Available online at www.sciencedirect.com

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0006-8993/$ - see frohttp://dx.doi.org/10.

nCorresponding aBeijing 100053, Chin

E-mail address: ji1Co-first author.

Research Report

Different expression of ubiquitin C-terminalhydrolase-L1 and αII-spectrin in ischemicand hemorrhagic stroke: Potential biomarkersin diagnosis

Changhong Rena,c,1, Susie Zoltewiczb,c,1, Joy Guingab-Cagmatb,c,John Anaglib,c, Mingqing Gaoa, Adam Hafeezd, Ning Lia,c, Jinqiang Caoa,Xiaokun Genga, Firas Kobeissyf, Stefania Mondellob, Stephen F. Larnerb,c,Ronald L. Hayesb,c, Xunming Jia,c,e,n, Yuchuan Dingc,d

aInstitute of Hypoxia Medicine, Xuanwu Hospital, Capital Medical University, Beijing 100053, ChinabBanyan Laboratories, Banyan Biomarkers Inc., Alachua, FL, USAcXuanwu-Banyan Biomarker Research and Assay Center, Beijing 100053, ChinadDepartment of Neurosurgery, Wayne State University School of Medicine, Detroit, MI, USAeDepartment of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing 100053, ChinafDepartment of Psychiatry, Center for Neuroproteomics and Biomarkers Research, University of Florida, Gainesville, FL, USA

a r t i c l e i n f o

Article history:

Accepted 30 September 2013

The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally

different mechanisms and thus different treatment options. These two stroke categories were

Available online 16 October 2013

Keywords:

Stroke

Biomarker

Ischemia

Hemorrhagia

Ubiquitin C-terminal hydrolase-L1

αII-Spectrin

nt matter & 2013 Elsevie1016/j.brainres.2013.09.05

uthor at: Department ofa. Fax: þ86 106 301 0085.xm@ccmu.edu.cn (X. Ji).

a b s t r a c t

applied to rat models to identify potential biomarkers that can distinguish between them.

Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion

while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain

hemispheres and biofluids were collected at two time points: 3 and 6 h after stroke. Known

molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF)

and Banyan's proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses

revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after

6 h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were

elevated after 6 h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels

were increased after 3 and 6 h of ischemia detected by ELISA. However, levels of those proteins

in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs

and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. Thesemolecules behaved

differently in the two stroke models and thus may be capable of being differentiated.

& 2013 Elsevier B.V. All rights reserved.

r B.V. All rights reserved.1

Neurosurgery, Xuanwu Hospital, Capital Medical University, Chang Chun Road 45,

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1. Introduction

Stroke is the third most common cause of death and a majorcause of disability worldwide. Of all strokes, 87% are ischemic (IS)and 13% are intracerebral hemorrhagic stroke (ICH) (Lloyd-Joneset al., 2010). Despite the significant advances in neuroimagingand clinical management, it is still challenging to establish adiagnosis and to make therapeutic decisions for patients withacute stroke. Currently, imaging is required to distinguishbetween IS and ICH. Once such a diagnosis is made, patientsbenefit from thrombolytic therapy administered within 6 h ofstroke onset (Kent et al., 2003). Unfortunately less than a third ofpatients with IS show characteristic changes on a CT scan within3 h of symptom onset (Chalela et al., 2007). Although MRI offersadvantages for the assessment of acute stroke within the first 3 hof symptom onset, the sensitivity of MRI to detect a laterclinically confirmed ischemic stroke is less than 73% (Chalelaet al., 2007). Thus current imaging technology is limited tempo-rally and hinders timely treatment. Furthermore, imaging can beinconvenient and cumbersome due to machine size and itsunavailability, such as in the ambulance, immediately afterstroke onset. This further delays the diagnosis and, ultimately,the treatment.

Biomarkers have been used for the rapid diagnosis of diseasesin organs other than the brain. Biomarkers are a useful toolbecause they guide treatment and assess its progression. Cur-rently researchers are dedicated to exploring the relationshipbetween biomarkers and stroke. Biomarkers may serve as apotential diagnostic tool in the pre-hospital setting because oftheir ability to rapidly and reliably distinguish between the twomajor categories of stroke (IS, ICH). This will be crucial inefficiently treating stroke patients because, unlike imaging tech-nology, biomarkers can be measured rapidly and convenientlyvia a simple blood test. This can be done en-route to the hospitaland provide vital information early on. Thus biomarkers have

Fig. 1 – Representative IS and ICH images. (A) IS 3 h after surgeryafter surgery.

the ability to allow cause-specific management and can poten-tially facilitate the hyperacute delivery of stroke therapies(Foerch et al., 2009).

Brain tissue, blood, and cerebrospinal fluid (CSF) from strokepatients and experimental animal models have been used toidentify novel diagnostic biomarkers (Cuadrado et al., 2010;Lescuyer et al., 2004; Whiteley et al., 2009). Following a strokemolecules are released into the blood and cerebrospinal fluid(CSF) by both brain injury and the body's physiological reactionto stroke. Analysis of the levels of certain molecules has distinctadvantages that could provide a relatively comprehensive cluefor the identification of potential biomarkers.

The main objective of this study was to identify biomarkersthat distinguish between IS and ICH stroke using brain, bloodand CSF within 6 h of stroke onset. We identified a handful ofbiomarker candidates that fulfill two criteria: brain specificityand high abundance in brain (Liu et al., 2010). In this study wefocused on two candidate biomarkers: ubiquitin C-terminalhydrolase-L1 (UCH-L1) and αΙΙ-Spectrin. UCH-L1 is a highlyabundant and brain-specific protein that is found in neurons(Wilkinson et al., 1992). αΙΙ-Spectrin, which expressed in neuronsand glia, is a valuable biomarker for tracking cell death proteaseactivity after traumatic and ischemic brain injury (Brophy et al.,2009; Liu et al., 2010). In the targeted approach, UCH-L1 and αΙΙ-Spectrin were tested on the rat samples via quantitative immu-noblotting (brain, CSF) and Banyan's proprietary ELISA assays(CSF, serum).

2. Results

2.1. CBF, neurological deficits, brain infarct and brainhemorrhage

To verify the effectiveness of the MCAO model, a totalnumber of 16 rats subjected to MCAO showed significant

. (B) IS 6 h after surgery. (C) ICH 3 h after surgery. (D) ICH 6 h

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(Po0.01) reduction in regional cerebral blood flow duringischemia, as monitored by laser Doppler flowmetry, with avalue that dropped to 21.674.1% of the pre-ischemic level.Ischemia induced infarct was clearly visualized in Fig. 1.Moreover, the neurological scores of the 3 h group (N¼8)and 6 h group (N¼8) are 2.2570.46 and 2.3870.52, respec-tively. To verify the accuracy of the ICH model, a totalnumber of 16 rats subjected to ICH showed hematomas inthe striatum (Fig. 1). Furthermore, the neurological scores ofthe 3 h group (N¼8) and 6 h group (N¼8) are 2.2570.46 and2.3870.52, respectively as compared to 0 in sham group.The above results indicated that the stroke model is viable.

CSF SBDP 150/145 (WB)

16

18

f

*

2.2. The expression of UCH-L1 and SBDP in injured ratbrain post stroke

The expression of αII-spectrin and UCH-L1 were examined byimmunoblotting on brain lysates (Fig. 2). Brain-blots showedthat spectrin was fragmented into its characteristic break-down products SBDP150 and SBDP145 primarily in ischemicbrains at both 3 h and 6 h after stroke (Fig. 2A). SBDP levelswere quantified from Western blots. This analysis revealedthat SBDPs were strongly increased at 3 h (Po0.01) and 6 h(Po0.001) after ischemic stroke (Fig. 2B). SBDPs were notelevated in the hemorrhagic brains. However, levels of UCH-L1, a biomarker of the neural cell body, did not differ amongthe brain samples and so was not quantified (Fig. 2A).

Brain SBDP 150/145 (WB)

0

2

4

6

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10

12

*

*

*

*

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ativ

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otei

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f SB

DP

150/

145

SBDP 150 SBDP 145

Fig. 2 – Quantitative immunoblotting analysis of biomarkers ininjured rat brain post stroke. (A) Representative Western blotsof brain lysates are shown, probed with antibodies for αII-spectrin and UCH-L1. UCH-L1 levels did not differ andwere notquantified. (B) Quantification of SBDP levels from immunoblotsis shown. SBDP150 and 145 were significantly elevated afterischemic stroke at 3 h and 6 h after ischemic stroke ascompared with the sham group. There is no differencebetween ICH group and sham group. *Po0.05; N¼8.

FCaReMtb(i

2.3. Quantitative immunoblotting of UCH-L1 and SBDP inrat CSF after stroke

When considering human patients, it would be much moreuseful to have biomarkers that are measurable in accessiblebiofluids such as CSF and blood. Therefore we extended theimmunoblotting analysis of spectrin and UCH-L1 to CSF(Fig. 3). Similar to the brain, SBDP150/145 were significantlyelevated in the CSF after 6 h ischemic stroke (Fig. 3B). It seemsthat SBDPs was slightly increased at 6 h after ICH. But thisincrease is not statistically different as compared to sham.However, in contrast to brain, UCH-L1 was also significantlyelevated in the CSF after 6 h ischemia (Fig. 3C). SBDP150/145and UCH-L1 were not elevated after 3 h ischemic stroke.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Rel

ativ

e pr

otei

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vel o

f UC

HL1 *

CSF UCHL1 (WB)

SBDP 150 SBDP 145

UCHL1

0

2

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*

ig. 3 – Quantitative immunoblotting of biomarkers in ratSF after stroke. (A) CSF samples (10 μL each) were blottednd probed with antibodies to spectrin and UCH-L1.epresentative immunoblots results show significantlylevated levels of UCH-L1 and SBDP in rat CSF 6 h afterCAO as compared with sham. (B) Quantification indicated

hat SBDP levels show a statistically significant differenceetween sham and 6 h ischemic stroke. nPo0.05, N¼8.C) Quantification indicated that UCH-L1 was elevated by 6 hschemic stroke as compared with sham. nPo0.05, N¼8.

b r a i n r e s e a r c h 1 5 4 0 ( 2 0 1 3 ) 8 4 – 9 1 87

2.4. Changes of UCH-L1 and SBDP in CSF and serummeasured by Banyan's quantitative ELISA assays

Biomarkers were also measured with Banyan's proprietaryELISA assays in both CSF and serum. As seen from Westernblots, SBDP145 was significantly increased in rat CSF at 6 hafter ischemic stroke (0.92 ng/mL) as compared with sham(0.11 ng/mL) (Po0.05)(Fig. 4A). CSF UCH-L1 levels were foundto be elevated at 3 h (3.34 ng/mL, Po0.05) and 6 h (9.63 ng/mL,Po0.001) after ischemic stroke (Fig. 4A and B). In clinicalsettings, compared with the CSF, serum is the conventionalbody fluid used in the clinic. Therefore, we further measuredUCH-L1 levels in serum with ELISA methods. We found thatUCH-L1 levels were significantly increased after 3 h (0.48 ng/mL)(Po0.001) and 6 h (0.46/ng/mL) (Po0.01) of ischemia as com-pared with sham (0.23 ng/mL) (Fig. 4C).

3. Discussion

We analyzed the expression of UCH-L1 and αΙΙ-Spectrin on ratsamples via quantitative immunoblotting (brain, CSF) and

Fig. 4 – Biomarker changes in CSF and serum measured by Banysignificant increase at 6 h after ischemic injury compared with sserum (C) at 3 h and 6 h after ischemic stroke. nnPo0.05, N¼8.

Banyan's proprietary ELISA assays (CSF, serum). In injuredbrain, we found that αII-spectrin breakdown products (SBDP150,SBDP145) were strongly increased after 3 and 6 h ischemia. InCSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) were elevated after 6 h ischemic stroke as detected byWestern blotting. In serum, UCH-L1 levels were increasedafter 3 and 6 h of ischemia as detected by ELISA. However,levels of those proteins in hemorrhagic stroke remainnormal.

rtPA has been demonstrated to improve outcomes in acuteischemic stroke when delivered within 3 h of symptom onset.However, patients were taken to hospital mostly more than3 h after symptom onset (Kent et al., 2003). And patients weretreated mostly between 3 and 6 h after symptom onset.In some clinical trials, candidate biomarkers were analyzedin patients with suspected stroke admitted within 24 hsymptom onset (Foerch et al., 2009). Within the first 6 h aftersymptom onset, some biomarkers best differentiated ICHfrom IS (Allard et al., 2004; Dambinova et al., 2003; Foerchet al., 2006). So as first step, we selected these two time points(3 and 6 h). In our parallel clinical study, most patients arrivedin hospital within this time range.

an's quantitative ELISA assays. (A) SBDP145 in CSF showed aham. nPo0.05, N¼8. (B,C) UCH-L1 was elevated in CSF (B) and

b r a i n r e s e a r c h 1 5 4 0 ( 2 0 1 3 ) 8 4 – 9 188

UCH-L1 is known as neuronal-specific protein gene pro-duct 9.5 (PGP9.5) and was previously used as a histologicmarker for neurons because of its high abundance andspecific expression in neurons (Jackson and Thompson,1981; Larsen et al., 1996; Wilkinson, 1997). UCH-L1 is presentin almost all neurons and represents 1–5% of total solublebrain protein. It is a 24-kDa protein with no known activetransport mechanism. It is likely that breakdown of the bloodbrain barrier (BBB) following brain ischemia or trauma isresponsible for its release into the circulating blood. In thisstudy, the potential of UCH-L1 as a biofluid-based proteinbiomarker for detection of ischemic and hemorrhagic strokewas evaluated. UCH-L1 was confirmed as being detectable inrat CSF as its levels were significantly elevated after MCAO,according to Western blot and ELISA analyses. Furthermore,UCH-L1 was present at elevated levels in serum at 3 h and 6 hafter ischemic stroke as determined by ELISA. This result issimilar with previous reports that UCH-L1 becomes elevatedin the ischemic/reperfusion rat model (Liu et al., 2010). SerumUCH-L1 was suggested as an excellent marker for BBB func-tion (Liu et al., 2010). The presence of UCH-L1 in serum fromischemic animals suggests that the BBB was disrupted inthese animal models.

This study showed that UCH-L1 became elevated in CSF at3 and 6 h after ischemic stroke as determined by ELISA.However, the elevated level of UCH-L1 was only detected inCSF at 6 h after ischemic stroke but not at 3 h as detected byWestern blot. There is a general correlation between CSFlevels determined by Western blot and by swELISA. This ispossibly due to the semi-quantitative and less sensitivenature of the Western blot technique. This result suggeststhat a swELISA was more sensitive to quantitatively detectUCH-L1 in the CSF. By measuring the UCH-L1 levels in rat CSFand serum, it was possible to readily distinguish ischemicstroke from controls and hemorrhagic stroke as early as 3 h.This also suggested that UCH-L1 levels can be used as anearly biomarker of ischemic induced injury.

The level of UCH-L1 is elevated in CSF and serum of theischemic stroke model. However, we did not find any changein UCH-L1 in the brain as determined by Western blot (Fig. 2).This is probably due to the immediate release of UCH-L1 intothe biofluids after brain injury causing us to miss the precisetime point for detection. Furthermore, another possible rea-son for not finding any change in the level of UCH-L1 is due tothe insensitivity of the Western Blot, thus it might still bepossible for early monitoring of brain UCH-L1 levels. Theconcentration gradient between the brain and CSF and serumshould decrease in the biofluids after injury and BBB leakage.Therefore, brain UCH-L1 levels are expected to remain thesame before and after ischemic and hemorrhagic stroke ifthere are no changes in protein expression levels or noproteolysis of UCH-L1 (see Fig. 2A). It is likely that UCH-L1crosses the compromised BBB after injury because UCH-L1 isdetectable in CSF and serum shortly after MCAO models (3 h).

αII-spectrin is primarily found in neurons and is abundantin axons and presynaptic terminals (Riederer et al., 1986).Importantly, αII-spectrin is a major substrate for cysteineproteases involved in necrotic (calpain) and apoptotic (cas-pase-3) cell death (Wang et al., 1998). There is considerableevidence that αII-spectrin is processed to cleavage products,

also known as spectrin breakdown products (SBDP), of mole-cular weight 150 kDa (SBDP150) and 145 kDa (SBDP145) bycalpain. In addition, it is cleaved to a major cleavage productof molecular weight 120 kDa (SBDP120) by caspase-3 (Weisset al., 2009). Calpain and caspase-3 are major factors causingnecrotic and apoptotic cell death, respectively, during ische-mia or TBI (Pike et al., 2004; Pineda et al., 2007). Furthermore,there is considerable evidence of the presence of SBDPsin vitro neuronal cell culture models of injury (Beer et al.,2000), in preclinical studies of TBI in mice (Hall et al., 2005),and in human studies of TBI (Cardali and Maugeri, 2006;Pineda et al., 2007) and subarachnoid hemorrhage (Lewiset al., 2007). Our data showed that SBDP150/145 was signifi-cantly up-regulated in ischemic stroke model, but SBDP120was not changed. Similar trends are also observed in CSF forischemic stroke group (Fig. 3). The results suggest thatnecrosis is the major pathological process in the early stageof stroke-induced brain injury, thus we propose that SBDPmight have the potential to be a mechanistic biomarker foracute brain injury. Comparing the expression of SBDP in thebrain and CSF of MCAO rat, we found that the elevated levelwas detected in brain at 3 and 6 h, but not in CSF at 3 h. Thisis probably due to the relative larger molecular mass (150/145 kDa) that may not allow the molecule to easily cross thedisrupted BBB. There is no ELISA kit to detect SBDP in theserum of rat. For further study of SBDP as a potentialbiomarker with proven clinical usefulness for diagnosis ofacute stroke, we will develop an ELISA assay kit to detectSBDP150/145 in rat serum. Based on the present study,spectrin breakdown products appear to be useful biomarkersto distinguish between ischemic and hemorrhagic stroke inrat brain tissue.

Our data suggest that UCH-L1 and SBDP might join agrowing list of candidate protein biomarkers that distinguishischemic stroke and intracerebral hemorrhagic stroke. Othermarkers include, but are not limited to, neuron-specificenolase (NSE) (Cunningham et al., 1991), astroglial proteinS-100β (Takahashi et al., 1999), myelin basic protein (MBP)(Matias-Guiu et al., 1986), and glial fibrillary acidic protein(GFAP) (Dvorak et al., 2009). It is a possibility that a panel ofmarkers might emerge as the best diagnostic tool (Whiteley,2011), but we caution that such a hypothesis will need to becarefully examined in clinical studies.

In conclusion, this study provides evidence for the use ofUCH-L1 and SBDP as potential biomarkers to distinguishbetween ischemic and hemorrhagic stroke.

4. Experimental procedures

4.1. Rat stroke models

The experimental design and all procedures are in accor-dance with the Animal Care Guidelines established by theAnimal Experimental Committee of Xuanwu Hospital Capi-tal Medical University and the National Institute of Health.280–300 g male Sprague-Dawley rats were used in this study.A total of 40 rats were used in this study. Eight rats wereused in each group. The sham rats only received anesthesiaand had their skull and neck muscles exposed without

b r a i n r e s e a r c h 1 5 4 0 ( 2 0 1 3 ) 8 4 – 9 1 89

any ischemic nor hemorrhagic surgery. For conducting focalischemic stroke, middle cerebral artery occlusion (MCAO)was used without reperfusion. MCAO was induced using theintraluminal filament model described by Longa et al. (1989).Successful MCAO was confirmed in vivo using DRT4 laserDoppler flowmetry (PERIMED 5000, Sweden) (Ding et al.,2002). Collagenase VII injections were used to induce intra-cerebral hemorrhage (ICH) for hemorrhagic stroke evalua-tion (Matsushita et al., 2000). Rats were sacrificed at two timepoints after injury 3 and 6 h.

4.2. Methods for CBF, neurological deficits, brain infarctand brain hemorrhage

To verify MCA occlusion, local cerebral blood flow (CBF) incortex supplied by the right MCA was measured by LDF(Vasamedics Inc.) before (baseline) and during occlusion. Asmall craniectomy was made to expose the right parietalcortex. The LDF probe was stereotaxically placed on theexposed and intact dura 3 mm posterior and 5 mm lateralto bregma. Changes in regional CBF during ischemia andreperfusion were calculated as a percentage of the pre-ischemic baseline (Ding et al., 2002).

Neurological deficits in rats were examined after ischemiaand intracerebral hemorrhagic surgery. The deficits werescored using a modified scoring system (Longa et al., 1989)and is as follows: 0, no deficits; 1, difficulty in fully extendingthe contralateral forelimb; 2, unable to extend the contral-ateral forelimb; 3, mild circling to the contralateral side; 4,severe circling; and 5, falling to the contralateral side. Ratsthat did not display neurologic deficit were removed from theexperiment.

Three or six hours after surgery, the rats were anesthe-tized with 1% chloral hydrate. Following the anesthetization,the brains were removed and sectioned coronally at the levelof the optic chiasm (2 mm posterior to the optic chiasma)generating one section. For IS stroke, the 2-mm-thick sectionwas stained with a 2% solution of 2,3,4-triphenytetrazolium-chloride (TTC). For ICH stroke models, the 2-mm-thick sectionwas used for observation of intracerebral hemorrhage. Thenthe brain slice was photographed using a camera.

The ipsilateral hemisphere was rapidly dissected (cerebel-lum was not included), rinsed in ice-cold PBS, snap-frozen inliquid nitrogen, and stored at �80 1C until used.

4.3. Rat CSF and serum collection

CSF was collected from rats as described previously by ourgroup (Liu et al., 2010). Briefly, the head of the rat was flexedso that the external occipital protuberance in the neck wasprominent, and a dorsal midline incision was made over thecervical vertebrae and occiput. The atlantooccipital mem-brane was exposed by blunt dissection, and a 25-gauge needleattached to polyethylene tubing was carefully inserted intothe cisterna magna. A proximately 0.1–0.15 mL of CSF wascollected from each rat. CSF samples were centrifuged at4000g for 4 min at 4 1C to clear any contaminating red bloodcells. Cleared CSF samples were stored at �80 1C until readyfor use. Blood (5 mL) was collected directly from the inferiorvena cava using a syringe equipped with a 21-gauge needle

placed in a polypropylene tube and allowed to stand for 2 h at4 1C to form clots. Tubes were centrifuged for 7 min at 3000gand then serum was removed and stored at �80 1C. Atharvest, brains were collected and divided hemisphericallyinto injured (right) sides and contralateral (left) sides andstored at �80 1C.

4.4. Western blotting

Frozen brain tissue was pulverized into powder using amortar and pestle on dry ice. Equal amounts of powder werethen transferred to 1.5 mL tubes and extracted with lysisbuffer (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mMEGTA, 1% TX-100, 1 mM DTT, 1� Roche complete proteaseinhibitors, Sigma phosphatase inhibitors) for 4 h at 4 1C.Primary antibodies used were mouse anti-αII-spectrin (a.k.a.α-fodrin, Biomol) and UCH-L1 (mouse monoclonal and rabbitpolyclonal, Banyan). Equal protein loading was confirmed andadjusted using β-actin (goat polyclonal anti β-actin antibody,1:1000; Santa Cruz Biotechnology, Inc.). Targeted antigenswere visualized using standard chemical luminescencemethods (Amersham ECL; GE Healthcare BioSciences, Piscat-away, NJ). Quantitative evaluation of biomarker levels onimmunoblots was performed via computer-assisted densito-metric scanning (Epson high resolution flatbed scanner)followed by image analysis with Image-J software. Therelative density of sham is expressed as 1. Other proteindensities were expressed as the relative value to sham.

4.5. Biomarker sandwich ELISA assays

CSF and plasma samples were analyzed with Banyan Bio-marker's proprietary sandwich enzyme-linked immunosor-bent assays (swELISA) for SBDP145 and UCH-L1. Selectedcapture and detection antibody pairs were used. Briefly, ELISAplates were passively coated overnight at 4 1C with captureantibody, then washed and blocked. CSF or serum sampleswere added and incubated at room temperature with shak-ing. After washing, peroxidase-conjugated detection antibodyor streptavidin-HRP conjugates were added which catalyzedthe reaction with a colorimetric substrate (TMB, Pierce), andthe product was quantified by absorbance at 450 nm in amicroplate spectrophotometer. Standard curves were gener-ated using recombinant proteins corresponding to the bio-marker measured in each assay. Four parameter-fit non-linear regression analyses were applied to determine biomar-ker quantities.

4.6. Statistical analyses

Primary quantitative data from immunoblotting and ELISAassays were compiled and charted in Excel and the statisticalanalysis was applied using GraphPad Prism 5.0. One-wayANOVA was used to determine significance followed by theBonferroni post test, charts display means and standarderrors. In all cases, Po0.05 was the criterion for significanceand P values were calculated relative to sham. In all casesasterisks were used to denote P values as *Po0.05.

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Acknowledgments

This work is supported by the Program of National NaturalScience Foundation of China (81171241) and the Program ofExcellent Talents of Beijing (2011D005018000007).

r e f e r e n c e s

Allard, L., Lescuyer, P., Burgess, J., Leung, K.Y., Ward, M., Walter,N., Burkhard, P.R., Corthals, G., Hochstrasser, D.F., Sanchez,J.C., 2004. ApoC-I and ApoC-III as potential plasmatic markersto distinguish between ischemic and hemorrhagic stroke.Proteomics 4, 2242–2251.

Beer, R., Franz, G., Srinivasan, A., Hayes, R.L., Pike, B.R., Newcomb,J.K., Zhao, X., Schmutzhard, E., Poewe, W., Kampfl, A., 2000.Temporal profile and cell subtype distribution of activatedcaspase-3 following experimental traumatic brain injury.J. Neurochem. 75, 1264–1273.

Brophy, G.M., Pineda, J.A., Papa, L., Lewis, S.B., Valadka, A.B.,Hannay, H.J., Heaton, S.C., Demery, J.A., Liu, M.C., Tepas III.,J.J., Gabrielli, A., Robicsek, S., Wang, K.K., Robertson, C.S.,Hayes, R.L., 2009. alpha II-Spectrin breakdown productcerebrospinal fluid exposure metrics suggest differences incellular injury mechanisms after severe traumatic braininjury. J. Neurotrauma 26, 471–479.

Cardali, S., Maugeri, R., 2006. Detection of alpha II-spectrin andbreakdown products in humans after severe traumatic braininjury. J. Neurosurg. Sci. 50, 25–31.

Chalela, J.A., Kidwell, C.S., Nentwich, L.M., Luby, M., Butman, J.A.,Demchuk, A.M., Hill, M.D., Patronas, N., Latour, L., Warach, S.,2007. Magnetic resonance imaging and computed tomographyin emergency assessment of patients with suspected acutestroke: a prospective comparison. Lancet 369, 293–298.

Cuadrado, E., Rosell, A., Colome, N., Hernandez-Guillamon, M.,Garcia-Berrocoso, T., Ribo, M., Alcazar, A., Ortega-Aznar, A.,Salinas, M., Canals, F., Montaner, J., 2010. The proteome ofhuman brain after ischemic stroke. J. Neuropathol. Exp.Neurol. 69, 1105–1115.

Cunningham, R.T., Young, I.S., Winder, J., O'Kane, M.J., McKinstry,S., Johnston, C.F., Dolan, O.M., Hawkins, S.A., Buchanan, K.D.,1991. Serum neurone specific enolase (NSE) levels as anindicator of neuronal damage in patients with cerebralinfarction. Eur. J. Clin. Invest. 21, 497–500.

Dambinova, S.A., Khounteev, G.A., Izykenova, G.A., Zavolokov,I.G., Ilyukhina, A.Y., Skoromets, A.A., 2003. Blood testdetecting autoantibodies to N-methyl-D-aspartateneuroreceptors for evaluation of patients with transientischemic attack and stroke. Clin. Chem. 49, 1752–1762.

Ding, Y., Li, J., Rafols, J.A., Phillis, J.W., Diaz, F.G., 2002.Prereperfusion saline infusion into ischemic territory reducesinflammatory injury after transient middle cerebral arteryocclusion in rats. Stroke 33, 2492–2498.

Dvorak, F., Haberer, I., Sitzer, M., Foerch, C., 2009.Characterisation of the diagnostic window of serum glialfibrillary acidic protein for the differentiation of intracerebralhaemorrhage and ischaemic stroke. Cerebrovasc. Dis. 27,37–41.

Foerch, C., Curdt, I., Yan, B., Dvorak, F., Hermans, M., Berkefeld, J.,Raabe, A., Neumann-Haefelin, T., Steinmetz, H., Sitzer, M.,2006. Serum glial fibrillary acidic protein as a biomarker forintracerebral haemorrhage in patients with acute stroke.J. Neurol. Neurosurg. Psychiatry 77, 181–184.

Foerch, C., Montaner, J., Furie, K.L., Ning, M.M., Lo, E.H., 2009.Invited article: searching for oracles? Blood biomarkers inacute stroke. Neurology 73, 393–399.

Hall, E.D., Sullivan, P.G., Gibson, T.R., Pavel, K.M., Thompson, B.M.,Scheff, S.W., 2005. Spatial and temporal characteristics ofneurodegeneration after controlled cortical impact in mice:more than a focal brain injury. J. Neurotrauma 22, 252–265.

Jackson, P., Thompson, R.J., 1981. The demonstration of newhuman brain-specific proteins by high-resolution two-dimensional polyacrylamide gel electrophoresis. J. Neurol. Sci.49, 429–438.

Kent, D.M., Ruthazer, R., Selker, H.P., 2003. Are some patientslikely to benefit from recombinant tissue-type plasminogenactivator for acute ischemic stroke even beyond 3 hours fromsymptom onset?. Stroke 34, 464–467.

Larsen, C.N., Price, J.S., Wilkinson, K.D., 1996. Substrate bindingand catalysis by ubiquitin C-terminal hydrolases:identification of two active site residues. Biochemistry 35,6735–6744.

Lescuyer, P., Allard, L., Zimmermann-Ivol, C.G., Burgess, J.A.,Hughes-Frutiger, S., Burkhard, P.R., Sanchez, J.C.,Hochstrasser, D.F., 2004. Identification of post-mortemcerebrospinal fluid proteins as potential biomarkers ofischemia and neurodegeneration. Proteomics 4, 2234–2241.

Lewis, S.B., Velat, G.J., Miralia, L., Papa, L., Aikman, J.M., Wolper,R.A., Firment, C.S., Liu, M.C., Pineda, J.A., Wang, K.K., Hayes,R.L., 2007. Alpha-II spectrin breakdown products inaneurysmal subarachnoid hemorrhage: a novel biomarker ofproteolytic injury. J. Neurosurg. 107, 792–796.

Liu, M.C., Akinyi, L., Scharf, D., Mo, J., Larner, S.F., Muller, U., Oli,M.W., Zheng, W., Kobeissy, F., Papa, L., Lu, X.C., Dave, J.R.,Tortella, F.C., Hayes, R.L., Wang, K.K., 2010. Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic andtraumatic brain injury in rats. Eur. J. Neurosci. 31, 722–732.

Lloyd-Jones, D., Adams, R.J., Brown, T.M., Carnethon, M., Dai, S.,De Simone, G., Ferguson, T.B., Ford, E., Furie, K., Gillespie, C.,Go, A., Greenlund, K., Haase, N., Hailpern, S., Ho, P.M.,Howard, V., Kissela, B., Kittner, S., Lackland, D., Lisabeth, L.,Marelli, A., McDermott, M.M., Meigs, J., Mozaffarian, D.,Mussolino, M., Nichol, G., Roger, V.L., Rosamond, W., Sacco, R.,Sorlie, P., Stafford, R., Thom, T., Wasserthiel-Smoller, S.,Wong, N.D., Wylie-Rosett, J., 2010. Executive summary:heart disease and stroke statistics—2010 update: a reportfrom the American Heart Association. Circulation 121,948–954.

Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989.Reversible middle cerebral artery occlusion withoutcraniectomy in rats. Stroke 20, 84–91.

Matias-Guiu, J., Martinez-Vazquez, J., Ruibal, A., Colomer, R.,Boada, M., Codina, A., 1986. Myelin basic protein and creatinekinase BB isoenzyme as CSF markers of intracranial tumorsand stroke. Acta Neurol. Scand. 73, 461–465.

Matsushita, K., Meng, W., Wang, X., Asahi, M., Asahi, K.,Moskowitz, M.A., Lo, E.H., 2000. Evidence for apoptosis afterintercerebral hemorrhage in rat striatum. J. Cereb. Blood FlowMetab. 20, 396–404.

Pike, B.R., Flint, J., Dave, J.R., Lu, X.C., Wang, K.K., Tortella, F.C.,Hayes, R.L., 2004. Accumulation of calpain and caspase-3proteolytic fragments of brain-derived alpha II-spectrin incerebral spinal fluid after middle cerebral artery occlusion inrats. J. Cereb. Blood Flow Metab. 24, 98–106.

Pineda, J.A., Lewis, S.B., Valadka, A.B., Papa, L., Hannay, H.J.,Heaton, S.C., Demery, J.A., Liu, M.C., Aikman, J.M., Akle, V.,Brophy, G.M., Tepas, J.J., Wang, K.K., Robertson, C.S., Hayes,R.L., 2007. Clinical significance of alphaII-spectrin breakdownproducts in cerebrospinal fluid after severe traumatic braininjury. J. Neurotrauma 24, 354–366.

Riederer, B.M., Zagon, I.S., Goodman, S.R., 1986. Brain spectrin(240/235) and brain spectrin (240/235E): two distinct spectrinsubtypes with different locations within mammalian neuralcells. J. Cell Biol. 102, 2088–2097.

b r a i n r e s e a r c h 1 5 4 0 ( 2 0 1 3 ) 8 4 – 9 1 91

Takahashi, M., Chamczuk, A., Hong, Y., Jackowski, G., 1999. Rapidand sensitive immunoassay for the measurement of serumS100B using isoform-specific monoclonal antibody. Clin.Chem. 45, 1307–1311.

Wang, K.K., Posmantur, R., Nath, R., McGinnis, K., Whitton, M.,Talanian, R.V., Glantz, S.B., Morrow, J.S., 1998. Simultaneousdegradation of alpha II- and beta II-spectrin by caspase 3(CPP32) in apoptotic cells. J. Biol. Chem. 273, 22490–22497.

Weiss, E.S., Wang, K.K., Allen, J.G., Blue, M.E., Nwakanma, L.U.,Liu, M.C., Lange, M.S., Berrong, J., Wilson, M.A., Gott, V.L.,Troncoso, J.C., Hayes, R.L., Johnston, M.V., Baumgartner, W.A.,2009. Alpha II-spectrin breakdown products serve as novelmarkers of brain injury severity in a canine model of

hypothermic circulatory arrest. Ann. Thorac. Surg. 88,543–550.

Whiteley, W., Chong, W.L., Sengupta, A., Sandercock, P., 2009.Blood markers for the prognosis of ischemic stroke: asystematic review. Stroke 40, e380–e389.

Whiteley, W., 2011. Identifying blood biomarkers to improve thediagnosis of stroke. J. R. Coll. Physicians Edinb. 41, 152–154.

Wilkinson, K.D., Deshpande, S., Larsen, C.N., 1992. Comparisonsof neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminalhydrolases. Biochem. Soc. Trans. 20, 631–637.

Wilkinson, K.D., 1997. Regulation of ubiquitin-dependentprocesses by deubiquitinating enzymes. FASEB J. 11,1245–1256.