Introduction: Dimethyl fumarate (DMF) has shown potential for protection in various animal models of neurological diseases. However, the impact of DMF on changes in peripheral immune organs and the central nervous system (CNS) immune cell composition after ischemic stroke remains unclear. Methods: Eight-week-old C57BL/6J mice with photothrombosis ischemia and patients with acute ischemic stroke (AIS) were treated with DMF. TTC staining, flow cytometry, and immunofluorescence staining were used to evaluate the infarct volume and changes in immune cells in the periphery and the CNS. Results: DMF reduced the infarct volume on day 1 after PT. DMF reduced the percentages of peripheral immune cells, such as neutrophils, dendritic cells, macrophages, and monocytes, on day 1, followed by NK cells on day 3 and B cells on day 7 after PT. In the CNS, DMF significantly reduced the percentage of monocytes in the brain on day 3 after PT. In addition, DMF increased the number of microglia in the peri-infarct area and reduced the number of neurons in the peri-infarct area in the acute and subacute phases after PT. In AIS patients, B cells decreased in patients receiving alteplase in combination with DMF. Conclusion: DMF can change the immune environment of the periphery and the CNS, reduce infarct volume in the acute phase, promote the recruitment of microglia and preserve neurons in the peri-infarct area after ischemic stroke.

Acute ischemic stroke (AIS) affects millions of people worldwide annually [1]. Most people who survive AIS are unable to live independently and experience neurological sequelae [1]. Timely reperfusion is considered to be the most effective treatment for patients with AIS [2]. However, a large proportion of the population still does not receive such treatment in time [2]. Therefore, researchers are trying to discover safe alternative drugs that might be beneficial for treating AIS through basic and translational experiments. Currently, stroke therapy focuses on inflammatory mechanisms [3, 4]. Inflammation and the immune response are the most important pathological mechanisms of acute injury [5]. To date, several clinical trials have investigated the efficacy of different immunomodulatory approaches for treating ischemic stroke, including enlimomab [6, 7], anakinra [8], fingolimod [9‒11], and natalizumab [12, 13]. However, use of these drugs for ischemic stroke treatment did not have significant therapeutic effects or were terminated due to complications. Although fingolimod reduced infarct volume after treating AIS patients, these three clinical trials are phase II proof-of-concept trials and need to be further verified in a large population.

Therefore, we are looking at another drug, dimethyl fumarate (DMF). DMF is an oral disease-modulating treatment drug for relapsing-remitting multiple sclerosis and is a small molecule that has anti-inflammatory, immunomodulatory, and antioxidant properties [14]. DMF can exert neuroprotective effects by activating the transcription factor nuclear factor erythroid-derived 2 (Nrf2) [15], further driving the expression of antioxidant genes [14]. DMF can also shift the immune balance from a pro-inflammatory to an anti-inflammatory state, inhibiting the activation, proliferation, expression and release of pro-inflammatory cytokines by T cells and B cells and promoting their apoptosis [14]. Previous studies have reported that DMF can improve neurological function, reduce infarct volume and cerebral edema [16‒18], inhibit neural apoptosis, suppress glial activation [17, 18], and reduce immune cell infiltration [18] through animal experiments. However, changes in different types of immune cells in peripheral immune organs and the central nervous system (CNS) in response to DMF in mice and humans after ischemic stroke have not been reported.

In this study, we examined the effects of DMF on various types of immune cells in experimental stroke using a photothrombosis model and AIS patients. By exploring the effects of DMF on infarct volume, changes in microglia and neurons, and changes in immune cells in peripheral immune organs and the CNS, our aim was to provide a new approach for the treatment of ischemic stroke, focusing on immune modulation.

Animals

In this study, two-month-old male C57BL/6J mice were used. We followed the guidelines for Animal Research: Reporting of In Vivo Experiments (ARRIVE) and obtained approval from the Animal Care and Use Committee of Xuanwu Hospital (XW-20211216-2).

PT-Induced Brain Ischemia Model

Focal cerebral ischemia was modeled using PT. The procedure was executed as previously described [19‒21]. In brief, the mice were anesthetized with 1.25% 2,2,2-tribromoethanol at a dose of 312.5 mg/kg (Sigma), followed by the injection of Rose Bengal (Sigma) through the retro-orbital vein at a dose of 30 mg/kg body weight. The skin over the midline of the skull was incised and pulled to the edge to expose the skull. The intact skull was subjected to focal illumination for 5 min using a KL 1600 LED cold light (SCHOTT) on an area 3 mm in diameter in the cortex. After the skin was repositioned, it was subsequently sutured back into its original position. One day after PT was regarded as the acute phase, while 3–7 days after PT were regarded as the subacute phase.

Treatment of Mice with DMF

DMF (Sigma) was dissolved in 10% dimethyl sulfoxide. DMF was given at 50 mg/kg body weight through intraperitoneal injection twice a day for 1, 3, or 7 consecutive days until the mice were sacrificed, with the first dose given 2–3 h after stroke. C57BL/6J mice were divided into 2 groups: the DMF (PT + 50 mg/kg DMF) group and the vehicle (PT + 10% dimethyl sulfoxide) group.

Infarct Volume Calculation

Mice were euthanized with 2,2,2-tribromoethanol, and brain tissue collected from the mice on days 1, 3, and 7 after ischemia. After the mice were sacrificed, the brain tissue was removed and manually sliced into 1-mm-thick coronal sections from the rostral to the caudal. Then, the brain sections were immersed in 2% TTC solution and incubated for 20 min at 37°C in the dark. The pale region indicates tissue infarction, and the infarct volume was calculated by ImageJ software. The cumulative infarct volume was determined by summing the individual section volumes of infarction. The percentage of infarct volume was determined as follows: ([total infarction volume in the ipsilateral hemisphere])/(total contralateral hemispheric volume + total ipsilateral hemispheric volume) × 100%.

Tissue Preparation

Mice were anesthetized and subjected to transcardial perfusion with chilled PBS at 1, 3 and 7 days after PT. For histological analysis, the entire brain was carefully removed from the skull and placed in a 4% paraformaldehyde solution for 6 h at 4°C. After undergoing sequential dehydration in sucrose solutions of 15% and 30%, the brains were embedded in Tissue-Tek® O.C.T. compound (Sakura® Finetek Inc., USA), rapidly frozen in liquid nitrogen, and then stored at −80°C for subsequent immunofluorescence staining.

Immunofluorescence Staining

Refrigerated sections (20 μm) were utilized for immunofluorescence staining. Frozen brain sections were permeabilized in PBST (PBS containing 0.3% Triton X-100). To prevent nonspecific staining, brain sections were incubated in 2% bovine serum albumin together with 5% goat serum in PBST for 1 h at RT. Afterward, the sections were incubated with diluted primary antibodies and left overnight at 4°C. Next, the sections were incubated with species-specific Alexa Fluor (488, 546 or 647)-conjugated secondary antibodies (Thermo Scientific, USA) for 1 h at RT in the dark. The following primary antibodies were utilized in this study: rabbit anti-Iba1 (1:500, Abcam), rat anti-GFAP (1:500, Invitrogen), and mouse anti-NeuN (1:500, Abcam). After DAPI nuclear counterstaining (Abcam), the sections were visualized using a confocal microscope (Zeiss, Germany) and Mica (Leica, Germany). The region located 200–300 µm away from the border of the infarct was identified as the peri-infarct area.

Flow Cytometry

Mice were anesthetized as previously described, and blood was collected via retro-orbital bleeding into anticoagulant tubes. After transcardial perfusion with cold PBS, the spleens and brains were quickly removed and immersed in cold PBS. RBC lysis buffer (BioLegend) was added to the whole blood for erythrocyte lysis. After centrifugation, the remaining pellets were washed with FACS buffer (pH 7.4; 0.1 m PBS; 1% 0.5 m EDTA; 1% FBS) and resuspended into single-cell suspensions. Spleens were subjected to mechanical homogenization through 100-µm cell strainers, followed by centrifugation. To induce erythrocyte lysis in spleen samples, RBC lysis buffer was added. Ultimately, splenocytes were processed to obtain single-cell suspensions. The brains were cut into small pieces and digested with collagenase D (1 mg/mL; Roche) and DNAse I (0.1 mg/mL; Roche) for 30 min at 37°C in FACS buffer before mechanical dissociation using a piston and 100-µm cell strainers. To isolate brain cells, 30% Percoll (Cytiva) was added to the cell pellets, which were subsequently centrifuged at 700 g for 20 min without braking. After the myelin layer and the supernatant were carefully aspirated, the remaining pellets were washed with FACS buffer and resuspended into single-cell suspensions. The cells were stained for extracellular markers with the following anti-mouse antibodies (BioLegend) diluted at 1:200: FITC-CD45, BV510-CD4, APC-CD8a, APC/Cy7-CD19, AF700-NK-1.1, BV785-CD11b, BV711-Ly-6C, PE/Cy7-CD11c, BV650-Ly-6G, BV605-F4/80, and 7-AAD Viability Staining Solution (BioLegend). Subsequently, the stained samples were analyzed by a FACSAria III flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software. The gating protocol is shown in online supplementary Figure S1 (for all online suppl. material, see https://doi.org/10.1159/000539589).

Patient Samples from the Clinical Trial

Blood samples from AIS patients were obtained from a clinical trial, which was designed as an open-label, 2-arm, evaluator-blinded, case-control study conducted at Xuanwu Hospital Capital Medical University, Beijing, China. The protocol and supporting documentation of this study were approved by the Xuanwu Hospital Capital Medical University Institutional Review Board. This research has been registered with the identifier NCT04890366 on ClinicalTrials.gov, and the inclusion and exclusion criteria can be found on the website. AIS patients who received rt-PA treatment within 4.5 h of symptom onset were divided into two groups: a control group (standard treatment according to the guidelines of the American Heart Association) and a DMF group (standard treatment plus DMF [Tecfidera, Biogen]). Each patient in the DMF group received an oral dose of 240 mg of the medication twice daily for 3 consecutive days, commencing within 1 h after baseline MRI and no later than 24 h after symptom onset. We confirmed that individuals from both groups exhibited comparable clinical and imaging characteristics (Table 1). Blood samples were collected at baseline and at days 1, 3, and 7.

Table 1.

Baseline characteristics of patients with AIS

VariableControl (n = 8)DMF (n = 3)p value
Age, years 61.8±5.6 66.3±2.4 0.65 
Sex, female, n (%) 2 (25) 0 (0) 
Previous stroke, n (%) 2 (25) 2 (67) 0.49 
Risk factors 
 Hypertensiona, n (%) 7 (87.5) 3 (100) 
 Diabetes mellitusb, n (%) 1 (12.5) 1 (33) 0.49 
 Blood glucose, mmol/L 10.0±1.9 8.7±2.3 
 Hyperlipidemiac, n (%) 2 (25) 0 (0) 
 Atrial fibrillation, n (%) 0 (0) 0 (0) 
 Current smoking, n (%) 2 (25) 2 (67) 0.49 
 Alcohol abuse, n (%) 3 (37.5) 1 (33) 
Medication, n (%) 
 Antiplatelet agent 1 (12.5) 1 (33) 0.49 
Etiology, n (%) 
 Subtype 1: atheromatosis 5 (62.5) 3 (100) 
 Subtype 2: embolus 1 (12.5) 0 (0) 
 Subtype 3: lacunar infarct 0 (0) 0 (0) 
 Subtype 4: other causes 2 (25) 0 (0) 
 Subtype 5: undetermined 0 (0) 0 (0) 
NIHSSd on admission 9.4±1.3 13.3±4.1 0.51 
Infarct volume on admission, mL 23.7±7.8 35.3±1.6 0.48 
Time, h 
 Time to alteplase treatment 2.6±0.6 2.0±0.5 0.58 
Occlusion site, n (%) 
 Anterior cerebral artery 1 (12.5) 0 (0) 
 Middle cerebral artery 3 (37.5) 3 (100) 
 Terminal internal carotid artery 4 (50) 0 (0) 
VariableControl (n = 8)DMF (n = 3)p value
Age, years 61.8±5.6 66.3±2.4 0.65 
Sex, female, n (%) 2 (25) 0 (0) 
Previous stroke, n (%) 2 (25) 2 (67) 0.49 
Risk factors 
 Hypertensiona, n (%) 7 (87.5) 3 (100) 
 Diabetes mellitusb, n (%) 1 (12.5) 1 (33) 0.49 
 Blood glucose, mmol/L 10.0±1.9 8.7±2.3 
 Hyperlipidemiac, n (%) 2 (25) 0 (0) 
 Atrial fibrillation, n (%) 0 (0) 0 (0) 
 Current smoking, n (%) 2 (25) 2 (67) 0.49 
 Alcohol abuse, n (%) 3 (37.5) 1 (33) 
Medication, n (%) 
 Antiplatelet agent 1 (12.5) 1 (33) 0.49 
Etiology, n (%) 
 Subtype 1: atheromatosis 5 (62.5) 3 (100) 
 Subtype 2: embolus 1 (12.5) 0 (0) 
 Subtype 3: lacunar infarct 0 (0) 0 (0) 
 Subtype 4: other causes 2 (25) 0 (0) 
 Subtype 5: undetermined 0 (0) 0 (0) 
NIHSSd on admission 9.4±1.3 13.3±4.1 0.51 
Infarct volume on admission, mL 23.7±7.8 35.3±1.6 0.48 
Time, h 
 Time to alteplase treatment 2.6±0.6 2.0±0.5 0.58 
Occlusion site, n (%) 
 Anterior cerebral artery 1 (12.5) 0 (0) 
 Middle cerebral artery 3 (37.5) 3 (100) 
 Terminal internal carotid artery 4 (50) 0 (0) 

The mean ± SE represents plus-minus values.

aHypertension was defined as systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg.

bDiabetes mellitus is diagnosed when fasting plasma glucose ≥7.0 mmol/L or 2-h postprandial blood glucose ≥11.1 mmol/L.

cHyperlipidemia is characterized by serum total cholesterol levels >5.72 mmol/L or triglycerides >1.7 mmol/L.

dNIHSS scores range from 0 to 42, with higher scores indicating more severe neurological deficits. NIHSS, National Institutes of Health Stroke Scale.

FACS Assessments of Human Blood

Lymphocyte subset changes were evaluated in all patients by analyzing whole-blood samples taken prior to the initial dose and at 1 day, 3 days, and 7 days following administration of the first dose. Routine blood examinations were also performed at the indicated time points. Whole blood was stained with the following antihuman antibodies (BioLegend) at a 1:100 dilution: FITC-CD3, PE-CD4, PE/Cyanine7-CD8a, APC-CD19, APC/Cyanine7-CD16, BV421-CD56, BV510-CD14, and PerCP-CD45. Then, RBC lysis buffer (BioLegend) was added for erythrocyte lysis. After centrifugation, the remaining pellets were washed with PBS. Then, Precision Count Beads (BioLegend) were added to calculate the absolute counts of the cells. The calculation formula is shown below. Finally, the samples were collected on a FACSAria III flow cytometer (BD Biosciences) and analyzed using FlowJo software.
AbsolutecellcountCells/μL=CellCount×PrecisionCountBeadsVolumePrecisionCountBeadsCount×CellVolume×PrecisionCountBeadsconcentrationBeads/μL

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8.0 software, and the data are presented as the mean ± SD. The normality of continuous variables was assessed using the Kolmogorov-Smirnoff test. Unpaired Student’s t tests were used to compare continuous variables between two groups with a normal distribution. Variables that did not follow a normal distribution or were discontinuous were presented as medians (ranges) and compared between groups using the Mann-Whitney U test. Multiple comparisons were carried out using two-way ANOVA followed by a Bonferroni post hoc correction or one-way ANOVA followed by a Tukey-Kramer post hoc test. Categorical variables were analyzed using the χ2 test (Fisher’s exact test when the expected value was <5). Statistical significance was determined at p < 0.05.

Treatment with DMF Reduces Infarct Volume at 1 Day after Ischemia

We first examined whether treatment with DMF could reduce infarct volume at the indicated time points after ischemic stroke. TTC staining was performed to measure the infarct volume. The infarct volume was significantly reduced in the DMF treatment group compared with that in the control group (infarct volume p = 0.0102; % infarct volume p = 0.0135) at 1 day after PT (Fig. 1a–c). However, the infarct volume at 3 days and 7 days after PT did not increase (Fig. 1d–i). The above results indicated that DMF might play a protective role in the acute phase of ischemic stroke.

Fig. 1.

TTC staining of brain tissue in DMF group and control group on 1-, 3-, and 7 days after PT. a-c TTC staining of brain sections from DMF treatment and vehicle treatment mice 1 day after PT. Six representative rostro-caudal brain sections are displayed (n = 7:6). d–f TTC staining of brain sections from DMF treatment and vehicle treatment mice 3 days after PT. Seven representative rostro-caudal brain sections are displayed (n = 9:9). g–i TTC staining of brain sections from DMF treatment and vehicle treatment mice 7 days after PT. Seven representative rostro-caudal brain sections are displayed (n = 6:7). *p < 0.05.

Fig. 1.

TTC staining of brain tissue in DMF group and control group on 1-, 3-, and 7 days after PT. a-c TTC staining of brain sections from DMF treatment and vehicle treatment mice 1 day after PT. Six representative rostro-caudal brain sections are displayed (n = 7:6). d–f TTC staining of brain sections from DMF treatment and vehicle treatment mice 3 days after PT. Seven representative rostro-caudal brain sections are displayed (n = 9:9). g–i TTC staining of brain sections from DMF treatment and vehicle treatment mice 7 days after PT. Seven representative rostro-caudal brain sections are displayed (n = 6:7). *p < 0.05.

Close modal

DMF Alters the Proportion of Peripheral Immune Cells and Reduces the Number of Monocytes in the CNS after Ischemia

Next, to determine the effects of DMF on immune cells involved in peripheral and CNS immunity after PT, flow cytometry was performed using blood, the spleen, and the brain at the indicated time points. One day after PT, the percentages of neutrophils, dendritic cells, macrophages, and monocytes in the spleen, which are involved in innate immunity, were significantly lower in the DMF group than in the control group (p = 0.0236, 0.0102, 0.0140, and 0.0027, respectively) (Fig. 2b). Next, the percentage of NK cells in the spleen of the DMF group was significantly decreased on day 3 after PT (p = 0.0204) (Fig. 2e), and the percentage of B cells in the spleen of the DMF group was also significantly decreased on day 7 after PT (p = 0.0010) (Fig. 2h); however, the percentage of CD4+ T cells in the spleen significantly increased in the DMF group both on day 3 (p = 0.0167) and day 7 (p = 0.0076) after PT. Furthermore, the percentage of monocytes in the brain parenchyma on day 3 after PT was significantly lower in the DMF group (p = 0.0464) (Fig. 2g). However, there were no significant changes in immune cells in the brain after treatment with DMF on days 1 or 7 (Fig. 2d, J) or in the blood after treatment with DMF on days 1, 3, or 7 (Fig. 2c, f, i). In addition, the absolute counts of these cells at each time point were also calculated, and there were no significant differences between the two groups (online suppl. Fig. S2). The above results indicated that DMF can reduce the number of innate immune cells in peripheral immune organs in the acute phase after PT and change the proportion of adaptive immune cells in peripheral immune organs in the subacute phase after PT. However, in the CNS, DMF can only reduce the number of monocytes in the brain in the subacute phase after PT.

Fig. 2.

Percentage of immune cells in the spleen, blood and brain of DMF-treated mice and vehicle-treated mice after 1-, 3-, and 7-day post-ischemia using flow cytometry. a-c The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 1 day after PT (n = 6: 7). d–f The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 3 days after PT (n = 7: 6). g–i The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 7 days after PT (n = 7: 7). *p < 0.05, **p < 0.01.

Fig. 2.

Percentage of immune cells in the spleen, blood and brain of DMF-treated mice and vehicle-treated mice after 1-, 3-, and 7-day post-ischemia using flow cytometry. a-c The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 1 day after PT (n = 6: 7). d–f The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 3 days after PT (n = 7: 6). g–i The percentage of CD4+ T cells, CD8+ T cells, B cells, NK cells, neutrophil, DCs, macrophages, monocytes, and microglia in DMF group and control group in the spleen, blood, and brain at 7 days after PT (n = 7: 7). *p < 0.05, **p < 0.01.

Close modal

DMF Recruits Microglia and Preserves Neurons in the Peri-Infarct Area after Ischemia

Then, we investigated how the number of resident microglia and neurons in the peri-infarct area around the ischemic core of the brain changed with DMF treatment after PT stroke via immunofluorescence staining (Fig. 3a, d, g). The number of microglia in the peri-infarct area was significantly greater in the DMF group than in the control group on day 1 (p = 0.0197) after PT (Fig. 3b), indicating that DMF can recruit microglia to the peri-infarct area in the acute phase after PT. In addition, the number of neurons in the peri-infarct area was significantly lower in the control group than in the DMF group on day 3 after PT (p = 0.0018) (Fig. 3f), indicating the protective role of DMF in preserving neurons in the peri-infarct area. However, the number of microglia on days 3 and 7 and neurons on days 1 and 7 after PT did not significantly change in the peri-infarct area after treatment with DMF (Fig. 3c, e, h, i).

Fig. 3.

Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated mice and vehicle-treated mice after 1-, 3-, and 7-days post-ischemia. a Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 1 day post-ischemia. b–c The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 1 day post-ischemia (n = 3:3, 3–5 slides per mouse). d Immunostaining of IBA1, GFAP and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 3 days post ischemia. e–f The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 3 days post-ischemia (n = 3:3, 3–5 slides per mouse). g Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 7 days post-ischemia. h–i The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 7 days post-ischemia (n = 3:3, 3–5 slides per mouse). *p < 0.05, **p < 0.01.

Fig. 3.

Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated mice and vehicle-treated mice after 1-, 3-, and 7-days post-ischemia. a Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 1 day post-ischemia. b–c The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 1 day post-ischemia (n = 3:3, 3–5 slides per mouse). d Immunostaining of IBA1, GFAP and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 3 days post ischemia. e–f The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 3 days post-ischemia (n = 3:3, 3–5 slides per mouse). g Immunostaining of IBA1, GFAP, and NeuN in the peri-infarction area in DMF-treated and vehicle-treated mice after 7 days post-ischemia. h–i The number of IBA1+ microglia and NeuN+ neurons in the peri-infarction area and the corresponding contralateral area of the two groups after 7 days post-ischemia (n = 3:3, 3–5 slides per mouse). *p < 0.05, **p < 0.01.

Close modal

DMF Reduces B Cells in AIS Patients Receiving Alteplase

A total of 11 AIS patients were enrolled in this study. There were 3 patients in the DMF group and 8 patients in the control group. The mean time from symptom onset to DMF treatment was 16.7 ± 3.4 h. The demographic, clinical, and imaging characteristics of the patients are shown in Table 1. The two groups did not differ with respect to age, stroke etiology, NIHSS score, infarct volume, or location (Table 1).

The absolute counts and percentages of lymphocytes, monocytes, neutrophils, eosinophil granulocytes, and basophilic granulocytes were measured through routine blood examinations. A steady decrease in lymphocytes was observed after DMF administration (Fig. 4a), while monocytes and neutrophils seemed to undergo an increase (Fig. 4b, c). Eosinophil granulocytes exhibited a significant decrease on day 7 after receiving DMF (p = 0.0121) (Fig. 4d). However, despite the above trends, the absolute counts and percentages of lymphocytes, monocytes, neutrophils and basophilic granulocytes in the DMF group were not significantly different from those in the control group at any time point (Fig. 4a–e).

Fig. 4.

Dynamics of counts and percentages of lymphocyte and its subsets, monocytes, neutrophil, eosinophil granulocyte, and basophilic granulocyte during DMF treatment in AIS patients. a-e Counts and percentages of lymphocytes, monocytes, neutrophil, eosinophil granulocyte, and basophilic granulocyte during DMF treatment in AIS patients. f–j Absolute numbers and percentages of T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells determined by flow cytometry during DMF treatment in AIS patients. Blood was drawn from patients at the baseline and at 1, 3, 7 days after first dose of DMF. Blood was drawn from control subjects at the same time points. *p < 0.05.

Fig. 4.

Dynamics of counts and percentages of lymphocyte and its subsets, monocytes, neutrophil, eosinophil granulocyte, and basophilic granulocyte during DMF treatment in AIS patients. a-e Counts and percentages of lymphocytes, monocytes, neutrophil, eosinophil granulocyte, and basophilic granulocyte during DMF treatment in AIS patients. f–j Absolute numbers and percentages of T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells determined by flow cytometry during DMF treatment in AIS patients. Blood was drawn from patients at the baseline and at 1, 3, 7 days after first dose of DMF. Blood was drawn from control subjects at the same time points. *p < 0.05.

Close modal

In addition, the absolute counts of CD3+ T, CD4+ T, CD8+ T, CD19+ B, and CD56+ NK cells were similar between the two groups at baseline (p > 0.05). These cell counts exhibited a variable yet consistent decrease on day 1, as well as on days 3 and 7 following the administration of DMF. The most significant change was observed in the percentage of B cells after 3 days of treatment with DMF (p = 0.0279). In addition, the cell counts of CD8+ T cell and the percentage of NK cells decreased on day 7 after treatment with DMF, but the differences were not significant (Fig. 4f–j).

In recent years, the treatment of ischemic stroke has focused on inflammation [3, 4]. Treatment of AIS with immunomodulatory drugs has been attempted for decades; however, no satisfactory results have been achieved. In the present study, the effect of DMF on stroke was further investigated, and the changes in immune cells after ischemic stroke caused by DMF treatment were evaluated both in mice and in humans for the first time.

To date, several articles have reported the role of DMF in ischemic stroke and the potential mechanism involved in cerebral protection [16‒18, 22‒25]. In animal experiments, treatment with DMF has been shown to improve neurobehavioral function and reduce infarct volume in a dose-dependent manner [17, 18]. Therefore, in the present article, after synthesizing the pharmacological properties of DMF and determining the effective dose and duration at which the application of DMF can significantly reduce the infarct volume after ischemic stroke, we administered a dose of 50 mg/kg DMF until sacrifice at 1, 3, and 7 days after stroke to observe the corresponding indicators. Although most of the previous studies involved oral administration, there are also studies supporting intraperitoneal injection, and the dose we used was the maximum dose described in the literature. In addition, the PT stroke model was selected for animal experimentation due to its ability to consistently produce infarction with minimal variation while also providing precise control over the size and location of the ischemic region [20, 26].

In the present study, 50 mg/kg DMF significantly reduced the infarct volume 1 day after PT-induced ischemic stroke; however, the difference in infarct volume 3 days after PT was not significant. To our surprise, the infarct volume 7 days after PT showed the opposite result. Several studies have shown that regardless of the method in which the drug is administered, 15 mg/kg ∼ 30 mg/kg DMF or MMF cannot significantly alter infarct volume after experimental stroke [16, 22, 25]. Because the concentrations of the drug used in the above studies were relatively low, they did not effectively protect against enlargement of the ischemic area. However, administering higher doses or prolonging the treatment duration of DMF may enhance its efficacy in reducing the infarct volume [17, 18].

Our research was a pioneering investigation to document alterations in immune cells within the spleen, circulating blood, and brain following ischemic stroke in mice treated with DMF and in the circulating blood of AIS patients receiving DMF therapy. We discovered that the effect of DMF mainly changes the immune environment of the spleen in the peripheral immune system; suppresses innate immune cells, such as neutrophils, dendritic cells, macrophages, and monocytes, first on day 1 after PT; and then switches to adaptive immune responses, such as decreased NK cells on day 3 and B cells on day 7 and increased CD4+ T cells on days 3 and 7. It is well known that immune cells gradually infiltrate the brain after stroke [5]. After treatment with DMF, the first response in the brain is a decrease in monocytes on day 3 after PT, which is consistent with previous findings on the effect of DMF on MS [27]. However, there was no difference in the number of monocytes in the brain parenchyma between the two groups on day 7 after PT. For blood tests for AIS patients treated with DMF, the most significant change was observed in B cells after 3 days of enrollment, followed by CD8+ T cells and NK cells. Previous research has indicated that the administration of DMF to MS patients leads to a decrease in most cell types [28], and the impact on absolute T-cell counts is more significant than that on B or NK cell counts. The reduction in CD8+ T cells is particularly pronounced compared to that in CD4+ T cells [29, 30], as DMF-induced cell death affects CD8+ T cells more severely [28]. However, there are some disagreement regarding changes in NK cells. Several studies have reported different changes in NK cells in MS patients after DMF application, with some showing increases [31, 32], others showing decreases [33], and others showing no significant change [34]. In our study, although the patients suffered from a different disease from MS and DMF was only administered for 3 days, the results showed that B cells significantly decreased after 3 days of treatment with DMF, and both CD8+ T cells and NK cells tended to decrease. The administration of DMF to treat MS can last for years to change the immune environment [30]; however, short-term treatment with DMF to treat ischemic stroke makes it difficult to change other proportions of immune cell components. Therefore, the use of DMF to treat ischemic stroke might prolong the treatment time for weeks or for months for a better prognosis.

In addition to its immunomodulatory properties, DMF can protect the CNS against autoimmunity through direct interactions with neurons, microglia, and astrocytes via both Nrf2-dependent and Nrf2-independent pathways [35]. Activation of the Nrf2 antioxidant pathway by DMF offers protection to neurons against cell death while also promoting the differentiation of anti-inflammatory M2 microglia [15]. In addition, the HCAR-2-mediated pathway can be activated by DMF to inhibit the activation of microglia and the infiltration of neutrophils into the CNS [36, 37]. Moreover, DMF has the ability to safeguard neurons and inhibit the pro-inflammatory activation of astrocytes through the NF-κB-mediated pathway [38, 39]. In this study, we found that on day 1 after PT, the number of microglia in the peri-infarct area in the DMF group was significantly greater than that in the control group, suggesting that DMF may promote the recruitment of microglia to the peri-infarct area. In addition, on day 3 after PT, the number of neurons in the peri-infarct area was significantly lower in the control group than in the DMF group, which indicates that the application of DMF may protect neurons from death during ischemic stroke. These results were also consistent with those of previous studies.

In conclusion, our results demonstrate that DMF is effective in reducing the infarct volume in the acute phase of experimental stroke, recruiting microglia, preserving neurons from loss in the peri-infarct area after PT, and changing the composition of the CNS and peripheral immune cells in both PT mice and AIS patients, which provides an immunological basis for DMF treatment in ischemic stroke.

The protocol of clinical trial of this study and supporting documentation of this trial were approved by Xuanwu Hospital Capital Medical University Institutional Review Board (No. 2021–091). The study was conducted in accordance with the ethical standards as laid down in the Declaration of Helsinki. The clinical trial has been registered with the identifier NCT04890366 on ClinicalTrials.gov on November 05, 2021. The animal study was reviewed and approved by the Animal Care and Use Committee of Xuanwu Hospital (XW-20211216-2). Written informed consent was acquired from each patient or a surrogate who is legally authorized. This clinical trial did not involve vulnerable participants.

The authors have no competing interests to declare that are relevant to the content of this article.

This work was supported by National Natural Science Foundation (No. 82104435, Sponsor: Mingyang Wang, role: execution and analysis), National Natural Science Foundation (No. 82171396, Sponsor: Lihua Wang, role: study design), and the Major Science and Technology Special Project of Yunnan Province (No. 202102AA100061, Sponsor: Lianmei Zhong, role: manuscript conception, planning, writing, and decision to publish).

Chunrui Bo, Lianmei Zhong, Lihua Wang, and Junjie Wang contributed to the study conception and design. Animal experiments were undergone by Chunrui Bo, Junjie Wang, Yaxin Zhang, and Shiyue Hou. The patient screening and enrollment of the clinical trial were completed by Chunrui Bo, Tao Wu, Jingkai Li, Yan Liang, and Shufang Zhao. Jianjian Wang and Huixue Zhang analyzed the data. Chunrui Bo, Mingyang Wang, and Xiyue Zhang visualized the data. The first draft of the manuscript was written by Chunrui Bo, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (L.M.Z.) upon reasonable request.

1.
Feigin
VL
,
Stark
BA
,
Johnson
CO
,
Roth
GA
,
Bisignano
C
,
Abady
GG
, et al
.
Global, regional, and national burden of stroke and its risk factors, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019
.
Lancet Neurol
.
2021
;
20
(
10
):
795
820
.
2.
Kim
J
,
Olaiya
MT
.
Global stroke statistics 2023: availability of reperfusion services around the world
.
Int J Stroke
.
2024
;
19
(
3
):
253
270
.
3.
Chamorro
Á
,
Meisel
A
,
Planas
AM
,
Urra
X
,
van de Beek
D
,
Veltkamp
R
.
The immunology of acute stroke
.
Nat Rev Neurol
.
2012
;
8
(
7
):
401
10
.
4.
Iadecola
C
,
Anrather
J
.
The immunology of stroke: from mechanisms to translation
.
Nat Med
.
2011
;
17
(
7
):
796
808
.
5.
Shi
K
,
Tian
DC
,
Li
ZG
,
Ducruet
AF
,
Lawton
MT
,
Shi
FD
.
Global brain inflammation in stroke
.
Lancet Neurol
.
2019
;
18
(
11
):
1058
66
.
6.
Schneider
D
,
Berrouschot
J
,
Brandt
T
,
Hacke
W
,
Ferbert
A
,
Norris
SH
, et al
.
Safety, pharmacokinetics and biological activity of enlimomab (anti-ICAM-1 antibody): an open-label, dose escalation study in patients hospitalized for acute stroke
.
Eur Neurol
.
1998
;
40
(
2
):
78
83
.
7.
Enlimomab Acute Stroke Trial Investigators
.
Use of anti-ICAM-1 therapy in ischemic stroke: results of the Enlimomab Acute Stroke Trial
.
Neurology
.
2001
;
57
(
8
):
1428
34
.
8.
Emsley
HC
,
Smith
CJ
,
Georgiou
RF
,
Vail
A
,
Hopkins
SJ
,
Rothwell
NJ
, et al
.
A randomised phase II study of interleukin-1 receptor antagonist in acute stroke patients
.
J Neurol Neurosurg Psychiatry
.
2005
;
76
(
10
):
1366
72
.
9.
Fu
Y
,
Zhang
N
,
Ren
L
,
Yan
Y
,
Sun
N
,
Li
YJ
, et al
.
Impact of an immune modulator fingolimod on acute ischemic stroke
.
Proc Natl Acad Sci USA
.
2014
;
111
(
51
):
18315
20
.
10.
Zhu
Z
,
Fu
Y
,
Tian
D
,
Sun
N
,
Han
W
,
Chang
G
, et al
.
Combination of the immune modulator fingolimod with alteplase in acute ischemic stroke: a pilot trial
.
Circulation
.
2015
;
132
(
12
):
1104
12
.
11.
Tian
DC
,
Shi
K
,
Zhu
Z
,
Yao
J
,
Yang
X
,
Su
L
, et al
.
Fingolimod enhances the efficacy of delayed alteplase administration in acute ischemic stroke by promoting anterograde reperfusion and retrograde collateral flow
.
Ann Neurol
.
2018
;
84
(
5
):
717
28
.
12.
Elkins
J
,
Veltkamp
R
,
Montaner
J
,
Johnston
SC
,
Singhal
AB
,
Becker
K
, et al
.
Safety and efficacy of natalizumab in patients with acute ischaemic stroke (ACTION): a randomised, placebo-controlled, double-blind phase 2 trial
.
Lancet Neurol
.
2017
;
16
(
3
):
217
26
.
13.
Elkind
MSV
,
Veltkamp
R
,
Montaner
J
,
Johnston
SC
,
Singhal
AB
,
Becker
K
, et al
.
Natalizumab in acute ischemic stroke (ACTION II): a randomized, placebo-controlled trial
.
Neurology
.
2020
;
95
(
8
):
e1091
104
.
14.
Montes Diaz
G
,
Hupperts
R
,
Fraussen
J
,
Somers
V
.
Dimethyl fumarate treatment in multiple sclerosis: recent advances in clinical and immunological studies
.
Autoimmun Rev
.
2018
;
17
(
12
):
1240
50
.
15.
Linker
RA
,
Lee
DH
,
Ryan
S
,
van Dam
AM
,
Conrad
R
,
Bista
P
, et al
.
Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway
.
Brain
.
2011
;
134
(
Pt 3
):
678
92
.
16.
Kunze
R
,
Urrutia
A
,
Hoffmann
A
,
Liu
H
,
Helluy
X
,
Pham
M
, et al
.
Dimethyl fumarate attenuates cerebral edema formation by protecting the blood-brain barrier integrity
.
Exp Neurol
.
2015
;
266
:
99
111
.
17.
Yao
Y
,
Miao
W
,
Liu
Z
,
Han
W
,
Shi
K
,
Shen
Y
, et al
.
Dimethyl fumarate and monomethyl fumarate promote post-ischemic recovery in mice
.
Transl Stroke Res
.
2016
;
7
(
6
):
535
47
.
18.
Lin
R
,
Cai
J
,
Kostuk
EW
,
Rosenwasser
R
,
Iacovitti
L
.
Fumarate modulates the immune/inflammatory response and rescues nerve cells and neurological function after stroke in rats
.
J Neuroinflammation
.
2016
;
13
(
1
):
269
.
19.
Clarkson
AN
,
López-Valdés
HE
,
Overman
JJ
,
Charles
AC
,
Brennan
KC
,
Thomas Carmichael
S
.
Multimodal examination of structural and functional remapping in the mouse photothrombotic stroke model
.
J Cereb Blood Flow Metab
.
2013
;
33
(
5
):
716
23
.
20.
Li
H
,
Zhang
N
,
Lin
HY
,
Yu
Y
,
Cai
QY
,
Ma
L
, et al
.
Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice
.
BMC Neurosci
.
2014
;
15
:
58
.
21.
Siret
C
,
van Lessen
M
,
Bavais
J
,
Jeong
HW
,
Reddy Samawar
SK
,
Kapupara
K
, et al
.
Deciphering the heterogeneity of the Lyve1(+) perivascular macrophages in the mouse brain
.
Nat Commun
.
2022
;
13
(
1
):
7366
.
22.
Clausen
BH
,
Lundberg
L
,
Yli-Karjanmaa
M
,
Martin
NA
,
Svensson
M
,
Alfsen
MZ
, et al
.
Fumarate decreases edema volume and improves functional outcome after experimental stroke
.
Exp Neurol
.
2017
;
295
:
144
54
.
23.
Safari
A
,
Fazeli
M
,
Namavar
MR
,
Tanideh
N
,
Jafari
P
,
Borhani-Haghighi
A
.
Therapeutic effects of oral dimethyl fumarate on stroke induced by middle cerebral artery occlusion: an animal experimental study
.
Restor Neurol Neurosci
.
2017
;
35
(
3
):
265
74
.
24.
Liu
L
,
Vollmer
MK
,
Ahmad
AS
,
Fernandez
VM
,
Kim
H
,
Doré
S
.
Pretreatment with Korean red ginseng or dimethyl fumarate attenuates reactive gliosis and confers sustained neuroprotection against cerebral hypoxic-ischemic damage by an Nrf2-dependent mechanism
.
Free Radic Biol Med
.
2019
;
131
:
98
114
.
25.
Owjfard
M
,
Bigdeli
MR
,
Safari
A
,
Haghani
M
,
Namavar
MR
.
Effect of dimethyl fumarate on the motor function and spatial arrangement of primary motor cortical neurons in the sub-acute phase of stroke in a rat model
.
J Stroke Cerebrovasc Dis
.
2021
;
30
(
4
):
105630
.
26.
Uzdensky
AB
.
Photothrombotic stroke as a model of ischemic stroke
.
Transl Stroke Res
.
2018
;
9
(
5
):
437
51
.
27.
Carlström
KE
,
Ewing
E
,
Granqvist
M
,
Gyllenberg
A
,
Aeinehband
S
,
Enoksson
SL
, et al
.
Therapeutic efficacy of dimethyl fumarate in relapsing-remitting multiple sclerosis associates with ROS pathway in monocytes
.
Nat Commun
.
2019
;
10
(
1
):
3081
.
28.
Dello Russo
C
,
Scott
KA
,
Pirmohamed
M
.
Dimethyl fumarate induced lymphopenia in multiple sclerosis: a review of the literature
.
Pharmacol Ther
.
2021
;
219
:
107710
.
29.
Mehta
D
,
Miller
C
,
Arnold
DL
,
Bame
E
,
Bar-Or
A
,
Gold
R
, et al
.
Effect of dimethyl fumarate on lymphocytes in RRMS: implications for clinical practice
.
Neurology
.
2019
;
92
(
15
):
e1724
38
.
30.
Longbrake
EE
,
Mao-Draayer
Y
,
Cascione
M
,
Zielinski
T
,
Bame
E
,
Brassat
D
, et al
.
Dimethyl fumarate treatment shifts the immune environment toward an anti-inflammatory cell profile while maintaining protective humoral immunity
.
Mult Scler
.
2021
;
27
(
6
):
883
94
.
31.
Longbrake
EE
,
Ramsbottom
MJ
,
Cantoni
C
,
Ghezzi
L
,
Cross
AH
,
Piccio
L
.
Dimethyl fumarate selectively reduces memory T cells in multiple sclerosis patients
.
Mult Scler
.
2016
;
22
(
8
):
1061
70
.
32.
Marastoni
D
,
Buriani
A
,
Pisani
AI
,
Crescenzo
F
,
Zuco
C
,
Fortinguerra
S
, et al
.
Increased NK cell count in multiple sclerosis patients treated with dimethyl fumarate: a 2-year longitudinal study
.
Front Immunol
.
2019
;
10
:
1666
.
33.
Spencer
CM
,
Crabtree-Hartman
EC
,
Lehmann-Horn
K
,
Cree
BA
,
Zamvil
SS
.
Reduction of CD8(+) T lymphocytes in multiple sclerosis patients treated with dimethyl fumarate
.
Neurol Neuroimmunol Neuroinflamm
.
2015
;
2
(
3
):
e76
.
34.
Wu
Q
,
Wang
Q
,
Mao
G
,
Dowling
CA
,
Lundy
SK
,
Mao-Draayer
Y
.
Dimethyl fumarate selectively reduces memory T cells and shifts the balance between Th1/Th17 and Th2 in multiple sclerosis patients
.
J Immunol
.
2017
;
198
(
8
):
3069
80
.
35.
Yadav
SK
,
Soin
D
,
Ito
K
,
Dhib-Jalbut
S
.
Insight into the mechanism of action of dimethyl fumarate in multiple sclerosis
.
J Mol Med Berl
.
2019
;
97
(
4
):
463
72
.
36.
Parodi
B
,
Rossi
S
,
Morando
S
,
Cordano
C
,
Bragoni
A
,
Motta
C
, et al
.
Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS
.
Acta Neuropathol
.
2015
;
130
(
2
):
279
95
.
37.
Chen
H
,
Assmann
JC
,
Krenz
A
,
Rahman
M
,
Grimm
M
,
Karsten
CM
, et al
.
Hydroxycarboxylic acid receptor 2 mediates dimethyl fumarate's protective effect in EAE
.
J Clin Invest
.
2014
;
124
(
5
):
2188
92
.
38.
Campolo
M
,
Casili
G
,
Biundo
F
,
Crupi
R
,
Cordaro
M
,
Cuzzocrea
S
, et al
.
The neuroprotective effect of dimethyl fumarate in an MPTP-mouse model of Parkinson's disease: involvement of reactive oxygen species/nuclear factor-κb/nuclear transcription factor related to NF-E2
.
Antioxid Redox Signal
.
2017
;
27
(
8
):
453
71
.
39.
Lin
SX
,
Lisi
L
,
Dello Russo
C
,
Polak
PE
,
Sharp
A
,
Weinberg
G
, et al
.
The anti-inflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1
.
ASN Neuro
.
2011
;
3
(
2
):
e00055
.