Dimethyl Fumarate Protects Brain From Damage Produced by Intracerebral Hemorrhage by Mechanism Involving Nrf2
Background and Purpose—Intracerebral hemorrhage (ICH) represents a devastating form of stroke for which there is no effective treatment. This preclinical study was designed to evaluate dimethyl fumarate (DMF), a substance recently approved for the treatment of multiple sclerosis, as therapy for ICH. We hypothesized that DMF through activating the master regulator of cellular self-defense responses, transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), would act as effective treatment for ICH-mediated damage.
Methods—Male rats and mice, including Nrf2 knockouts, were subjected to intracerebral injection of blood (to mimic ICH) and then treated with DMF. Neurological deficit, brain edema, gene induction profile and hematoma resolution were evaluated. Phagocytic functions of primary microglia in culture were used to study hematoma resolution.
Results—Treatment with DMF induced Nrf2-target genes, improved hematoma resolution, reduced brain edema, and ultimately enhanced neurological recovery in rats and wild-type, but not Nrf2 knockout, mice. Most importantly, the treatment of ICH with DMF showed a 24 h window of therapeutic opportunity.
Conclusions—A clinically relevant dose of DMF demonstrates potent therapeutic efficacy and impressive 24 h therapeutic window of opportunity. This study merits further evaluation of this compound as potential treatment for ICH in humans.
Spontaneous intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality and poor prognosis that affects an estimated 37 000 to 52 000 people in the United States annually1 and for which no effective therapy is currently available.2
At first, progressive accumulation of blood within the brain matter leads to increased intracranial pressure and cell/tissue damage that is primarily related to the mechanical injury.3,4 In addition, it is considered that hematoma-derived toxic products (eg, hemolysis products), oxidative stress, and proinflammatory responses3,5–7 in brain areas around the hematoma robustly aggravates the initial damage (a process often referred to as secondary brain injury) for many days after the ICH onset.3,4,8
We and others have recently identified that the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), a master regulator of antioxidative and detoxification processes, plays a central role in inducing various defense processes, which represent attractive targets in treating diverse aspects of the secondary injury after ICH.9–12
The objective of this study was to demonstrate that dimethyl fumarate (DMF), a substance that was recently approved for the treatment of multiple sclerosis (MS), by mechanisms involving mobilization of anti-inflammatory, antioxidative, and immunomodulatory responses mediated by Nrf213 could also be effective in treating ICH.
Material and Methods
All animal studies followed the guidelines outlined in Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and were approved by the Animal Welfare Committee of University of Texas Health Science Center at Houston. All studies were performed using randomization approach, and all analyses were performed by the investigators blinded to treatment assignments.
ICH in Rat and Mice
ICH in rat and mouse was induced by intrastriatal injection of autologous blood as has been described previously.4,11,14 Briefly, male Sprague–Dawley rats (250–350 g) or Nrf2+/+ and Nrf2−/− mice11 (both C57BL/6 background; 25–30 g) under chloral hydrate anesthesia (0.35 g/kg; IP) were immobilized onto a stereotaxic frame. A 1-mm-diameter burr hole was drilled in the skull and a 26-gauge stainless steel cannula was inserted for blood infusion (collected from femoral artery; 15 μL/5 min for mice or 35 μl/5 min for rats) into the left corpus striatum. Stereotactic coordinates with respect to bregma were for Sprague–Dawley rat, 0.5 mm anterior, 2 mm lateral, and 3.5 mm deep; for mouse, 0.5 mm anterior, 1.2 mm lateral, and 2.5 mm deep. Core body temperature was maintained at 37±0.5°C during entire surgery and for 2 h afterward.
Animals were anesthetized with chloral hydrate (0.5 g/kg; IP) and intracardially perfused with ice-cold PBS. The whole brains or the subdissected tissues representing hematoma-affected striatum were snap frozen by submersion in –80°C 2-methylbutane and stored in −80°C freezer before cryosectioning, RNA isolation, or protein analyses.
Hematoma Size Measurement
Hematoma resolution was assessed by measuring the amount of hemoglobin (Hb) remaining in the hematoma-affected brain on d7 after ICH, as we detailed previously.14
We also used immunoblotting for Hb level determination. Brain homogenates from ICH-affected hemisphere were separated on SDS-PAGE and processed for immunoblot. Rabbit anti-Hb antibody (Santa Cruz, sc-31332) followed by goat anti-rabbit Ig-HRP (Zymed) and ECL (Pierce, Rockford, IL) were used to visualize Hb. Luminescence signal intensity was determined by analyses of optical density on x-ray film.
Dimethyl Fumarate or Sulforaphane Treatment
For the animal experiments, 15 mg/kg DMF15 (Sigma) dissolved in 10% dimethyl sulfoxide (DMSO) was administered. In the Sprague–Dawley rats, the DMF was injected intraperitoneally (IP) at 2 h after ICH and then administered orally twice a day on d1, d2, and d3. In the mice experiment, the DMF was injected IP at 24 h after ICH and then at d2 and d3. 10% DMSO in saline was used as the vehicle control.
For the cell culture phagocytosis experiments, 1 to 100 μM DMF was directly applied into the culture medium at 16 h before the cells were exposed to RBC. Sulforaphane, a prototypic Nrf2 activator, prepared in 10% DMSO (LKT Laboratories, Inc. S8044) was directly applied to the cell using the same conditions as for DMF. 0.5% DMSO (equal to the final concentration of DMSO in the experimental groups) in culture media was used as the vehicle control for sulforaphane and DMF.
Nrf2 Knockout Mice
We isolated microglia using p1-p2 rat pups as we described.14 Briefly, the cells from brain tissue were seeded in 75 cm2 TC flasks and cultured for 14 days. The loosely adherent microglia were harvested, centrifuged, and replated onto poly-l-lysine–coated TC plates, with or without 12-mm diameter German-glass, at a density of 2≈5×105 cells/mL. 96% of all the cells were positive for CD11b.
The immunohistochemistry for CD68 and RBC double labeling was performed using the procedure as we described.14 Briefly, the microglia cells grown on German glass were fixed with 95% methanol containing 5% acetic acid for 10 min at −20°C and incubated in mouse anti-CD68 (ED1; Serotec) and rabbit anti-rat RBC antibody (20R-RR012; Fitzgerald) overnight at 4°C. Goat-antimouse IgG-Alexa Fluor 488 and goat anti-rabbit IgG-Alexa Fluor-546 (Invitrogen, USA) was used to visualize CD68-labeled microglia and the phagocytosed RBCs in the microglia. The nuclei were visualized with DAPI.
Image Capture and Cell Counting
A Zeiss Axioskop-2 microscope equipped with CCD camera and operated by MetaMorph 7.4 software was used for image acquisition. The fluorescence-labeled cells were visualized using Ex/Em of 490/520 nm for Alexa 488, Ex/Em of 550/575 nm for Alexa 546, and Ex/Em of 365/480 nm for DAPI.
RBC Isolation and Labeling for Phagocytosis Assay
The RBC were purified using density gradient centrifugation (BD Vacutainer CPT). After washing with PBS, the purified RBC was labeled with the fluorescent dye 5(6)-carboxyfluorescein diacetate (Molecular Probes). The labeled RBCs were diluted to 109cells/mL and used as an indicator/target of phagocytosis, as we have previously reported.14
Phagocytosis In Vitro
The phagocytosis assay was performed as we described earlier.14 At 2 h after adding the 5(6)-carboxyfluorescein diacetate–labeled RBC to the cultured microglia (20:1 ratio), we separated the unphagocytosed free floating RBC from phagocytes that are attached to the plastic by aspiration. The microglia containing engulfed RBC were lysed in distilled water, and fluorescence intensity in the supernatant from the cell lysate was measured using a fluorometer with a 490/520 nm filter set. The fluorescence intensity (OD) was referred as phagocytosis index.
In experiments with DMF or sulforaphane, we preincubated microglia in DMF or sulforaphane for 16 h before adding the 5(6)-carboxyfluorescein diacetate–labeled RBC, the targets of phagocytosis.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
The ipsilateral corpus striatum (hematoma and peri-hematoma areas) was dissected on ice, snap-frozen, and processed for mRNA extraction using Trizol-Reagents. Reverse transcription-polymerase chain reaction analyses were done as we described.14,17 We used glyceraldehyde-3-phosphate dehydrogenase gene as an internal standard. The sequences of primers are listed in Table I in the online-only Data Supplement. Measurements of the gene products were normalized to the optical density of glyceraldehyde-3-phosphate dehydrogenase bands. The results were calculated as percentage change over the control (naïve animal, in vivo).
Neurological Deficits Score
All behavioral tests were conducted in a quiet and low-lit room by an experimenter blinded with respect to the treatment groups. The neurological deficits score was determined by a battery of behavioral tests, including Postural Flexing, Circling test, Footfault, Forelimb Placing, and Wire, as we have previously described.11 All animals were pretested and then evaluated behaviorally at time points as indicated.
The brain edema was measured using the wet-weight/dry-weight method.18 Briefly, the brains were removed without perfusion and the ICH-affected left brain hemispheres were dissected. A brain coronal section (4 mm-thick) at 2 mm anterior and 2 mm posterior to the blood injection site was excised. The tissue weight was determined before and after drying in a 95°C oven for 48 h. The brain edema was expressed as percent of water content as (wet weight–dry weight)/wet weight×100.
All results are expressed as mean±SEM. For the in vitro experiments, we pooled the samples from 3 culture wells and repeated the experiments 3 times. We performed statistical analyses using the GraphPad and InStat programs. One-way analysis of variance followed by Newman–Keuls post-test was used for multiple group comparisons. Nonpaired t test was used when 2 groups were compared.
DMF Reduced Functional Deficit and Brain Edema in Rats After ICH
Our initial objective was to determine whether DMF, at doses that are recognized to ameliorate dysfunction in animal model of MS,15 reduced neurological deficit and brain edema caused by ICH. The rats were treated with DMF at 2 h after ICH and then behaviorally tested at day 1 and day 3, followed by brain edema determination. This study demonstrated that DMF significantly ameliorates the neurological deficit at both day 1 and day 3 after ICH (Figure 1A) and that this functional improvement corresponded with robust reduction in brain edema in DMF-treated rats (Figure 1B).
DMF Induces Expression of Gene Profile Suggesting Activation of Nrf2 and Anti-Inflammatory Activities
By working under the assumption that DMF protects brain by activating Nrf2, we first decided to establish that therapeutic doses of DMF could indeed induce the expression of Nrf2 gene targets in ICH-affected brain tissue. To make sure that the DMF-mediated gene induction in fact represents Nrf2 activation, we contrasted the gene expression pattern between Nrf2-KO and the wild-type mice. Mice were subjected to ICH and treated with DMF, and the gene profile was assessed at 48 h. Our study established that DMF was indeed able to upregulate the prototypic Nrf2-responsive genes, including heme oxygenase-1, CD36, catalase, or quinolone oxidoreductase-1, as well as Nrf2 itself in the ICH-affected brain (Figure 2). Among many genes with potential roles in ICH, we found that DMF induced the expression of haptoglobin (Hp) (key hemoglobin/Hb detoxifying protein18) and also CD163 (Hb–Hp scavenger receptor; Figure 2). The upregulation of all the above genes by DMF was reduced (or even neutralized) in the Nrf2-KO mice, stressing the underlying role of Nrf2 in the induction process. In addition, we found that DMF was also effective in inhibiting the expression of proinflammatory mediators, as probed with iNOS and IL-1β expression, although increasing the expression of anti-inflammatory IL-10 (Figure 2). Interestingly, DMF-mediated responses toward the expression of inflammatory genes were similar in WT and Nrf2-deficiency mice, suggesting an Nrf2-independent mechanism of DMF in this process.
DMF Stimulates Phagocytic Activities of Microglia Toward RBC and Improves Hematoma Resolution
Our earlier research suggests that the effective cleanup process conducted by microglia/macrophages after ICH, including in response to the Nrf2 activator sulforaphane, may improve hematoma resolution and improve post-ICH recovery.10 Thus, in a proof-of-concept experiment, we now demonstrated that DMF (similar to sulforaphane) activates microglia in culture toward more effective engulfment of RBC (Figure 3). We next used the ICH model in rat and showed that treatment with DMF that reduced neurological dysfunction (Figure 4A) led to improved hematoma resolution, as measured by comparing the amount of brain’s residual hemoglobin using an immunoblot (Figure 4B and 4D; P<0.05) or Drabkin’s reagent (Figure 4C; P=0.057) at days 7 after ICH.
DMF Reduces Neurological Deficit After ICH in Wild-Type, But Not Nrf2 Knockout, Mouse With 24 h Therapeutic Window
In this experiment, WT and Nrf2-KO mice were subjected to ICH and then treatment with DMF starting at 24 h after the onset of ICH. Despite this marked delay with the intervention, DMF significantly improved the neurological performance of animals, as judged based on the composite neurological score (postural flexing, forward placing, wire hanging, footfault, and circling tests; Figure 5), as well as based on individual test performance (Figure I in the online-only Data Supplement), as assessed on day 3 after ICH. In addition, we repeated this experiment using separate group of animals, and this time established that the therapeutic benefit of DMF lasts for at least 7 days (Figure II in the online-only Data Supplement). Importantly, the beneficial effect of DMF was lost in Nrf2-KO mice, again suggesting an important function of Nrf2 in the DMF-mediated therapy. Finally, Nrf2-KO mice as compared with WT mice showed less neurological improvement over time, again implicating endogenous Nrf2 as critical component of self-protection in this insult.
This study shows for the first time that DMF potently attenuates neurological deficit and improves hematoma resolution in a mouse and rat model of ICH, even when administered as late as 24 h after the onset of ICH. The beneficial effect of DMF was lost in the Nrf2-KO animals, suggesting that its therapeutic effect is through activating Nrf2. The treatment of WT, but not Nrf2-KO, mice with DMF indeed induced brain expression of many signature Nrf2 genes and also selected Nrf2-target genes of the detoxification-cleanup system (eg, CD36, CD163, and haptoglobin). Furthermore, DMF reduced expression of genes associated with proinflammatory responses. In vitro, DMF activated the prophagocytic function of microglia, suggesting that DMF-mediated hematoma resolution may in part involve improved effectiveness of phagocytosis mediated by microglia/macrophages.
DMF has been extensively studied in animal models of MS15 and recently was approved by FDA as a possible treatment for people with relapsing-remitting MS.19,20 Based on mechanistic studies with cell culture and experimental autoimmune encephalomyelitis, an animal model of MS, it was proposed that the underlying therapeutic and immunomodulatory mechanism of action of DMF, at a cellular level, is activation of Nrf2.15
Nrf2 is a pleiotropic transcription factor and a key genomic homeostatic regulator that, through antioxidant response elements within the regulatory region of many target genes, coordinates antioxidative and detoxification processes, allowing all types of cells in the neurovascular unit to adapt to detrimental conditions caused by intracellular or extracellular stress.11,21,22 We and others have proposed that Nrf2 by its pleiotropic mechanism of action resulting in reduction of oxidative damage, reduction of inflammation, blood product detoxification, and improved microglia/macrophages-mediated cleanup process may represent an excellent target for the treatment of ICH.9–12 In agreement with this assumption, we now demonstrate that DMF, at clinically relevant doses, benefits the brain by improving the antioxidative milieu of the brain through enhancement of HO-1, catalase, or NQO1 genes expression. DMF enhanced expression of CD36, a scavenger receptor that plays a prominent role in the phagocyte-mediated hematoma cleanup process.14 In addition, DMF was effective in amplifying the expression of mRNA for Hp and CD163 in ICH-affected brain. Hp, by forming virtually irreversible complexes with Hb, is known to neutralize the cytotoxicity of Hb (including after ICH),18 whereas CD163 serves as a scavenger receptor on microglia/macrophages for endocytosis of Hp–Hb complexes, a process that is necessary for the permanent removal of Hb from brain parenchyma.23,24 Finally, DMF reduced expression of IL-1β and iNOS, 2 prototypic proinflammatory products, and upregulated the expression of anti-inflammatory IL-10. In contrast to the gene products mentioned earlier that required Nrf2 for their induction, DMF-mediated change in IL-1β, iNOS, or IL-10 could be detected in both Nrf2-proficient and Nrf2-deficient mice. This may suggest that the regulation of inflammation (at least the components measured in this study) by DMF may not directly involve Nrf2 activation. As such, because Nrf2 proficiency was a prerequisite for the therapeutic/neurological benefit with DMF (Figure 5), it could furthermore be concluded that the mechanisms underlying the therapeutic effect of DMF could include enhanced antioxidative mechanisms, neutralization of hemolytic products, and cleanup and are likely not directly based on the regulation of inflammatory responses.
One unique aspect of the ICH-mediated damage is chemical insult caused by lysis of red blood cells within hematoma. This process does not start until 1 to 3 days after ICH6 and escalates over time, leading to buildup of toxic levels of intraparenchymal Hb, heme, and iron, ultimately inflicting oxidative stress, cellular damage, BBB disruption, and edema.3,5 We have recently proposed that the progressive nature of the chemical insult may be combated with a therapy that may precondition cells around the hematoma, so that they acquire resistance to forthcoming chemical insult.9 We also suggested that because chemical insult starts 1 to 3 days after the ictus, this approach may show a particularly long therapeutic window. We now demonstrate that DMF can ameliorate ICH-mediated injury with a therapeutic window of at least 24 h. We suggest that DMF may represent a viable treatment for ICH.
Sources of Funding
Supported by National Institute of Health, National Institute of Neurological Disorders and Stroke, grants NS060768 and NS064109.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.115.009398/-/DC1.
- Received March 11, 2015.
- Revision received April 9, 2015.
- Accepted April 23, 2015.
- © 2015 American Heart Association, Inc.
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