Neddylation inhibition protects against ischemic brain injury

Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating NEDD8 to target proteins. However, the roles of neddylation in stroke, remain elusive. Here, we report that NEDD8 conjugation increased after ischemic stroke and was abundantly present in neutrophils, whereas cullin-1, a key substrate of neddylation, was upregulated in endothelium. Inhibition of neddylation by MLN4924, inactivated cullin-RING E3 ligase (CRL), reduced brain infarction and improved functional outcomes. MLN4924 treatment induced accumulation of the CRL substrate NF1. Knockdown of NF1 abolished MLN4924-dependent inhibition of neutrophil tracking. These effects were mediated through activation of endothelial P-selectin and ICAM-1. Moreover, NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of PKCδ, MARCKS and MLC in cerebral microvessels. Our results demonstrate that increased neddylation promoted neutrophil tracking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be benecial in ischemic stroke.


Introduction
Stroke continues to be a leading cause of death and the most frequent cause of disability in adults.
Despite signi cant advances in decoding the pathophysiology of cerebral ischemia, therapeutic options for stroke are still limited. Ischemic injury to the brain rapidly triggers adhesion molecule expression on the activated endothelium 1 , resulting in rolling, adhesion, and extravasation of blood-derived in ammatory cells 2 . In ltrating in ammatory cells, including neutrophils, result in irreversible impairment of blood-brain barrier (BBB) function and tissue damage through the release of reactive oxygen species, proteolytic enzymes, and proin ammatory mediators [3][4][5] . However, our understanding of the links between BBB breakdown and peripheral neutrophils in ltrating the ischemic brain, is still incomplete.
Neddylation is the process of posttranslational protein modi cation by conjugating the ubiquitin-like protein, NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8), to target proteins 6 . This process is catalyzed by NEDD8-activating enzyme E1 (NAE1 and UBA3), NEDD8conjugating enzyme E2 (UBC12), and NEDD8 E3 ligase 7 . The best characterized substrates of NEDD8 are cullins 6 , which are scaffold proteins for the cullin-RING E3 ubiquitin ligase (CRL) 8 . The conjugation of NEDD8 to cullins leads to the activation of CRL 9 , which ubiquitinates a multitude of different proteins for targeted degradation 10 . Recently, the neddylation pathway was reported to contribute to growth of a variety of cancer cells and in ammatory responses 11,12 . In contrast, inhibition of neddylation by MLN4924, which is a small molecule inhibitor of NAE, suppressed tumor growth, reduced in ammation and prevented atherogenesis 13,14 . However, the role of neddylation in ischemic stroke has not been addressed so far. Using a mouse model of focal transient cerebral ischemia, we show that neddylation was upregulated in the peri-infarct cortex after stroke and was abundantly expressed in neutrophils. Treatment with the neddylation inhibitor MLN4924 reduced brain infarction and improved neurological functions. We also demonstrated that MLN4924-afforded neuroprotection was mediated via anti-in ammatory and BBBprotective effects involving the accumulation of CRL substrate neuro bromatosis 1 (NF1).

Results
Neddylation pathway is activated in the brain after ischemic stroke. To explore the function of the neddylation pathway in ischemic stroke, we subjected mice to 1-hour transient focal cerebral ischemia and examined brains at 3, 6, 12 and 24 hours. We found that the global protein neddylation in brain lysates were increased 3 hours post stroke, reached a peak around 12 hours, and continued over 24 hours ( Fig. 1a, b). Using a speci c NEDD8 antibody that recognizes NEDD8-conjugated proteins, we con rmed that NEDD8 was upregulated in the peri-infarct cortex at 12 hours after stroke compared to shamoperated brains (Fig. 1c). In the ischemic cortex, NEDD8 was abundantly expressed on Ly6G + neutrophils inside the blood vessels and parenchyma (Fig. 1d), suggesting that NEDD8 may play a role in neutrophil extravasation from blood vessels and consequent BBB impairment after ischemia. The increased protein level of NEDD8 was accompanied by upregulation of the NEDD8-activating enzyme E1-NAE subunits (NAE1 and UBA3) and NEDD8-conjugating enzyme E2 (UBC12) at 12 hours after stroke ( Fig. 1e-h). The best characterized substrates of neddylation are cullin-family proteins 7 . Therefore, we asked whether cullin neddylation might change after stroke. We observed a marked 5.5-fold increase in NEDD8-cullin-1 conjugation in the ischemic cortex ( Fig. 1i-j). Immunostaining of brain sections showed that cullin-1 was upregulated in CD31-positive microvessels (Fig. 1k). Taken together, these data indicate that ischemic stroke induced protein neddylation.
Inhibition of neddylation by MLN4924 reduces ischemic brain damage. To address whether neddylation plays an active role in ischemic stroke, we inhibited the neddylation pathway by the speci c NAE inhibitor MLN4924 15 . Treatment with MLN4924 signi cantly reduced cullin-1 neddylation ( Fig. 2a and Supplementary Fig. 1a), showing the inactivation of CRL. Compared with the vehicle controls, injection of MLN4924 resulted in a 43.8% reduction of infarct volume at 24 hours after stroke (Fig. 2b, c). MLN4924treated mice had signi cant improvements in functional outcomes, as shown by forelimb force and rotarod latency (Fig. 2d, e). Next, we investigated whether the neuroprotective effect of MLN4924 is mediated by an apoptotic pathway. Western blot analysis showed that MLN4924 treatment induced expression of the antiapoptotic protein Bcl-2 and inhibited expression of the proapoptotic proteins p53, Bax, and caspase-3 ( Fig. 2f and Supplementary Fig. 2a-d). Moreover, there was a substantial reduction of TUNEL + neurons in the MLN4924-treated group compared with the control group (Fig. 2g, h).
MLN4924 blunts BBB damage after ischemic stroke. Increase in BBB permeability greatly in uenced the outcome of stroke 16 . To investigate the role of MLN4924 on BBB permeability, we rst analyzed Evans blue dye extravasation at 24 hours after MCAO. This experiment showed that there was a large decrease in BBB permeability in the ischemic brain in MLN4924-treated animals compared with vehicle-treated mice (Fig. 3a, b). Using in vivo multiphoton microscopy of intravenously injected FITC-dextran, we found an intact BBB in sham-operated mice and an increased BBB permeability to uorescent dextran in mice subjected to stroke (Fig. 3c, d). Treatment with MLN4924 signi cantly reduced BBB damage as compared with vehicle-treated animals. Similarly, mice treated with MLN4924 exhibited a substantial reduction in the extravasation of injected uorescent BSA (Fig. 3e, f). We next studied leakage of IgG, an endogenous blood-derived protein, in the brain. Western blot analysis of IgG in vascular-depleted brain homogenates revealed that MLN4924-treated mice had a 61.8% reduction in IgG accumulation in the brain parenchyma compared with vehicle-treated mice (Fig. 3g, h). Immunostaining for IgG and the endothelial cell marker CD31 further con rmed great reduction in perivascular IgG deposits in MLN4924-treated mice ( Fig. 3i and Supplementary Fig. 3a).
The permeability of BBB is impeded by endothelial junctions, which are reduced in stroke leading to the BBB breakdown 16 . We studied whether the expression of the BBB junctional proteins is altered by MLN4924 treatment in the ischemic brain. Immunoblotting of isolated brain microvessels showed that the loss of tight junction proteins ZO-1, occludin and claudin-5 as well as the adherens junction protein VE-cadherin caused by ischemia was abolished by MLN4924 treatment (Fig. 3j and Supplementary  Fig. 3b-e), suggesting that MLN4924 may play an important role in the maintenance of vascular integrity.
MLN4924 reduces cerebral neutrophil invasion. Based on these ndings, we searched to elucidate the underlying cause for the protective effect of MLN4924 on BBB breakdown. Because we showed that NEDD8 is abundantly expressed in neutrophils (Fig. 1d) inside the blood vessels, we determined the effect of MLN4924 on neutrophil invasion. In vivo multiphoton microscopy analysis of intravenously injected PE-Ly6G showed that neutrophils adhered to the microvascular endothelium and migrated into the injured brain in mice subjected to stroke (Fig. 4b), whereas neutrophils rarely adhered or extravasated in shamoperated mice. We observed that the number of adherent neutrophils and extravasation of neutrophils from blood vessels into the brain parenchyma at 12 hours after stroke were both substantially reduced in MLN4924-treated mice compared with vehicle-treated mice (Fig. 4c, d). We also found that the neutrophil rolling velocity was signi cantly increased in mice injected with MLN4924 ( Fig. 4e).
Consistent with these observations, immunohistochemical quanti cation revealed signi cantly decreased numbers of neutrophils in the ischemic hemispheres at 24 hours after ischemic stroke in mice treated with MLN4924 ( Fig. 4f, g). These results were further con rmed by western blot using an anti-Ly6G antibody (Fig. 4h). Signi cantly lower amount of neutrophil was observed in the ischemic brains in mice treated with MLN4924 ( Fig. 4i).
Quanti cation of the neutrophil-speci c enzyme MPO further con rmed a signi cant reduction in neutrophil in ux into MLN4924-treated murine brain (Fig. 4j). Together, these data suggest that MLN4924 controls both the intravascular adhesion and intraparenchymal migration of neutrophils.
In line with these ndings, we found that the mRNA expression levels of neutrophil chemotactic chemokines and chemokine receptor, including CXCL1, CX3CL1, CCL2, and CCR1 17-20 , were signi cantly lower in MLN4924-treated mice than in controls ( Fig. 4k-n), suggesting that the reduced accumulation of neutrophils in MLN4924-treated mice may cause by decreased recruitment. In addition, proin ammatory cytokine concentrations of IL-6, IL-1β and TNFα were also reduced in mice treated with MLN4924 ( Fig. 4oq).
MLN4924 reduces neutrophil in ltration via NF1. NF1, a tumor suppressor, is a key substrate of CRL 21 .
NF1 loss is associated with in ammation and vascular disease 22 . We tested the hypothesis that NF1 is critical for MLN4924-mediated inhibition of neutrophil tra cking. Western blot analysis showed that ischemic stroke signi cantly reduced NF1 expression compared with the sham-operated group ( Fig. 5a and Supplementary Fig. 4a). In contrast, injection of MLN4924 preserved the loss of NF1 caused by ischemia. We then hypothesized that NF1 silencing by adenoviral short hairpin RNA (shRNA) administration ( Supplementary Fig. 5a, b) could abolish the inhibitory effect of MLN4924 on neutrophil in ltration. In vivo multiphoton microscopy indicated that NF1 silencing decreased rolling velocity, and increased neutrophil adhesion and transmigration (Fig. 5b-e) in MLN4924-treated mice at 12 hours after stroke. At 24 hours, we observed that injection of NF1 shRNA into MLN4924-treated mice caused a signi cant increase in MPO activity (Fig. 5f). The MLN4924-mediated decrease in proin ammatory cytokines was also reversed by NF1 silencing (Supplementary Fig. 6a-c).
Extravasation of neutrophils during in ammation is mediated through interactions between adhesion molecules on endothelium and neutrophils 23,24 . Because NF1 was primarily present on cerebral vasculature in the ischemic brain (Fig. 5g), we hypothesized that NF1 loss may increase neutrophil extravasation in MLN4924-treated mice by regulating adhesion molecule expression on endothelia cells.
Indeed, immunoblotting of isolated brain microvessels showed that the expression of P-selectin and ICAM-1 was signi cantly reduced in MLN4924-treated mice, whereas this reduction was reversed by NF1 silencing (Fig. 5h-k). In addition, MLN4924 treatment did not change the expression of vascular cell adhesion molecule-1 (VCAM-1) ( Supplementary Fig. 7a, b). When using blocking antibodies against Pselectin or using anti-ICAM-1 antibodies, we observed a signi cant reduction in the numbers of neutrophils in the ischemic brain of mice treated with MLN4924 and NF1 shRNA (Fig. 5l, m). NF1 mediates MLN4924-afforded BBB protection via activation of PKCδ signals. We next studied the role of NF1 inhibition by shRNA silencing on the protection of BBB with MLN4924 treatment. We observed that NF1 silencing increased BBB permeability (Fig. 6a, b) and extravascular accumulation of serum IgG (Fig. 6c, d) in mice treated with MLN4924; this was accompanied by extended infarct volume (Fig. 6e, f) and exacerbated neurological functions (Fig. 6g, h).
As NF1 was reported to regulate PKCδ activity 25 , and PKCδ can be activated by ischemia 26 , we next studied whether NF1 silencing abolished the protective effect of MLN4924 against BBB damage through PKCδ. Western blot analysis of isolated brain microvessels showed that ischemia-induced phosphorylation of PKCδ was inhibited by MLN4924 treatment, and these effects were reversed by silencing NF1 (Fig. 6i, j). Similarly, NF1 silencing e ciently reversed MLN4924-mediated inactivation of MARCKS protein (Fig. 6k, l), a well-recognized substrate for PKC 27 . Because MARCKS is a CaM-binding protein 28 , we further studied the effects of MLN4924 treatment and NF1 silencing on the CaM-dependent phosphorylation of MLC (pMLC), which has been implicated in endothelial barrier integrity 29 . Isolated microvessels from the ischemic brain of MLN4924-treated mice exhibited decreased pMLC compared with vehicle-treated mice (Fig. 6m, n). However, the decrease in pMLC caused by MLN4924 treatment was abolished by silencing NF1. We then injected the PKCδ inhibitor rottlerin in mice subjected to ischemia and coadministration of MLN4924 with NF1 shRNA. Rottlerin substantially reduced NF1 silencingmediated increase in BBB permeability in MLN4924-treated mice (Fig. 6o, p). Together, these data indicate that NF1 silencing blunted MLN4924-provided BBB protection by inducing PKCδ activation.

Discussion
In this study, we found that neddylation was upregulated in the brain and active in intravascular and intraparenchymal neutrophils after transient focal ischemia in mice. We demonstrated that inhibition of neddylation by the NAE inhibitor MLN4924 improved stroke outcomes by reducing neutrophil in ltration, attenuating BBB damage and infarct volume and improving neurological functions. Furthermore, we show that MLN4924 reduced both neutrophil extravasation and BBB breakdown through attenuation of NEDD8 conjugation to cullin-1, and our data suggest that this is the result of a marked upregulation of the NF1 signals.
Neutrophils are the rst line of innate immune defense against invading pathogens 30 , but they also contribute to endothelial damage and tissue destruction by releasing reactive oxygen species, proteases, and proin ammatory mediators 3 . Our data show expression of vascular adhesion molecules and the accumulation of NEDD8 + neutrophils in the ischemic brain after stroke, suggesting that NEDD8-mediated neutrophil tra cking may cause BBB damage and in ammation. Inhibiting neddylation using a smallmolecule inhibitor MLN4924 reduced neutrophil in ltration and proin ammatory cytokine production.
MLN4924 was shown to cause accumulation of a multitude of different proteins by inactivating CRL 31 . In this study, we found that MLN4924 signi cantly increased the accumulation of NF1, a CRL substrate, whereas NF1 silencing by adenoviral short hairpin RNA administration induced the expression of Pselectin and ICAM-1 in brain microvessels to increase neutrophil extravasation in MLN4924-treated mice, suggesting that the anti-in ammatory effect of MLN4924 may involve NF1. Importantly, we show here that NF1 silencing-induced increase in neutrophil in ltration in MLN4924-treated mice could be rescued by either blocking antibodies against P-selectin or anti-ICAM-1 antibodies, suggesting the need of a speci c interaction between activated neutrophils and injured endothelial cells for induction of neutrophil transmigration.
In ammation in cerebral vessels contributes to BBB disruption 32,33 , and high BBB permeability correlates with infarction growth 34 and poor clinical prognosis after stroke 35,36 . The present study demonstrated that MLN4924 treatment preserved BBB integrity and thereby reduced BBB permeability after cerebral ischemia. These BBB-protective effects of MLN4924 were accompanied by reduced apoptotic neurons and smaller brain infarctions and less severe neurologic de cits. Furthermore, we show that MLN4924offered protection against BBB breakdown is depended on its action on NF1. We then attempted to provide information regarding the signaling mechanisms by which NF1 mediated the BBB protection of MLN4924. Our data indicated a robust downregulation of ischemia-induced activation of PKCδ-MARCKS-MLC pathway upon MLN4924 treatment, and silencing NF1 promoted the activation of PKCδ pathway again and blocked MLN4924-afforded BBB protection. However, in addition to NF1-mediated inactivation of PKCδ pathway, multiple other CRL substrates may also contribute to the effects of MLN4924.
In summary, our study has uncovered a crucial role for protein neddylation in regulating cerebral ischemia. Because its impressive anticancer e cacy, MLN4924 is currently in phase I/II clinical trials for the treatment of several cancers 37 . Our data demonstrated that the neddylation inhibitor MLN4924 protected the brain against ischemic injury by attenuating neutrophil extravasation into brain and maintaining BBB integrity. We conclude that MLN4924 could represent a novel therapeutic option for ischemic stroke. Methods Animal stroke model. Male C57BL/6 mice (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China) weighing 23-26g were used in this study. All protocols for these studies were approved by the Animal Care and Use Committee of the Shanghai Medical College of Fudan University according to National Institutes of Health Guidelines Mice were anesthetized with 1-1.5% iso urane in 30% oxygen and 70% nitrous oxide. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCA) for 60 minutes with a siliconized lament 38 . Cerebral blood ow was monitored by continuous laser doppler owmetry (Perimed, Stockholm, Sweden) to con rm induction of ischemia and reperfusion. Body temperature was maintained at 37 ± 0.5°C using a temperature control unit (World Precision Instruments, Florida) during surgery. MLN4924 (10mg/ml, 60mg/kg) or vehicle (10% 2-hydroxypropyl-β-cyclodextrin) was injected subcutaneously twice at 1 and 12 hours after MCA occlusion 39 . Adenoviral vector expressing NF1 shRNA (pDKD-CMV-Puro-U6-(NF1)-shRNA, produced by Obio Technology, Shanghai, China) or control shRNA (pDKD-CMV-Puro-U6-shRNA) (2 µl of 1.34 × 10 11 plaque-forming-unit/ml) was injected into three points of cortex (coordinates: 0.3 mm anterior, 0.8 mm and 1.9 mm posterior to bregma, 3.0 mm lateral to midline, and 2.0 mm ventral to skull surface) in the right hemisphere 3 days before stroke 40 . Blocking antibodies against P-selectin (1.6 mg/kg, 553742, BD Pharmingen, San Jose, CA), anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (200 µg/mouse, YN1/1.7.4, BE0020-1, Bioxcell, NH) or isotype control antibody was administered intravenously immediately before MCAO 41,42 . Protein kinase C δ (PKCδ) inhibitor rottlerin (10 mg/kg) or vehicle (2% dimethyl sulfoxide) was injected intraperitoneally 30 minutes before stroke 43,44 .
Cranial window surgery and multiphoton microscopy. Cranial windows were prepared as we previously described 45,46 . Mice were anaesthetized with 1-1.5% iso urane in 30% oxygen and 70% nitrous oxide.
Body temperature was maintained at 37 ± 0.5°C during surgery. After xation in a stereotaxic head holder, a craniotomy (5 mm diameter) was created above the right somatosensory cortex (centered 2.5 mm lateral and 2.5 mm posterior to the bregma) using a high-speed micro drill. The window was closed with a sterile cover glass. For multiphoton imaging, Olympus FluoView FVMPE-RS upright multiphoton laserscanning system with an Olympus XL Plan N 25 × /1.05 WMP ∞/0-0.23/FN/18 dipping objective was used. Multiphoton excitation was performed using MAITAI eHPDS-OL and Spectra Physics InSight DS-OL lasers (Mai Tai, Spectra-Physics, CA). Emitted uorescence was detected through 495-540 nm and 575-645nm bandpass lters.
Time-lapse images at 6 µm steps were acquired from 100 to 150 µm below the surface every 6 seconds for 25 minutes. The area scanned was at 900 nm excitation wavelength in a 508 × 508 µm with 512 × 512 pixel resolution. Neutrophil movement was determined by imaging of blood vessels with a diameter between 20 and 40 µm. Images reconstruction was carried out using Olympus FV 10-ASW software. Neutrophil movement analysis was performed using the IMARIS image analysis software (Bitplane AG, Zurich, Switzerland). Forty cells per animal were tracked. Adherent neutrophils were de ned as the cells that were remained arrested in the microvessels for at least 30 seconds. The intravascular rolling velocity for neutrophils was calculated by determining the distance neutrophils moved between a certain time. Transmigrated neutrophils were determined in an area reaching out 75 µm to each side of a 100-µmlength vessel (representing 1.5 × 10 4 µm 2 tissue area).
Cerebrovascular permeability based on FITC-dextran (MW = 40 KDa, Sigma-Aldrich) leakage was analyzed as described previously 47 . In brief, time lapse imaging of FITC-dextran was acquired every 3 minutes for 30 minutes. The uorescence of randomly chosen 20 × 20 µm 2 regions of interest within the vessel and corresponding areas within the perivascular brain parenchyma were recorded.
Measurements of Evans blue and BSA vascular leakage. At 23 hours after MCAO, mice were intravenously injected with 4 ml/kg of 4% Evans blue dye (Sigma-Aldrich). After 1 hour, mice were perfused transcardially with phosphate buffer saline (PBS), and ischemic hemispheres were weighted and placed in formamide for 72 hours. After centrifugation, the amount of extravasated Evans blue dye in the supernatants was evaluated by spectrophotometry (Thermo Scienti c, MA) at 620 nm 48 . Alexa uor 488-conjugated bovine serum albumin (BSA; 66 kDa, 50 µl of 100 mg/ml) was intravenously injected into mice 1 hour before killing 49 . Brains were collected, xed in 4% paraformaldehyde and cryoprotected in 30% sucrose in PBS. Coronal brain sections (20 µm thick) were used for uorescent detection. Images were acquired using an Olympus FV1000 confocal microscope and an Olympus BX 63 microscope, and extravascular BSA uorescence in tissue sections was quanti ed using NIH Image J software.
Analysis of extravascular IgG deposition. Coronal brain sections were blocked with 1% BSA in PBS and incubated with goat anti-CD31 antibody (AF3628, R&D Systems, MN) overnight at 4˚C. The sections were washed and incubated with Alexa Fluor 488-conjugated donkey anti-mouse immunoglobulin G (IgG) and Alexa Fluor 594-conjugated donkey anti-goat IgG (Invitrogen, Carlsbad, CA). To quantify extravascular deposits of IgG, the images were contrast enhanced to clearly differentiate positivity from background and quanti ed using the NIH Image J integrated density analysis tool.
Neurobehavioral test. Forelimb force and rotarod test were carried out by an investigator blinded to the experimental groups as described previously 46,48 . In the forelimb force test, a grip strength meter (Bio-Seb, Vitrolles, France) was used to assess the peak force exerted by a mouse when the mouse released the forepaws from a grid. In the rotarod test, mice were placed on an accelerating rotating rotarod cylinder (Ugo Basile, Varese, Italy), and the time the mice remained on the rotarod was recorded. The speed was increased from 5 to 40 rpm within 5 minutes. Before surgery, mice were trained for 3 days.
Measurement of infarct volume and neuronal death. At 24 hours after MCAO, mice were sacri ced. The infarct area was detected by 2% triphenyl-2,3,5-tetrazolium chloride (TTC) staining and measured using the NIH Image J software in a blinded manner. Neuronal cell death in peri-infarct regions was detected using an In Situ Cell Death Detection Kit (11684795910, Roche, Mannheim, Germany) and mouse antineuronal nuclei (NeuN, MAB377, Millipore, MA) immunostaining.
Immunoblotting. Brain capillaries and capillary-depleted brain homogenates were prepared as we described previously 46,50 . Isolated protein from brain tissues, capillaries, and capillary-depleted brain homogenates was detected by immunoblotting according to standard procedures 46,50  MPO activity assay. Mice were sacri ced, perfused with ice cold PBS and the brains were removed. Ipsilateral brain hemispheres were homogenized in 50 mM potassium phosphate buffer, centrifuged, and resuspended in 0.5% cetyltrimethylammonium bromide (Sigma-Aldrich) in potassium phosphate buffer.
The suspensions were sonicated for 30 seconds with 3 cycles of freeze-thaw in liquid nitrogen. After centrifugation, 40 µl of supernatant was mixed with 100 µl tetramethylbenzidine solution (Sigma-Aldrich) in a 96-well plate in duplicates. The reaction was stopped with 100 µl 2N HCl after 15 minutes. The absorbance was measured at 450 nm in a microplate reader (Bio-Tek, Vermont). Myeloperoxidase (MPO) activity was calculated using puri ed MPO (Sigma-Aldrich) and was expressed as units of MPO per mg protein.
Immunohistochemistry. Mice were deeply anaesthetized with iso urane and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS. Brains were removed and immersed in 4% paraformaldehyde and cryoprotected in 30% sucrose. Coronal sections of 20 µm thickness were prepared on a cryostat and collected on glass slides. Sections were stained according to standard immunohistochemistry procedures with the following primary antibodies: Fluor 594-conjugated donkey anti-goat IgG, Alexa Fluor 647-conjugated donkey anti-goat IgG and biotindonkey anti-mouse IgG (all from Invitrogen). DNA was stained with Hoechst 33342 (1:10000, H3570, Invitrogen). For each animal, three elds from the peri-infarct cortex in each section were obtained under × 40 objective. Images were traced (quantitative analyzed) using Image J 1.48v software. The numbers of Ly6G + neutrophils in the traced area were counted.
Statistics. All values are presented as means ± standard deviation (SD). Statistical analysis for multiple comparisons were performed in Prism 7 software using one-way ANOVA followed by Bonferroni multiple comparison test. Differences between the two groups were assessed by unpaired Student's t-test. A value of P < 0.05 was considered statistically signi cant.