Fumarate modulates the immune/inflammatory response and rescues nerve cells and neurological function after stroke in rats

Abstract

Background: Dimethyl fumarate (DMF), working via its metabolite monomethylfumarate (MMF), acts as a potent antioxidant and immunomodulator in animal models of neurologic disease and in patients with multiple sclerosis. These properties and their translational potential led us to investigate whether DMF/MMF could also protect at-risk and/or dying neurons in models of ischemic stroke in vitro and in vivo. Although the antioxidant effects have been partially addressed, the benefits of DMF immunomodulation after ischemic stroke still need to be explored.

Methods: In vitro neuronal culture with oxygen-glucose deprivation and rats with middle cerebral artery occlusion were subjected to DMF/MMF treatment. Live/dead cell counting and LDH assay, as well as behavioral deficits, plasma cytokine assay, western blots, real-time PCR (Q-PCR) and immunofluorescence staining, were used to evaluate the mechanisms and neurological outcomes.

Results: We found that MMF significantly rescued cortical neurons from oxygen-glucose deprivation (OGD) in culture and suppressed pro-inflammatory cytokines produced by primary mixed neuron/glia cultures subjected to OGD. In rats, DMF treatment significantly decreased infarction volume by nearly 40 % and significantly improved neurobehavioral deficits after middle cerebral artery occlusion (MCAO). In the acute early phase (72 h after MCAO), DMF induced the expression of transcription factor Nrf2 and its downstream mediator HO-1, important for the protection of infarcted cells against oxidative stress. In addition to its antioxidant role, DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T cells and the number of activated microglia/macrophages in the infarct region by more than 50 % by 7-14 days after MCAO. Concomitantly, the levels of potentially harmful pro-inflammatory cytokines were greatly reduced in the plasma and brain and in OGD neuron/glia cultures.

Conclusions: We conclude that DMF is neuroprotective in experimental stroke because of its potent immunomodulatory and antioxidant effects and thus may be useful as a novel therapeutic agent to treat stroke in patients.

Keywords: Dimethyl fumarate; Inflammation; Stroke.

Fig. 1

MMF rescues forebrain neurons from OGD-induced cell death in culture. Cultures of E15 rat forebrain were treated with MMF (25, 50, or 100 μM) beginning 12 h prior to OGD deprivation and throughout the remaining culture period. Cultures treated with MMF contained significantly fewer dead/dying cells at 24 h (a–d, i) and 48 h (e–h) than untreated controls as assessed by LIVE/DEAD assay (a–i, k) or assay of LDH released into the media by dead cells (j). *p < 0.05; **p < 0.01, Student’s t test

Fig. 2

Infarction size and behavioral assessment after MCAO and DMF treatment. Control (vehicle treated, n = 8) and DMF (25 or 50 mg/kg, n = 8 of each group)-treated rat brains were sectioned and stained for TTC 72 h after MCAO (a). Infarction volume was then calculated as described in the “Methods” section using ImageJ and expressed as a percentage of total hemisphere (b). mNSS was assessed at 24 h, 72–84 h, 7 days, or 14 days after MCAO during which rats received either vehicle or 25 or 50 mg/kg DMF by oral gavage beginning 2–3 h after MCAO (n = 8 of each group). A significant and long-lasting decline in neurobehavioral deficits was seen 72 h, 7 days, and 14 days after the initiation of DMF treatment as compared to controls (c). *, ^# p < 0.05, Mann-Whitney U test

Fig. 3

Western analysis of Nrf-2 and HO-1 after MCAO and DMF treatment. Protein levels of Nrf-2 (a) and HO-1 (b) were measured by Western analysis after vehicle (veh) or 50 mg/kg DMF treatment for 72 h, 7 days, or 14 days after MCAO (n = 4 of each group). All values were expressed as percent change HO-1 expression over normal rat brain (100 %). Significant differences are shown for MCAO/DMF as compared to MCAO/veh. *p < 0.05; **p < 0.01, Mann-Whitney U test

Fig. 4

Cytokines and growth factors in mixed neuron/glia culture after OGD, brain and plasma after MCAO with or without MMF/DMF pretreatment. Cultures were processed for RT-qPCR and analyzed for cytokine mRNA levels 24 h after OGD (a). The side of the brain ipsilateral to MCAO was isolated 72 h, 7 days, and 14 days after surgery for RT-qPCR and analyzed for cytokine and growth factor mRNA levels (b, n = 4 of each group). In animals treated as in b, blood levels of factors were measured by multiplex array (see the “Methods” section) in normal uninjured brain or in MCAO rats after 72 h or 7 days of vehicle or 50 mg/kg DMF treatment; protein levels were expressed as pg/ml (c, n = 3–4 per group). All values were compared to normal uninjured control brain. *p < 0.05; **p < 0.01, Mann-Whitney U test

Fig. 5

Analysis of immune cell infiltration into the infarct region 72 h, 7 days, and 14 days after MCAO (n = 6 of each group). Neutrophil cells in the infarct region were stained for MPO in control (a–c) and DMF treated (d–f) MCAO rats and quantified (M). T cells were stained for CD3 in control (g–i) and DMF treated (j–l) MCAO rats and quantified (N). *p < 0.05, **p < 0.01, Mann-Whitney U test

Fig. 6

Analysis of activated microglia/macrophages in penumbra 72 h, 7 days, and 14 days after MCAO (n = 6 of each group). Activated microglia/macrophages were stained for CD68 in control (a–c) and DMF treated (d–f) MCAO rats, and cell number was quantified (g). iNOS staining was also used to demonstrate activated microglia/macrophages at 72 h (h, i, arrows). *p < 0.05, **p < 0.01, Mann-Whitney U test