 4 days of consuming the KD, motor (Figures 7B, C) and visual (Figures 7D, E) deficits were significantly mitigated in both sexes and continued to improve such that motor and visual function were restored to near baseline levels by the termination of the study. Notably, these studies spanned 26-30 days from the day of MOG immunization until termination but the data are graphed with the x-axis representing "days postsymptom onset and diet switch" to normalize for all mice not .
Frontiers in Neurology frontiersin.org . /fneur. . manifesting their respective initial functional deficits on the same day. These results complement the above prevention studies and importantly, confer translational relevance and feasibility to this dietary strategy.

Discussion
Previous studies established that dietary interventions including caloric restriction (20), a fast-mimicking diet (21,22), intermittent fasting (23, 24), and a KD (22,25,26) can ameliorate EAE motor and cognitive deficits in EAE mice. Our data expand on these findings by demonstrating that a KD preserves visual acuity ( Figure 3) and myelination of the optic nerve, reduces Iba1 + and CD3 + infiltrates (Figure 4), and spares RGCs (Figure 3). We further show that the benefits extend comparably to female and male mice and that the KD increased circulating levels of the anti-inflammatory ω3 fatty acids 18:3n3, 20:4n3, 20:5n3, and 22:5n3 while decreasing the pro-inflammatory fatty acids 20:3n9 and 20:4n6 ( Figure 5). We also detected a KD-mediated decrease in circulating cytokines implicated in EAE and MS, namely G-CSF, CXCL2, CCL11, and IL-6 ( Figure 6B). When tested as an intervention, the KD promoted rapid and nearly complete recovery of motor and visual function (Figure 7). This finding is particularly promising for establishing the clinical feasibility of acutely implementing the KD in response to symptom relapse.
The diet composition of the KD used in the present study (19) differs with previous EAE studies that used KDs containing butter, corn oil, and lard [e.g., (22,26)], sources of fat associated with hepatic dysfunction and inflammation (67, 68). The different KDs were efficacious in mitigating EAE sequelae, but a consideration of the cardiometabolic health risks of chronically consuming high fat diets is of relevance to people with MS as these individuals will likely implement long-term nutritional interventions. In this regard, our studies support that the use of a KD enriched in ω3 fatty acids including α-linolenic acid (ALA, 18:3n3) can elicit therapeutic benefits without promoting weight gain or increasing inflammation (Supplementary Figure 1 and Figure 6).
Because different KD formulations mitigate EAE pathologies despite containing various pro-and anti-inflammatory fats (22,26), the therapeutic efficacy of KDs may derive from the production of ketones and/or the dramatic limitation of simple sugars and starch consumption common to these regimens. Consistent with these hypotheses, high dietary glucose increases disease severity in the EAE model by promoting T H 17 cell differentiation via ROSmediated TGF-β activation (69). In clinical work, a KD improved glycemic control and reduced the medications of people with obesity and diabetes more effectively than a 55% carbohydrate low glycemic index diet (70). For people with (pre)diabetes, a KD reduced glycosylated hemoglobin (HbA1C), fasting glucose, fasting insulin, weight, blood pressure, triglycerides, alanine aminotransferase, and high-density lipoprotein, consistent with safety and tolerability for long-term adherence. Remarkably, 53% of enrolled participants achieved disease resolution (30). Individuals that are overweight and implemented a KD also had decreased body weight, insulin resistance, and serum markers of inflammation (e.g., TNF-α, IL-6, IL-8, MCP-1) (71)(72)(73). These normalizing effects on insulin sensitivity and insulin resistance benefit people with metabolic syndrome and diabetes [reviewed in (74)] and likely extend to people with MS, as insulin resistance is associated with elevated disability scores (16,17). Clinical trials with small cohorts of people with MS have also shown promising responses using (modified) paleolithic diets that eliminate or dramatically reduce the consumption of simple carbohydrates, ultra-processed foods, and other putative disease aggravators (e.g., gluten, dairy, legumes) (13,(75)(76)(77)(78)(79)(80)(81).
A potential mechanistic contribution mediating the efficacy of the KD in the present study comes from the anti-inflammatory actions of resolvins, a family of SPMs derived from ALA. In alignment with this idea, elevated dietary consumption of 18:3n3 has been inversely linked with the risk of developing MS (82). ALA was enriched in the KD (Supplementary Figure 4C) as well as in the plasma of EAE mice fed the KD ( Figure 5B). Moreover, the ω3 fatty acids in the plasma of mice fed the KD may directly contribute to lower levels of inflammatory infiltrates in the MA optic nerves (Figure 4) as ω3 fatty acids enhanced lesion recovery by decreasing phagocytic infiltration to the corpus callosum following demyelination (83).
The plasma of KD EAE females and males was enriched in ETA (20:4n3) and DPA (22:5n3; Figure 5 and Supplementary Figures 4A, B) and males were also enriched with EPA (20:5n3; Supplementary Figure 4B). 20:5n3 and 22:5n3 are intermediate biosynthetic precursors of E-series and T-series resolvins, respectively. DHA (22:6n3) and arachidonic acid (20:4n6), the intermediates from which the Resolvin D-series and the lipoxins are synthesized, respectively, were not increased and arachidonic acid levels were reduced. However, the lack of elevated DHA and arachidonic acid does not necessarily preclude contributions from their respective SPMs as efficient synthesis reactions could limit accumulation of these intermediates. A study using the MOG-EAE model and standard chow over a 45-day course showed PUFA metabolism is compromised during disease progression and that daily administration of exogenous Resolvin D1 decreased EAE pathologies (84). Clinical work has identified imbalances in pro-inflammatory eicosanoids and SPM levels in the plasma of people with MS as a function of disease progression and severity. The expression of multiple biosynthetic enzymes and receptors for SPMs were impaired in peripheral blood mononuclear cells from people with MS and their monocytes were less responsive to SPMs in culture (85). Such deficiencies in the SPM synthetic machinery may underlie the observation that elevated EPA in the serum of people with MS tracks with increased severity on the expanded disability status scale (EDSS). This study additionally reported that circulating arachidonic acid levels associate with relapse-free status (86). Notably, study participants were taking IFN-β or other disease-modifying therapies, which confounds comparing these data directly to our study or other stand-alone dietary intervention studies.
Fitting with the KD promoting a systemic anti-inflammatory milieu, we detected reductions in four pro-inflammatory cytokines and chemokines in the circulation as a function of diet: G-CSF, CXCL2, CCL11, and IL-6 ( Figure 6). G-CSF and CXCL2 were the two pro-inflammatory cytokines reduced in both sexes fed the KD (Supplementary Figure 5). G-CSF levels are typically kept low but rapidly increase in response to stress and inflammation to stimulate the production and maturation of granulocytes and neutrophils.
G-CSF governs early signaling necessary for EAE disease induction (87) with the neutrophils produced driving multiple steps of EAE and MS progression [e.g., (88)(89)(90)(91)(92)], fitting with the KD reducing G-CSF in the circulation and blocking symptom onset. CXCL2 levels were also reduced by the KD (Figure 6B). This factor is also known as macrophage inflammatory protein 2-α (MIP2-α), is produced by macrophages and neutrophils at sites of inflammation, and functions to recruit neutrophils during inflammation (93). Transient receptor potential melastatin 2 knockout mice also have reduced CXCL2 and a suppression of neutrophil infiltration into the CNS during EAE (94). Thus, reduced levels of circulating CXCL2 are consistent with disease mitigation (95-97) and fit logically with lower G-CSF levels limiting neutrophil maturation.
KD fed mice had reduced CCL11, also known as eosinophil chemotactic protein (eotaxin-1; Figure 6B). This chemokine is a putative biomarker of disease duration in people with secondary progressive MS (98). Curiously, in a rat EAE model, increased CCL11 expression was associated with a T H 2 anti-inflammatory response but did not correlate with eosinophil recruitment. Elevated CCL11 was detected in the CSF and lymph nodes (but not in the serum) and correlated with decreased ED1 + /Iba1 + macrophages in the spinal cord and with protecting the integrity of the blood-brain barrier (99). In contrast, CCL11 was found to be elevated in the spinal cords of mice undergoing MOG 35−55 EAE (100). As CCL11 has been implicated in systemic inflammation and as a pathogenic factor in a range of neurodegenerative and neuroinflammatory diseases [reviewed in (101)], reduced CCL11 in the serum by the KD is consistent with an anti-inflammatory milieu.
The KD reduced circulating levels of IL-6 ( Figure 6B), a pro-inflammatory cytokine secreted by astrocytes, macrophages, and microglia as well as other cell types in the CNS (e.g., neurons and endothelial cells) (102). IL-6 is detectable in brain lesions (103) and in the CSF from people with MS (104) and plays critical roles in MS and EAE pathophysiology that include compromising blood-brain barrier integrity in combination with IL-17A (105), cooperating with transforming growth factor-β to drive the differentiation and expansion of auto-reactive T H 17 cells (106)(107)(108)(109), and damaging myelin (102). Reduced circulating IL-6 by a KD has been reported [e.g., (110-116)] although studies in select populations [e.g., (117,118)] and some mouse models have also reported that a KD may not change or may even increase levels of this cytokine [e.g., (119)]. Reduced levels of IL-6 in the serum of KD-fed EAE mice are consistent with this dietary approach blunting CNS disease burden and maintaining motor and visual functions.
In conclusion, our work demonstrates the efficacy of a KD to preserve motor and visual function in mice undergoing autoimmune demyelinating disease. This dietary strategy limits systemic inflammation by reducing key cytokines involved in mediating the infiltration, activation, and differentiation of auto-reactive T cells and neutrophils into the CNS. We further posit that this KD provides an abundance of ω3 fatty acids for SPM biosynthesis, the products of which restrict acute inflammatory responses to self-antigens from transitioning to chronic inflammation and tissue damage.
The observation that initiating the KD beginning at the time of symptom onset can resolve both motor and visual deficits (Figure 7) supports the potential of this diet for direct translational application and improved patience compliance, a current barrier for nutrition-based therapeutic strategies (120)(121)(122).

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.

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SUPPLEMENTAL FIGURE
The KD preserves optic nerve myelination and reduces infiltrates. Mean values and % confidence intervals for CNPase (A), Iba (B), and CD (C) labeling of optic nerve sections. Data shown for females and males separately ("F"; "M") as well as combined ("F + M"). Whisker bars depict % confidence intervals for each variable and horizontal bars with accompanying p-values above the bars indicate statistical significance.

SUPPLEMENTAL FIGURE
Fatty acids analyzed in the plasma and diets. (A, B) Graphs depicting nmols of individual fatty acids per mg of protein in the plasma that are statistically significantly di erent ( * p ≤ . , * * p < . , * * * p < . , * * * * p < . as determined by Mann-Whitney test with Bonferroni correction) between EAE mice fed the KD vs. the CD. Graphs represent data from females (A) and males (B). (C) Graph showing micrograms (µg) of each fatty acid per milligram (mg) of diet comparing the KD, CD, and standard chow (SC). For each graph, the fatty acid omega designations are listed on the x-axis. n ≥ mice per diet from ≥ independent experiments and diet samples.

SUPPLEMENTAL FIGURE
Cytokines analyzed in the plasma. Graphs showing concentrations of the indicated cytokines and chemokines in the plasma dpi as a function of both sex and diet. Female and male EAE mice consumed either the KD (blue for females and green for males) or a CD (red for females and purple for males), -mice per diet per sex from ≥ independent experiments. Statistical significance was determined by Mann-Whitney test with 'ns' indicating not significant. * p < . , * * p < . , * * * p < . , * * * * p < . .  Table  showing the incidence of "breakthrough" mice on the KD that developed EAE motor and visual deficits within days of harvest (i.e., -dpi). Data compiled ≥ independent experiments. (B) Kaplan Meier graph showing the percent of mice (females + males) that developed EAE symptoms as a function of diet and time post-immunization. Mice on the KD were followed for days after immunization. N = mice on KD (green trace) and n = mice on CD (orange trace). Statistical significance determined by log-rank (Mantel-Cox) test; * * * * p < .
. (C) Graph of mean EAE motor scores as a function of diet using symptomatic mice from (B). N = mice on KD and n = mice on CD that manifested symptoms. Statistical significance determined by unpaired T-test; * * * p < . .