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Journal of Neuroimmunology xxx (2013) xxx–xxx

JNI-475654; No of Pages 8

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Journal of Neuroimmunology

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The immunomodulatory effect of bone marrow stromal cells (BMSCs) on interleukin(IL)-23/IL-17-mediated ischemic stroke in mice

Shuainan Ma a,1, Di Zhong a,1, Hongping Chen a, Yi Zheng a, Yanyan Sun a, Jian Luo a, Hulun Li c,Guozhong Li a,⁎, Yanhong Yin b,⁎⁎a Department of Neurology, The First Affiliated Hospital of Harbin Medical University, 23 YouZheng Street, Harbin, 150001, HeiLongJiang, PR Chinab Department of Neurology Chinese People Liberation Army (PLA) General Hospital 28 Fuxing Road, Haidian District, Beijing 100854, PR Chinac Department of Neurobiology, Harbin Medical University, 157 BaoJian Street, Harbin, 150086, Heilongjiang, PR China

Abbreviations: BMSC, Bone marrow stromal cell; IL, Imiddle cerebral artery occlusion; PBS, Phosphate-buftranscriptase-polymerase chain reaction; ELISA, Enzymet-PA, Tissue-plasminogen activator; IFN-γ, Interferon-gamT cell; HE, Hematoxylin and eosin; DAB, Diaminobenzidin⁎ Corresponding author. Tel.: +86 451 85555886; fax

⁎⁎ Corresponding author. Tel.: +86 010 66937234; faxE-mail addresses: [email protected] (G. Li)

(Y. Yin).1 Shuainan Ma and Di Zhong contributed equally to t

0165-5728/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.jneuroim.2013.01.007

Please cite this article as:Ma, S., et al., The imischemic stroke in mice, J. Neuroimmunol. (2

a b s t r a c t
a r t i c l e i n f o
Article history:Received 5 September 2012Received in revised form 15 January 2013Accepted 20 January 2013Available online xxxx

Keywords:Bone marrow stromal cell (BMSC)IL-23/IL-17 axisImmunomodulatoryIschemic cerebral stroke

Ischemic cerebral infarction is a leading cause of death and disability, but the key inflammatory cytokines werenot fully understood and no successful therapy has been established. We have used the methods of reversetranscriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), westernblot, and immunohistochemical staining to detect the dynamic changes of IL-23/IL-17 axis in brain infarctionand BMSC treatment on the sixth day. BMSC transplantation could reduce the infarct size (Pb0.05) and improvefunctional deficits (Pb0.001) in brain infarction. The level of IL-23/IL-17 axis expression is higher (Pb0.05) inpMCAO-operated group than that in sham operated group. BMSC treatment could reduce IL-23 expression(Pb0.05) and attenuate IL-17 expression (Pb0.01) both in serum and around infarct lesion. So we have drawnthe conclusion that IL-23/IL-17 axis induces inflammation in the pathophysiological process of cerebral infarc-tion. BMSC treatment plays therapeutic role by immunomodulating the expression of IL-23/IL-17 axis. Thesefindings may help to understand the cytokines in cerebral infarction and open up a possible new way of immu-nological treatment.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Stroke, especially ischemic cerebral infarction, is a leading cause ofdeath and disability, but no successful therapy has been establishedthat can benefit patient beyond narrow timewindow of tissue plasmin-ogen activator (t-PA) thrombolysis (Sacco et al., 2007; Donnan et al.,2008). Ischemic cerebral infarction is associated with complex patho-logical process involving many aspects of mechanism, of which inflam-matory responses play an important role in exacerbating brain tissuedamage (Barone and Feuerstein, 1999; Yilmaz et al., 2006). Whenbrain infarction happens, inflammatory cells infiltrate and secrete in-flammatory cytokines in brain tissue that can lead to local edema andnerve cell damage. In the immediate phase of brain infarction, macro-phages infiltrate and promote rapid inflammatory responses, while in

nterleukin; pMCAO, Permanentfered saline; RT-PCR, Reverse-linked immunosorbent assay;ma; Th17 cell, Type 17 Helpere tetrahydrochloride.: +86 010 68918451.: +86 010 68918451., [email protected]

he work.

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munomodulatory effect of bon013), http://dx.doi.org/10.101

the delayed phases of brain infarction, various types of inflammatorycells, including macrophages, neutrophils and lymphocytes, and cyto-kines exacerbate ischemic injury (Barone and Feuerstein, 1999;Matsui et al., 2002; Charriaut-Marlangue et al., 1996; Fink et al.,1999). However, such mechanism and the regulation have not beenfully understood. Because of the paradoxical role of interferon-gamma(IFN-γ) (Lambertsen et al., 2004), IL-17, the typical effector cytokineof Type 17 Helper T (Th17) cells, has became the focus of importantstudies in immunology (Miossec et al., 2009). IL-17 acts as aproinflammatory cytokine, a potent inducer of autoimmunity and tissueinflammation (Iwakura et al., 2011; Furuzawa-Carballeda et al., 2007).Other studies also showed that IL-17 could promote blood–brain barrierdisruption and central nervous system inflammation (Kebir et al.,2007). IL-23, a heterodimer of IL-23P19 and IL-23P40, is a key factorfor the induction of IL-17-producing cells, including γδ T cells, Th17cells and so on (Langrish et al., 2005). It has been demonstrated thatIL-23/IL-17 axis is pivotal in the onset and progression of autoimmuneencephalomyelitis (EAE) (Chen et al., 2006; Aranami and Yamamura,2008; Komiyama et al., 2006). In the studies of genetically deficientmice, the IL-23KOmice were highly resistant to the development of in-flammation rather than IL-12 (Cua et al., 2003). IL-23 is the key player inexpanding the population of IL-17-expressing cells, so IL-23/IL-17 axisis a predominant pathway in inducing inflammation (Shichita et al.,2009). A better understanding of the effects of the cytokines in cerebralinfarction may lead to an improved therapy.

emarrow stromal cells (BMSCs) on interleukin (IL)-23/IL-17-mediated6/j.jneuroim.2013.01.007

http://dx.doi.org/10.1016/j.jneuroim.2013.01.007

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2 S. Ma et al. / Journal of Neuroimmunology xxx (2013) xxx–xxx

The therapeutic application of BMSCs by intravenous administrationwas suggested from the early observations in MCAO model, in whichBMSCs entered the brain and reduced neurological functional deficits(Chen et al., 2001; Borlongan et al., 2004). The mechanism by whichBMSCs mediated tissue repair was that transplanted cells underwentdifferentiation to replace injured cells. However, the levels of improve-mentmediated by BMSCs do not always depend on the levels of cellularengraftment and differentiation, so other mechanisms, including theactivation of tissue-resident stem cells, stimulation of vasculogenesisand inhibition of inflammation, have been reported widely (MeirellesLda et al., 2009; Prockop, 2009; Caplan, 2009; Xue et al., 2010). BMSCssecreted bioactive factors to regulate the diverse disease-associatedprocess (Bunnell et al., 2010). It has been proved that BMSCs producedifferent kinds of inflammatory factors which have effects on multipleprocesses, such as apoptosis, angiogenesis and inflammation (Weiet al., 2012). BMSCs may inhibit neutrophil apoptosis to make therelease of inflammatory cytokines relatively mild and mediate the bal-ance of proinflammatory cytokines and anti-inflammatory cytokinesto reduce the inflammatory damage to brain tissue (Raffaghello et al.,2008). Although the mechanisms underlying the modulating roleof BMSCs are not fully understood, some factors have been provedto be associated with their anti-inflammatory effects. For example,transforming growth factor (TGF)— β, a critical factor for IL-17 produc-tion in mouse, is constitutively expressed by BMSCs (Di Nicola et al.,2002; Schnyder et al., 2005). Studies showed that BMSCs could normal-ize the distribution of Th1, Th2, Th17, Treg cells in experimental autoim-mune myasthenia gravis, a typical disease which demonstratedalternation in the balance of the four subsets (Kong et al., 2009). Onthese grounds, we proposed the hypothesis that BMSCs could playimmunomodulatory role in the pathological progression of ischemic ce-rebral infarction by regulating IL-23/IL-17 axis.

2. Materials and methods

2.1. Animals

Adult male C57BL/6 mice weighting 25–30 g were used in ourexperiment (n=48, Vital River Beijing Co., China) and housed inpathogen-free conditions. Mice were randomly divided into pMCAOexperimental group (pMCAO group, n=12), sham-operated group(sham pMCAO group, n=12), BMSC administration group (pMCAO+BMSC group, n=12) and PBS control group (pMCAO+vehicle group,n=12). Every effort was made to minimize the number of animalsused and alleviate their sufferings.

2.2. BMSC preparation

C57BL/6 mouse BMSCs and all growth medium with 10% qualifiedfetal bovine serum (FBS) and 1% penicillin streptomycin were providedby Cyagen Biosciences Co. (USA). All normal cultured cells weremaintained in a humidified tissue culture incubator at 37 °C with 5%carbon dioxide. When dense colonies of spindle-shaped cells coveredabout 80%–90% of the dish, the cells were passaged into the secondaryculture. Bone marrow stromal cells of passage 11 (P11) were used inthis study.

2.3. Stroke surgery and BMSC implantation

The animals were anesthetized intraperitoneally with Hypnorm(fentanyl citrate 0.315 mg/ml and flu-anisone 10 mg/ml, JanssenPharmaceutica, Titusville, USA). The right common carotid artery(CCA), external carotid artery (ECA), and internal carotid artery (ICA)were exposed. In pMCAO-operated group, middle cerebral artery(MCA) occlusion was induced by inserting nylon monofilamentthrough the right ECA. A length of 6–0 surgical nylon suture (8.5–9.5 mm) with its tip rounded by heating near a flame was advanced

Please cite this article as:Ma, S., et al., The immunomodulatory effect of bonischemic stroke in mice, J. Neuroimmunol. (2013), http://dx.doi.org/10.101

from the ECA into the lumen of the ICA until it blocked the origin ofthe MCA. In sham-operated group, all procedures were the same as inpMCAO-operated group, however, no suture was inserted into MCA.In BMSC treatment group, first of all we built pMCAO models, andthen 3 h later, 1×106/ml BMSCs in 100 μl PBS were slowly injectedinto each mouse via the tail vein in about 5 min. In PBS control group,pMCAO models were established as well, but 100 μl PBS withoutBMSCs were injected to the mice.

2.4. Functional tests

Behavioral measurements included adhesive-removal test and ele-vated body swing test to evaluate the extent of functional recovery.Both tests were conducted by two individuals who were blind to thetreatment of animals. Data were collected from animals in pMCAO,sham pMCAO, pMCAO+BMSCs, and pMCAO+vehicle groups (n=12)before surgery and 6, 12, and 24 h, and 3 and 6 days after surgery andtreatment.

2.4.1. Adhesive-removal testAdhesive-removal test required training in advance. Two small ad-

hesive paper dots were attached to each wrist of the forelimbs as bilat-eral tactile stimuli. The time needed to remove both paper dots fromeach limb was recorded in five trials per day for three days. All micecan remove the dots within ten seconds (s) at the end of training. Theanimals were familiarized with the testing environment before opera-tion. After operation, the time, to a maximum of 180 s, for each mouseto remove the left paper dotswas recorded. Three trialswere conductedper day, with at least five-minute interval time. Data are presented asthe mean time for removal of the left dot.

2.4.2. Elevated body swing testElevated body swing test is a reflex test that examines the extent

and direction of the swing of body when the mouse is held elevatedby the tail. The animals were hold in vertical axis about five centimetersabove the surface. A swing was recorded whenever the head of the an-imal was moved out of the vertical axis more than 10° either to the leftor to the right side. The numbers of swings were recorded for 1 minduring each of 3 trials per day. Normal mouse swung to the left orright equally. Mouse swung predominantly to the left after rightpMCAO. Data were shown as the incidence of body swinging to theleft as a fraction of the total number of body swings.

2.5. Semi-quantitative RT-PCR

Six days after operation and injection, the mice were deeply anesthe-tized and quickly decapitated. Brain samples of pMCAO, sham pMCAO,pMCAO+BMSCs, and pMCAO+vehicle groups (n=6) were collectedandwere frozen in liquid nitrogen for 24 h and then stored at−80 °C be-fore being processed for total RNA and protein extraction. Total cellularRNA was isolated using Trizol reagents (Invitrogen, USA) according tothe manufacturer's guidelines and reverse-transcribed into cDNA withTianscript RT Kit (Beijing Co., China). Polymerase chain reaction (PCR)was performed using specific primers. Primers for the amplification ofeach gene are as follows: IL-23, sense: 5′-ATGCTGGATTGCAGAGCAGTA-3′,antisense:5′-ACGGGGCACATTATTTTTAGTCT-3′;IL-17,sense:5′-CTGCTGAGCCTGGCGGCTAC-3′,antisense:5′-GGCGGCACTGAGCTTCCCAG-3′;GAPDH,sense: 5′-TGGAGAAACCTGCCAAGTATG-3′, antisense: 5′-CCCTGTTGCTGTAGCCGTAT-3′. PCR products of 10 μl were separated by 2% aga-rose gel electrophoresis and visualized using ethidium bromide staining.RT-PCRwas performed at least three independent experiments. The den-sity of each band was measured with Image J software (NIH, Bethesda,MD, USA), and mRNA levels for different were normalized to GAPDH.



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Fig. 1. Functional outcomes and lesion volume. (a) In adhesive-removal test, themean timeneeded to remove a sticky tape from forepaw was significantly reduced in pMCAO+BMSCgroup (n=12) from 1-day after stroke, compared with pMCAO+vehicle group (n=12)(two-way ANOVA with Bonferroni's post hoc test). (b) The ischemic-induced unilateralbody swing preference was significantly reduced in animals receiving BMSC implantation(n=12) (two-way ANOVA with Bonferroni's post hoc test). (c) Infarct volume ofpMCAO+BMSC group decreased from that of pMCAO+vehicle group (n=6) (two-sampleT test). # Pb0.05, ### Pb0.001, between pMCAO+BMSC group and pMCAO+vehiclegroup.

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2.6. Western blot

To determine whether the expression of IL-23/IL-17mRNA wastranslated into protein in the brain, protein was extracted from wholemice brains and homogenized in ice cold RIPA buffer containing 1% pro-tein inhibitor cocktail, 10 mg/ml phenylmethylsulphonyl fluoride and100 mM sodium othovanadate. After incubating for 30 min on ice, themixturewas centrifuged at 4 °C at 10,000×g. Supernatantwas collectedas total cell lysate and the total protein concentration was determinedby Lowry method (using a detergent-compatible protein assay with abovine serum albumin standard), so that IL-23/IL-17 could be subjectedto12% SDS-PAGE. Proteins were transferred onto polyvinylidenedifluoride membranes (Millipore, Temecula, CA). Membranes wereblocked in 5% milk in TBS-T (20 mmol/L Tris pH 7.5, 150 mmol/LNaCl, 0.1% Tween-20) and probed with antibodies specific for IL-23(Rabbit, 1:500, Abcam, Cambridge, MA, USA) and IL17 (Rat, 1:1,000,Santa Cruz Biotechnology, Inc., USA), and GAPDH (Rabbit, 1:1,000,Santa Cruz Biotechnology, Inc., USA). Respectivemembraneswere incu-bated with rabbit anti-rat and goat anti-rabbit IgG horseradish peroxi-dase secondary antibodies (Santa Cruz Biotechnology, Inc., USA). Forsemi-quantitative analysis of the protein, Image J software (NIH,Bethesda, MD, USA) was used on the bands of the Western blots, andthe protein ratios were calculated by comparison with GAPDHexpression.

2.7. IL-23 and IL-17 ELISA

To detect the concentration of IL-23 and IL-17 in serum of pMCAO,sham pMCAO, pMCAO+BMSCs, and pMCAO+vehicle groups (n=6),ELISA Quantikine Kits (R&D Systems, Minneapolis, MN, USA) were pur-chased. All procedures were done according to the instructions of themanufacturer. Standard curve and sample concentrations were calcu-lated based on the mean of triplicates for each dilution or sample.

2.8. Quantitative analysis of infarct volume

The rest of animals in pMCAO, sham pMCAO, pMCAO+BMSCs, andpMCAO+vehicle groups (n=6) were sacrificed by transcardial perfu-sion with saline, followed by 4% paraformaldehyde perfusion on thesixth day. The entire brain was removed and immersed in 4% parafor-maldehyde overnight, and then tissue blocks were embedded in paraf-fin. One coronal paraffin slide (2–4-μm-thick) from each of the 7 brainblocks (1-mm-thick) of each mouse was stained with hematoxylinand eosin (HE) in order to observe inflammatory responses aroundthe infarct lesion and calculate the volume of cerebral infarction.Infarction-induced cerebral tissue loss was estimated as a fraction oftotal contralateral hemisphere.

2.9. Tissue preparation and immunohistochemical staining

The brain tissues, within the center of the lesion of pMCAO, werestained with polyclonal antibody against IL-23 and monoclonal anti-body against IL-17. All sections were dewaxed in xylene and takenthrough ethanol. Methanol containing 0.3% hydrogen peroxide wasused to block endogenous peroxidase. Sectionswere incubated in rabbitanti-mouse IL-23 antibody (1:200, Abcam, Cambridge, MA, USA) andrat anti-mouse IL17 antibody (1:100, Santa Cruz Biotechnology, Inc.,USA) overnight and were washed three times with PBS. After that, sec-tions were incubated with avidin-biotin-horseradish peroxidase com-plex and developed in diaminobenzidine tetrahydrochloride (DAB).Control experiments consisted of staining brain coronal tissue sections,but the primary antibodies were omitted. In all tissue sections of posi-tive cells, the number of positive cells per unit area (10 mm2) under100× and 400× microscope was calculated. Each slice (100× magnifi-cation) was calculated at least ten times.


2.10. Statistical analysis

Data were expressed as mean value±standard deviation. The func-tional outcomes were evaluated between the pMCAO+BMSC groupand pMCAO+vehicle group with two-way analysis of variance(ANOVA) followed by Bonferroni's post hoc test. Two-sample T testwas used to analyze the difference in lesion volume. We performedone-way ANOVA followed by Dunnett's post hoc multiple-comparisontests to analyze differences among four groups of mice. Between twogroups of mice, a two-tailed Student's t-test was performed to deter-mine statistical significance. Statistical analysis was performed usingGraphPad Software Version 5.00 (CA, USA). A value of Pb0.05 wastaken as statistically significant.

3. Results

3.1. Neurological functional outcome and lesion volume

Behavior function was determined with adhesive-removal test andelevated body swing test. Prior to pMCAO, neurological function amongthe test animals was similar. When animals were tested with tape re-moval, pMCAO-operated animals took longer than 180 s in 12 h after



Fig. 2. IL-23 and IL-17 ELISA. Mice from sham pMCAO group (n=6), pMCAO group (n=6), pMCAO+BMSC group (n=6) and pMCAO+vehicle group (n=6) were sacrificed on6-day after ischemic stroke. Blood samples were obtained and clot for 2 h at room temperature. We removed the serum sample to assess IL-23 and IL-17 production by ELISA. Datawere shown as mean±SD and results of IL-23 were as follows (pg/ml): 4.7±0.3 (sham pMCAO group), 44.0±0.9 (pMCAO group), 29.0±0.7 (pMCAO+BMSC group), 43.2±0.7(pMCAO+vehicle group); results of IL-17 were as follows (pg/ml): 56.3±4.4 (sham pMCAO group),190.8±7.0 (pMCAO group), 132.5±5.0 (pMCAO+BMSC group), 190.3±7.2(pMCAO+vehicle group) (one-way ANOVA with Dunnett's post hoc test). *** Pb0.001, between sham pMCAO and pMCAO groups. ### Pb0.001, between pMCAO+BMSCs andPMCAO+vehicle groups. о P>0.05, between pMCAO and pMCAO+vehicle groups.


stroke. However, mice with BMSC transplantation had improved perfor-mance from 1-day after stroke compared with pMCAO+vehicle group(Pb0.001) (Fig. 1, a). In the elevated body swing test, infarct animalsshowed a strong and persistent tendency to turn their upper bodies tothe left. At postoperative 6 h, the performance of pMCAO+BMSCgroup was superior to that of pMCAO+vehicle group (Pb0.001)(Fig. 1, b). The phenomenon lasted for the next six days, the longestsurvival that we have examined. Improvement was limited and failedto restore behavior in these animals before surgery. Size of infractvolume was evaluated by the results of HE staining at 6-day after

Fig. 3. Semi-quantitative determination of IL-23/IL-17 mRNA expression. Total RNA waspMCAO+vehicle groups (n=6). RT-PCR was conducted and results were normalized to Ggroup than in sham pMCAO group, while the expression decreased in pMCAO+BMSC grohoc test). *** Pb0.001, between sham pMCAO and pMCAO groups. ### Pb0.001, betwepMCAO+vehicle groups.


pMCAO operation. Infarct volume of BMSC group decreased significantlycompared with that of control group (Pb0.05) (Fig. 1, c).

3.2. Expression of IL-23/IL-17 in mice after ischemic cerebral infarction

To test the hypothesis that IL-23/IL-17 may contribute to inflamma-tion after experimental ischemic stroke, the expression of IL-23/IL-17was compared among pMCAO and sham pMCAO groups. The serumsamples were obtained from pMCAO group (n=6) and sham pMCAOgroup (n=6). We tested the expression of IL-23/IL-17 in serum and

extracted from sham pMCAO (n=6), pMCAO (n=6), pMCAO+BMSCs (n=6) andAPDH. RT-PCR revealed that the expression of IL-23/IL-17 mRNA is higher in pMCAOup compared with in pMCAO+vehicle group (one-way ANOVA with Dunnett's posten pMCAO+BMSCs and PMCAO+vehicle groups. о P>0.05, between pMCAO and




found that the expression was extremely significantly lower in shampMCAO group than in pMCAO group (Pb0.001). The whole brain sam-ples were collected from pMCAO (n=6) and sham pMCAO (n=6)groups 6-day after operation. Total RNA and protein were extracted toassess the expression of IL-23/IL-17 in mRNA and protein level. Thelevels of expression were normalized to GAPDG in order to obtainsemi-quantitative results of RT-PCR and Western blot. For sham-operated animals, the expression of IL-23/IL-17 was lower than that ofpMCAO-operated animals in mRNA level (Pb0.001). The expression ofIL-23/IL-17 in protein level was also compared between the two groupsand showed the similar trend (Pb0.05 for IL-23 and Pb0.001for IL-17).Immunohistochemistry was employed to measure the expression ofIL-23/IL-17 in mouse ischemic brain tissue. The brain tissue sectionswere stained with IL-23 and IL-17 antibodies. The results of immuno-chemical staining were calculated as the numbers of IL-23-positivecells and IL-17-positive cells. In sham pMCAO group (n=6), obviouspositive cells could hardly be seen, while the number of positive cellsof pMCAO group was more than that of the sham pMCAO group(Pb0.001) significantly (Fig. 2).

3.3. BMSC treatment of stroke regulates IL-23/IL-17 expression

In order to investigate whether BMSCs have immunomodulatoryeffects on IL-23/IL-17 axis in ischemic cerebral infarction, we alsocollected serum samples, total brain samples and brain tissue slicesfrom pMCAO+BMSC group (n=6, respectively) and pMCAO+vehiclegroup (n=6, respectively). To guarantee the effects of BMSCs,

Fig. 4. Western blot analysis of IL-23/IL-17 axis. Cell lysates from four distinct groups: sham plane 3), pMCAO+vehicle (n=6, lane 4) group were separated by electrophoresis and tranti-GAPDH antibodies. The results of IL-23/IL-17 expression were normalized to GAPDH expshowed increased expression of IL-23/IL-17 in pMCAOgroup comparedwith shampMCAO grougroup significantly (one-way ANOVA with Dunnett's post hoc test). * Pb0.05, *** Pb0.001, betand PMCAO+vehicle groups. о P>0.05, between pMCAO and pMCAO+vehicle groups.


pMCAO+vehicle group was also compared with pMCAO group. BMSCsattenuated the expression of IL-23/IL-17 in serum (Pb0.001), andpMCAO+vehicle group showed no significance (P>0.05). The densitiesof IL-23/IL-17 RT-PCR and Western blot bands were also calculated. Theexpression of IL-23/IL-17 in pMCAO+BMSC group is lower than that inpMCAO+vehicle group (Pb0.001) in mRNA level. The protein level ofIL-23/IL-17 showed the same tendency (Pb0.05 for IL-23 and Pb0.01for IL-17). In pMCAO+BMSC group, the numbers of IL-23-positve cellsand IL-17-positive cells decreased extremely significantly frompMCAO+vehicle group (Pb0.001). Data of pMCAO+vehicle groupshowed no significance (P>0.05), so the lowering effects resulted fromthe BMSC treatment (Fig. 3).

4. Discussion

The present study corroborated that BMSC treatment could improvefunctional recovery and reduce infarct lesion in mice after experimentalischemic stroke. BMSCs delivered to ischemic brain tissue through an in-travenous route have the capacity to enter the brain and restore nervoustissue (Chen et al., 2001). However, the mechanisms by which BMSCsenhance functional recovery are more complex than the strong prolifer-ative capacity and multilineage differentiation potential. We focused onthe immunomodulatory activities associated with BMSCs. We also paidour attention to inflammatory IL-23/IL-17 axis, which has been provedto be associated with the progression of ischemia–reperfusion injury(Shichita et al., 2009). Our present study detected the expression ofIL23 and IL-17 six days after stroke and BMSC treatment, and our

MCAO group (n=6, lane 2), pMCAO group (n=6, lane 1), pMCAO+BMSC group (n=6,ansferred to PVDF membranes. Proteins were blotted with anti-IL-23, anti-IL-17 andression. Data shown are representative of three independent experiments. Western Blotp. The IL-23/IL-17 expression of pMCAO+BMSCswas lower than that of PMCAO+vehicleween sham pMCAO and pMCAO groups. # Pb0.05, ## Pb0.01, between pMCAO+BMSCs




findings showed IL-23/IL-17 axis played proinflammatory role in thedevelopment of cerebral infarction especially in later phase and BMSCtreatment had therapeutic effects by downregulating IL-23/IL-17 expres-sion and attenuated inflammatory damage (Fig. 4).

When induction of experimental stroke happens, not only produceslocal lesion, but also activates peripheral immune responses and resultsin proliferation of inflammatory cells and secretion of cytokines whichcan infiltrate through blood-brain barrier, play pro-inflammatory rolein the pathophysiological process of experimental stroke, and aggravatethe tissue edema and neurological damage. IL-23 is mostly producedfrom antigen-presenting cells, such as dendritic cells and macrophage,and drives a T cell population characterized by IL-17 production,which was found to be elevated in both ischemic human brain andpMCAO-operated rat in our previous experiment (Li et al., 2005). Inthis study, we detected both IL-23 and its downstream effector IL-17in the sera, discovered IL-23/IL-17 in infarct tissue and verified their

Fig. 5. Immunohistochemical staining for IL-23. The brain sections were stained with polycsections, IL-23-positive cells were calculated and each slice was calculated at least ten timsham pMCAO group. (b) In pMCAO group, the infiltration of IL-23-positive cells could bepMCAO+BMSC group, the number of IL-23-positive cells reduced dramatically indicatinggroup showed no significant reduction. Data represents mean±sd for six individual miceand pMCAO groups. ### Pb0.001, between pMCAO+BMSCs and PMCAO+vehicle groups


pathophysiological role in cerebral infarction. According to the results,we could infer that the brain damage results in elevated IL-23/IL-17 ex-pression in peripheral circulation and proliferation of macrophages andIL-17-producing cells. These cells and cytokines could penetrate into thebrain which can cause the evolution of infarct volume and neurologicaldeficits. BMSC treatment could play immunomodulatory role on thesechanges in the pathological process of brain infarction. However,whether IL-23 and IL-17 are direct factors in brain injury and in whichlevel IL-23 plays a role in brain tissue needs further study. The applica-tion of IL-23 and IL-17 monoclonal antibodies may lead to a better un-derstanding of the mechanism. We will also focus on specificimmunomodulating factor produced by BMSCs and reveal the relation-ship between recovery of function and BMSC treatment (Fig. 5).

One of the advantages of BMSC treatment of cerebral infarction is along therapeutic time window. There is no unified standard of BMSCtransplantation time after cerebral ischemic injury. Whether there is a

lonal antibody against IL-23 and the arrows indicated IL-23-positive cells. In all tissuees. The magnification of the images is 100×. (a) Low levels of IL-23-positive cells inidentified indicating that IL-23 may involve in the pathological damage. (c) While inthat BMSC implantation could downregulate IL-23 expression. (d) pMCAO+vehicle(one-way ANOVA with Dunnett's post hoc test). *** Pb0.001, between sham pMCAO. о P>0.05, between pMCAO and pMCAO+vehicle groups.




best time window remains controversial, however early cell transplant ismore beneficial for recovery of function (Kawabori et al., 2012). In thisstudy, BMSCs were administrated in the tail vein 3 h after stroke, consid-ering early toxic neurotransmitters and oxygen free radical release. Therewere large numbers of animal experiments about transplantation celldoses for cerebral infarction treatment. Studies have shown that thetotal number of cells (1–5)×106 of intravenous injection could improvethe symptoms of neurological deficit. We used the dose of 1×106, butwhether there may exist relatively better dose is still under investigation(Fig. 6).

Taken together, BMSC treatment can restore brain tissue and im-prove functional outcomes by immunomodulating IL-23/IL-17 axis,

Fig. 6. Immunohistochemical staining for IL-17. The brain tissueswere stainedwithmonoclonaIL-17-positive cells were calculated. Each slice was calculated at least ten times. Themagnificatibe identified. (b)While in pMCAO group, IL-17-positive cells were located in cortex surroundinnumber of IL-17-positive cells reduced obviously in pMCAO+BMSC group. (d) pMCAO+vehictherapeutic role by regulating IL-17 expression. Data representsmean±sd for six individualmicpMCAO groups. ### Pb0.001, between pMCAO+BMSCs and PMCAO+vehicle groups. о P>0


which involves in immunopathological damage in ischemic cerebralinfarction. The experimental studies of BMSC transplantation in im-munological treatment of cerebral infarction may help prevent theprogression of tissue damage and open up a possible new therapeuticway.

Acknowledgments

This program was supported by the National Natural ScienceFoundation (no. 30971015) and Harbin Science and Technology Inno-vation Talents of Special Fund Project (2009RFXXS021).

l antibody against IL-17 and the arrows indicated IL-17-positive cells. In all tissue sections,on of the images is 100×. (a) In sham pMCAO group, no significant IL-17-positive cells cang infarction, indicating that IL-17 may play role in the pathophysiological process. (c) Thele group showed no significant reduction, indicating that BMSC transplantation may playe (one-way ANOVAwithDunnett's post hoc test). *** Pb0.001, between shampMCAO and.05, between pMCAO and pMCAO+vehicle groups.




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